Note: Descriptions are shown in the official language in which they were submitted.
~o 92/nx80~ ~ 1 ~ 2 0 9 ~ ~ 2 5 PCT/~S9~/08430
ERYTHROCYTES AND TEROMBO-ERYTHROCYTES
AS TARGET SPECIFIC AGENT8
The invention disclosed in this specification was
partially made with Government Support under research
grant NHLBI 19278 awarded by The National Institutes
of Health. The Government has certain rights in this
invention.
. :
1. FIELD OF THE INVENTION
The present invention is directed to a new
composition of matter called thrombo-erythrocytes,
which have the ability to bind selectively to
activated platelets but not to unactivated platelets.
The thrombo-erythrocytes are useful in controlling
bleeding in thrombocytopenic (blood platelet
deficient) mammals, and for the uptake and delivery of
labels, therapeutic agents and genetic materials to
selected targets. The invention further relates to
targeted erythrocytes and their uses in the uptake and
delivery of compounds.
2. BACKGROUND OF THE RELATED ART
2.1. CONTROL OF BLEEDING
Mammals control bleeding by producing platelets,
which are activated with agents that are produced or
released at the site of a wound. Activation is
necessary for the platelets to aggregate into clumps
or clots.
The activation of platelets is a complicated
process, which includes producing or exposing
receptors for the plasma protein fibrinogen on the
platelet surface. Fibrinoqen has multiple binding
sites, and binds two or more platelets simultaneously,
initiating the aggregation. A platelet receptor that
is present on the surface of platelets and becomes
exposed during the activation process is GPIIb/IIIa.
~092/OX80~ 2 0 9 5 9`2 5 PCT/~S91/08430 ~
Patients with low platelet counts often require
transfusions of platelets in order to control
bleeding. -
In 1910, Duke provided data suggesting that
transfusion of whole blood containing platelets could
arrest hemorrhage due to thrombocytopenia (Duke, 1910,
J. Am. Med. Assoc. 50:1185-1192). It was not,
however, until the 1950's that unequivocal data on the
efficacy of platelet transfusions were obtained in
animals made thrombocytopenic by treatment with total
body irradiation (Cronkite et al., 1959, in Progress
in Hematoloay, Vol. 2, Tocantins, ed, Grune and
Stratton, NY, pp. 239-257). The difficulties in
platelet procurement and storage led investigators to
seek alternatives to fresh platelets soon thereafter.
Studies performed with lyophilized platelets and
disintegrated platelets indicated that these products
failed to arrest bleeding (Cronkite et al., supra;
Jackson et al., 1959, J. Clin. Invest. 38:1689; Hjort
et al., 1959, Proc. Soc. Exp. Biol. Med. 102:31-35).
When phospholipids were found capable of substituting
for platelets in accelerating coagulation reactions,
the cephalin fraction of soy bean phosphatides was
investigated as a platelet substitute in
2~ thrombocytopenic children (Shulman et al., 1959, Ann.
N.Y. Acad. Sci. 75:195, Abstr.). Although a
preliminary report suggested clinical improvement in
some patients (Schulman et al., su~ra) animal studies
did not identify a benefit and this approach was
eventually abandoned (Kahn, et al., 1985 Blood 66:1-
12, Abstr.). Cryopreservation of autologous platelets
has been successfully utilized in patients with
problems related t~ alloimmunization and
isoimmunization (Schiffer et al., N. Enql. J. Med.
295:7-12), but the platelet yield is less than with
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~ 09~088~ 20`9`.~92.~ PCT/~S91/08~30
~ _ 3 -
fresh platelets, and the technique is limited by the
need for extra processing and the availability o~
storage space (Murphy, 1991, In Principles of
Transfusion Medicine, Williams & Wilkins, Baltimore,
Pp. 205-213).
Improved understanding of platelet physiology
has led to additional approaches to obtain a platelet
substitute. Several investigators have been able to
introduce platelet glycoproteins into liposomes for in
vitro experiments (Parise and Phillips, 1985, J. Biol.
Chem. 260:1750-1756; Baldassare et al., 1988, J. Clin.
Invest. 75:35-39; Rybak, 1986, Thromb. Haemostas.
55:240-245). More recently, Ryback and Renzulli
incorporated a deoxycholate extract of platelet
l~ membranes containing 15 proteins, including GPIb,
GPIIb/IIIa, and GPIV, into small (50-200 nm)
unilamellar liposomes prepared from either
sphingomyelin:phosphatidylcholine:monosialyloganglio-
side or egg phosphatide (Blood Suppl. 1:473a, abstr).
Intra-arterial injections of both preparations
decreased bleeding in thrombocytopenic rats to the
same extent as human platelets did, but neither
produced complete normalization of the bleeding time.
Interestingly, liposomes containing GPIIb/IIIa alone
were ineffective (Rybak and Renzulli, 1990, Blood
Suppl. 1:473a, Abstr.). This approach may provide
important mechanistic information but as a therapeutic
intervention it potentially suffers from the generic
problems of liposomes, including the possibility of
short in vivo survival and potential blockade of the
reticuloendothelial system (Kahn et al., 1985, Blood
66:1-12, Abstr.). Moreover, since platelets remain
the starting material, problems of platelet
procurement and the risks of transmitting infectious
diseases may not be eliminated. Finally, if whole
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~'092/08X04 ^` ` PCT/~iS9l/0843()~
- 4 - :
platelet extracts are required, immunogenicity may
limit the opportunity for repeat therapy because
platelets have class I HLA antigens (McFarland and
Aster, 1991, In Principles of Transfusion Medicine,
5 Williams & Wilkins, Baltimore, pp. 193-204), and some
platelet glycoproteins are polymorphic (Lopez and
Ludwig, 1991, Clin. Res. 39:327 a.s.).
Agam and Livne took an approach based on their
observations that passive, fixed platelets coated with
fibrinogen could function to augment platelet
aggregation of native, fresh platelets (Agam and -
Livne, 1983, Blood 61:186; A~am and Livne, 1984,
Thromb. Haemostas. 51:145-149; Agam and Livne, 1988,
Thromb. Haemostas. 59:504-506). They concluded that
the activated platelets had to undergo the release
reaction and èxpose thrombospondin on their surface in
order for the interactions to occur, with the final
interaction between the fibrinogen on the fixed
platelets and the thrombospondin on the activated
platelets (Agam and Livne, 1983, Blood 61:186; Agam
and Livne, 1984, Thromb. Haemostas. 51:145-149; Agam
and Livne, 1988, Thromb. Haemostas. 59:504-506). This
suggests that the fixation process alters the
fibrinogen so that it cannot interact directly with
GPIIb/IIIa, but leaves intact portions of the
fibrinogen molecule that can interact with
thrombospondin. This approach involves a significant
limitation in that it relies on the purification of
fibrinogen from plasma, and thus has the risk of
transmitting blood-borne disease. Moreover,
formaldehyde is a cytotoxic agent that may have
carcinogenic potential (Feron et al., 1991, Mutation
Res. 259:363-385) and so it may not be the most
desirable crosslinking reagent for in vivo use.
.: . . ,, , . , : .,
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,~.'0 92/08804 `2 0 ~`~ 9 2 5 PCI/~S91/08430
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Until the present invention, platelet transfusion
was the only effective therapy for the prevention and
treatment of hemorrhage due to thrombocytopenia
(Heyman et al., 1991, in Principles of Transfusion
5 Medicine, William & Wilkens, Baltimore, pp. 223-231).
The number of units of platelets transfused each year
in the United States has grown rapidly since the
widespread introduction of platelet transfusion
therapy in the 1960's; in fact, just between 1980 and
1987 the number nearly doubled, reaching in excess of
6 million units per year (Surgenor et al., 1990,
N. Enql. J. Med. 322:1646-1651). Despite its enormous
success, platelet transfusion therapy has a number of
very serious limitations and drawbacks: 1) supplies
are often limited due to difficulties in procurement
and the relatively short shelf life (5-7 days)
(Murphy, 1991, in Principles of Transfusion Medicine,
Williams ~ Wilkens, Baltimore, pp. 205-213); 2) there
is a risk of transmitting blood-borne pathogens such
as the viruses that produce hepatitis and AIDS,
especially since multiple units are usually
administered with each transfusion (Heyman et al.,
supra); 3) febrile reactions, presumably due to white
blood cell contaminants, are common in patients
receiving repeated transfusions (Snyder et al., 1991,
in PrinciPles of Transfusion Medicine, Williams &
Wilkensl Baltimore, pp. 641-648); 4) alloimmunization
results in patients becoming refractory to random
donor platelets, necessitating a switch to single
donor platelets matched for HLA antigens, and even HLA
matched platelet transfusions are not universally
successful (Hevman, et al., supra).
The interaction between fibrinogen and platelets
has been the subject of prior investigations. For
example, platelets interact with fibrinogen-coated
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~092/08804 2 0 9 S~2~ PCT/US91/08430 ~
6 -
polyacrylonitrile beads via a mechanism involving
fibrinogen receptors on platelet surfaces (see the
paragraph bridging pages 177 and 178 in Coller et al.,
1980, Blood, 55:169-178). Agam and Livne (1983, Blood
61:186-191) disclosed that fixed platelets to which
fibrinogen had been bound participated in the
aggregation of activated platelets, by selective
reaction with activated platelets. Ruoslahti et al.,
U S. Patent No. 4,792,525 suggested that the ability
of proteins such as fibrinogen to interact with cells
is associated with the amino acid sequence Arg-Gly-
Asp-Ser within the fibrinogen structure. The
Ruoslahti et al. patent further discloses that a
tetrapeptide consisting of the sequence Arg-Gly-Asp-
Ser, when properly immobilized on a substrate, has theproperty of causing cell attachment to the substrate.
The tetrapeptide could be extended with additional
amino acids at either end, and the possibility of very
limited substitution for the amino acids constituting
the tetrapeptide was suggested. A practical
application envisioned by Ruoslahti et al. was
platelet aggregation.
Despite the known role of platelets in
controlling bleeding and of the interaction between
fibrinogen and platelets, there are no current
procedures for controlling bleeding in
thrombocytopenic patients other than platelet
transfusions, with all the disadvantages of such
transfusions, as discussed above.
The availability of an abundant and safe
alternative to human platelets would, therefore, be of
considerable benefit. It is vital, however, that such
an alternative retain the platelet's specificity for
forming thrombi at sites of vascular injury, to be
certain that indiscriminate thrombus formation does
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W092/0~8~4 ~ ~ 9 ~ ~ 2 ~ PCT/ ~S91/08430
-- 7
not occur. Prior to the instant invention, no
a~undant safe alternative to human platelets has been
found.
Alternative procedures are needed in order to
5 reduce the large amount of blood necessary to obtain
sufficient platelets. The ideal procedure would
utilize a patient's own blood cells in order to reduce
the possibility of blood borne disease.
In addition, the precise delivery of radiolabeled
molecules, diagnostic, and therapeutic agents to
specific target tissues is an important laboratory and
clinical problem.
Accordingly, one objective of the present
invention is to solve the problems of obtaining cells
from a small amount of blood, particularly autologous
blood, that can be used to deliver precisely agents to
specific target issues.
Another objective is to provide compositions of
matter that are able to bind selectively to activated
platelets but not to unactivated platelets in vivo.
Activation refers to the process by which platelets
become more susceptible to aggregation. The process
by which platelets become activated is poorly
understood, especially, in vivo. It appears that the
activation process is induced by a number of agonists,
such as ADP, epinephrine, collagen, thrombin and
thromboxane A2. Indiscriminate binding of an agent to
both activated and unactivated platelets exposes the
patient to the risk of thrombosis (blood clots) that
can lead to the death of tissues in vital organs,
including the heart and brain.
3. SUMMARY OF THE INVENTION
The present invention provides new compounds and
methods for promoting platelet aggregation, and
.
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~092/0880~ ~ 9 ~ 9 2 ~ PCT/ ~S91/08430
preventing hemorrhage. The present invention is based
on the surprising discovery that erythrocytes
conjugated to certain peptides and polypeptides
containing an R-G-D (Arg-Gly-Asp) sequence
(collectively termed herein "RGD peptides"~ according
to the invention, selectively bind to activated
platelets but not to unactivated platelets. In
recognition of the dual nature of the derivatized
erythrocytes, they are termed herein "thrombo-
erythrocytes". The methods and compounds of theinvention overcome the problems with prior art
platelet substitutes by providing abundant, safe
material to promote platelet aggregation, specific for
sites of injury. By following the methods of the
; 15 instant invention, thrombo-erythrocytes are produced
which surprisingly, have no significant change in
their rheological properties. In addition, contrary
to expectations, and in a preferred aspect, the
thrombo-erythrocytes have the majority of RGD peptide
cross-linked specifically to glycophorin A and
glycophorin B on the surface of the erythrocyte,
producing a thrombo-erythrocyte that has an altered
membrane surface that can interact selectively with
activated platelets via the platelet GPIIb/IIIa
receptor. In the thrombo-erythrocytes of the
invention, preferably, the N-terminal Arg of the R-G-D
sequence should be spaced within g-50 Angstroms, more
prefera~ly 10-40 Angstroms, and most preferably 11-25
Angstroms, from the erythrocyte protein to which the
RGD peptide is conjugated. As a result, the activated
platelets aggregate with the erythrocytes, forming
clumps or clots. When such clumps or clots form in
vivo in mammals, including humans, they are helpful in
controlling kleeding, and are especially helpful in
controlling bleeding from small wounds.
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W092/08804 2 0 ~ ~ 9 2 ~ PCT/US91/08430
..~
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The invention is further directed to erthrocytes
mod~fied by replacement of their intracellular
contents with a composition comprising a label or
agent. Such modified erythrocytes are termed herein
"carrier erythrocytes". The carrier erythrocytes have
use in delivery of such labels or biologically active
agents to specific tissues by conjugation to a
targeting agent. In one embodiment, the carrier
erythrocytes are thrombo-erthrocytes, and are thus
targeted to a specific tissue, in particular an
activated platelet, by conjugation with an RGD peptide
in accordance with the present invention. In other
embodiments, different targeting molecules, such as
peptides, proteins, antibodies, antibody fragments,
lectins, carbohydrates, or steroids can be conjugated
to a carrier erythrocyte or, in particular, a carrier
thrombo-erythrocyte.
- 4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure l. Osmotic fragility of control
erythrocytes and thrombo-erythrocytes. The lysis of
control erythrocytes and thrombo-erythrocytes was
measured after a 20 min incubation in solutions
containing various salt concentrations. The results
are expressed in comparison to the hemolysis produced
by distilled water, which was defined as 100%. Data
are from 3 separate experiments.
Figure 2. Ektacytometer analysis of thrombo-
erythrocytes and control erythrocytes. Thrombo-
; 30 erythrocytes were prepared as described, with samples
removed after 15 min, 30 min, 60 min, and 120 min of
incubation. The thrombo-erythrocytes were then washed
in 0.15 M NaCl, l0 mM Tris/HCl, 5 mM KCl, l0 mM
glucose, 1% bovine serum albumin, pH 7.4, and
resuspended to a hematocrit of ~33%. Three different
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W092/0~80~ PCT/US91/08430
;~;'9;~:~2~ - lo ~
erythrocyte controls were prepared: 1) erythrocytes
that were just washed in the above buffer, 2)
erythrocytes incubated with peptide, but no
mal-sac-HNSA, and 3) erythrocytes incubated with
5 mal-sac-HNSA, but no peptide. The deformability index
of each sample was measured as a function of shear
rate in an isotonic medium of 22 cp viscosity. All of
the thrombo-eryt~rocyte samples and control samples
gave virtually superimposable curves, and so for
simplicity only the washed erythrocyte control and the
120 min thrombo-erythrocyte sample are shown. The
plateau deformability index (mean + SD) of more than
200 normal samples is also shown, indicating that all
of the samples tested were within this normal range.
Figure 3. Analysis of thrombo-erythrocyte
membrane proteins involved in peptide crosslinking.
After crosslinking the 3H-peptide to erythrocytes, the
cells were washed and lysed. The thrombo-erythrocyte
ghosts were then solubilized in sodium dodecyl sulfate
and electrophoresed in a 12.~% polyacrylamide gel.
The gel was sequentially stained with the periodic
acid-Schiff technique (P.A.S.), photographed, stained
with Coomassie blue, photographed, and then prepared
for fluorography by reactions with precipitating and
aqueous fluorescent solutions. The gel was
subsequently dried and placed a cassette with X-ray
film at -70C for 7 days. The P.A.S. stain revealed 3
major bands of Mr 87,000, 4~,000, and 22,000, which
corresponded to the radioactive bands identified by
fluOrography-
Figure 4. Platelet-thrombo-erythrocyte -
co-aggregation assay. Thrombo-erythrocytes and
control erythrocytes were prepared as described in
Section 8.1 and adjusted to a 10% hematocrit.
Citrated platelet-rich plasma was prepared (~500,000
WO92/OR804 2~9`5:~`25 PCT/~'S91/~8430
platelets/~l) and incubated with antibody 7E3 (anti-
GPIIb/IIIa + anti-~v~3 vitronectin receptor; 40 ~g/ml
final concentration), EDTA (lO mM final
concentration), RGDF (300 ~g/ml final concentration)
or buffer (0.15 M NaCl, O.Ol M Tris/HCl, 0.05~ Na
azidej pH 7.4) for 30 min at 22C. The assay was
performed by adding 50 ~l of PRP to microtiter wells,
followed by lO ~l of ADP to selected wells, and
finally 5 ~l of the thrombo-erythrocytes. The
microtiter plate was then rotated at 270 rpm at 22~C
for approximately 6 min and then the plate was
photographed. Note the absence of platelet
aggregation or platelet-erythrocyte co-aggregation in
the samples without ADP. With ADP treatment, the
thrombo-erythrocytes enter into mixed aggregates with
the platelets. ~areful inspection of the sample of
control erythrocytes with ADP stimulation shows small
white aggregates of platelets, indicating that
platelet activation and aggregation occurred, but the
control erythrocytes did not enter into the
aggregates.
Figure 5. Platelet-thrombo-erythrocyte
interactions. After performing the platelet-thrombo-
erythrocyte co-aggregation assay, samples were spread
on a glass slide, air-dried, and stained with a Wright
stain. Light microscopy was performed at l,OOOX
magnification with an oil immersion lens. Note the
intimate association between the platelets and the
thrombo-erythrocytes, with the platelets
interdigitated between the thrombo-erythrocytes. With
PRP + control erythrocytes, activation with ADP led to
platelet aggregates, but the control erythrocytes did
not enter into the aggregates.
Figure 6. Interactions of control erythrocytes
and thrombo-erythrocytes with gel-filtered platelets.
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U'092/08804 PCT/US9~tO8430
209~92~ - 12 - ~
Gel-filtered platelets (450 ~l; 340,000/~l), prepared
as described in Section 8.1, and control erythrocytes
or thrombo-erythrocytes (20 ~l; 10% hematocrit) were
stirred in an ag~regometer cuvette and then ADP (4.3
~M final concentration) was added. In this assay, the
erythrocytes contribute significantly to the optical
density. Control erythrocytes do not enter into
platelet aggregates and so there is only a slight
decrease in optical density in this sample. In
contrast, thrombo-erythrocytes do interact with the
ADP-activated platelets, resulting in a dramatic
decrease in optical density. The thrombo-erythrocytes
do not, however, interact with unactivated platelets
despite stirring at 37C. Finally, preincubating the
platelets with antibody lOE5, which reacts with
GPIIb/IIIa, blocks the platelet-platelet and
platelet-thrombo-erythrocyte-interactions. A mixture
of gel-filtration buffer (450 ~l) and control
erythrocytes (20 ~l) was used to establish the full
; 20 scale deflection.
Figure 7. Interactions of control erythrocytes
and thrombo-erythrocytes with platelets adherent to
collagen. Gel-filtered platelets were allowed to form
a dense lawn on collagen-coated microtiter wells and
then, after washing, control erythrocytes or thrombo-
erythrocytes (50 ~l; 10% hematocrit) were added to the
wells for 1 hour at 22C. Finally, non-adherent
control erythrocytes and thrombo-erythrocytes were --
remoYed by washing. With control erythrocytes, the
dense lawn of platelets can be seen with only a single
adherent erythrocyte in the field. In sharp contrast,
the thrombo-erythrocytes bound extensively to the
adherent platelets. The binding of thrombo-
erythrocytes to the adherent platelets was inhibited
by antibody lOE5 (20 ~g/ml), which is specific for
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~O 92/0880~ 2 0 9 ~9 2 ~ PC~/~is91/ox~3~
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GPIIb/IIIa, or the peptide RGDF (400 ~g/ml). The
experiment shown is representative of more than 12
separate experiments.
Figure 8 . A. Rating of (G) ~,~ RGDF bead
5 agglutination. Platelet-rich plasma (PRP; 70 ,ul) was
reacted with Gg-RGDF beads (5 ~1 containing 0 . 22 mg
beads) as described in Section 9.1 and roiated at 260
rpm. With increasing time, the agglutination became
more extensive. The examples shown were selected at
10 different time intervals to demonstrate the
semiquantitative scale used for judging the extent of
agglutination. Also shown are the platelet counts in
the supernatant fluid after allowing the bead
agglutinates to settle for 3 minutes.
B . (G) D-RGDF bead agglutination in PRP . The
experiment was conducted as above using G~-RGDF,
G3-RGDF, and Gg-RGDF beads. The reaction was stopped
at the time points indicated on the left and the
microtiter plate was photographed. The grading of the
20 extent of agglutination is indicated on the right of
each well. Note the minimal agglutination with the
G~-R&DF beads, the modest agglutination with the
G3-RGDF beads, and the extensive agglutination of the
G9-RGDF beads over the f irst 8 min.
Figure 9 . A. Agglutination of (G) n-RGDF beads by
PRP. The experiments were carried out with PRP and
Gl -PGDF ( n= 1 5 ), G3-RGDF ( n= 1 7 ), G5-RGDF ( n= 1 5 ), and
G9-RGDF (n=16) beads as indicated in the text. The
values plotted are the mean + SEM.
B. Agglutination of ~G) "-RGDF beads by PRP. The
experiments were carried out with PRP and G~3-RGDF
(n=9), G~7-RGDF (n=7), and G~g-RGDF (n-7) beads as
indicated in the text. The values plotted are the
35 mean + SEM.
., . . ~ .
W092/OX804 29S92S 14 PCr/US91/OX430
Figure lO . A. Agglutination of (G) ~-RGDF beads
by PRP pretreated with Al)P. PRP was incubated with
ADP (6.7 ~M) for 30 sec at 22C before adding G~-RGDF
(n=8), G3-RGDF (n=7), Gs-RGPF (n=8), or Gg-RGDF (n=9)
5 beads. The values plotted are the mean + SEM~
B. Agglutination of (G)n-RGDF beads by PRP
pretreated with PGE~. PRP was incubated with PGE~,
(0. 14 ~M) for 30 min before adding G~-RGDF (n=7),
G3-RGDF (n-7), G5-RGDF (n=7), or G9-RGDF (n=7) beads.
10 The values plotted are the mean + SEM.
C. Agglutination of G3-RGDF beads by native
platelets, platelets pretreated with ADP, and
platelets pretreated with PGE~. Experiments were
conducted as described in A and B above. The values
15 plotted are the mean + SEM.
Figure ll. A. Effect of decreasing the bead
surfat:e density of (G)9-RGDF peptides on agglutination
by PRP. The G9-RGDF peptide was coupled to beads at
the different millimolar concentrations indicated on
20 the graph. The efficiency of coupling was similar for
all of the peptides (see Table IV for coupling
efficiencies and Table V for maximal mean distances
between peptides). The agglutination of the beads by
PRP was then tested as indicated in the text. The
25 values plotted are the results of a single experiment.
B. Effect of decreasing G9-RGDF peptide density
on agglutination by PRP pretreated with ADP.
Experiment was conducted as indicated in A except that
the PRP was pretreated with ADP (6.7 ,uM) for 30 sec at
22 C before the beads were added. The values plotted
are the results of a single experiment.
Figure 12. Agglutination of G5-RGDF and Gl7-RGDF
beads by gel-filtered platelets (GFP) in the presence
35 and absence of ADP. Native GFP (70 ~1) was reacted
with either 5 ~l of Gs-RGDF beads ( n=5 ), or G~7-RGDF
, . . .. .. : .: ., .: -
U092/0880~ 2 0 9 ~;9~ 5 PCT/~S91/08430
- 15 -
beads (n=3). In other experiments, the GFP was
pretreated wlth ADP (6.7 ~M) for 30 sec at 22C before
adding the G5-RGDF beads (n=3) or G~7-RGDF beads (n=2).
The values plotted are mean + SEM.
Figure 13. A. Agglutination of (G)9-RGDF beads
by PRP pretreated with monoclonal antibodies 10E5,
A2~, and 7E3. Experiments were carried out with PRP
pretreated with 20 ~g/ml 10E5 (n=5), 20 ~g/ml A
(n=5), or 20 ~g/ml 7E3 (n=4) for 30-60 min. The
values plotted are the mean + SEM.
B. Agglutination of (G)g-RGDF beads by PRP
pretreated with monoclonal antibodies 10E5, A2~, or
7E3, followed by preactivation with ADP. Experiments
were carried out as in A except that the platelets
were pretreated with ADP (6.7 ~M) for 30 sec before
adding the beads. The values plotted are the mean +
SE~ .
Figure 14. Scheme 1: Preparation of thrombo-
erythrocytes.
Figure 15. Scheme 2: Preparation of thrombo-
erythrocytes.
Figure 16. Scheme 3: Two-step method for the
preparation of thrombo-erythrocytes.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to thrombo-
erythrocytes, which are erythrocytes conjugated as
provided herein to a RGD-containing peptide or
polypeptide ("the RGD peptide"), and which are able to
bind selectively to activated, but not to unactivated,
platelets, causing co-aggregation of the activated -
platelets and thrombo-erythrocytes. This highly
selective binding to activated platelets is in
contrast to the behavior of RGD peptides in solution
or long RGD peptides on the surface of beads, which
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~092/0~80~ ~ ~ l6 - ~~
bind to both activated and non-activated platelets
with much less selectivity. The specificity of the
thrombo-erythrocytes for activated platelets can
exhibited in vitro in the absence of an exogenous
activating agent (see Section 8, infra). The thrombo-
erythrocytes of the invention overcome the problems
associated with prior art platelet substitutes by
providing an abundant, safe material to promote
platelet aggregation ln vivo, specific for sites of
injury. Thus, bleeding can be controlled, and
hemorrhage can be prevented. In a preferred aspect
involving the administration of autologous thrombo-
erythrocytes, the possibility of infectious agent
transmittal and adverse allo-immune reactions present
in prior art methods are thus avoided.
The thrombo-erythrocytes bind to activated
platelets via a specifically spaced R-G-D sequence in
a peptide conjugated to the erythrocytes. The
distance from the erythrocyte to the N-terminal end of
Arg within the RGD peptide influences the binding
profile, and is preferably about 9 to about 50
Angstroms, more preferably about lO to about 40
Angstroms, and most preferably about ll to about 25
Angstroms. The distance is estimated by considering
the crosslinker and peptide sequence at the N-terminus
of Arg as a linear molecule using standard bond
lengths, assuming an extended conformation for the
amino acids in the polypeptide. The distance
represents the length of the segment from the covalent
bond between the erythrocyte and linker molecule,
including the bond length, to the N-terminal end of -
Arg in the RGD peptide.
Surprisingly, the thrombo-erythrocytes also
exhibit rheological properties which do not
significantly differ from those of untreated red blood
:
, ~ : ,
~ ' , . . .. :
~09~/OX~0~ 2 0 9 ~ 9 2 5 PCr/~s9l/0~3~ 1
. . ;
- 17 -
cells. Furthermore, in a preferred aspect of the
invention, the thrombo-erythrocytes surprisingly have
the majority of the RGD peptides cross-linked to
glycophorin A and glycophorin B on the erythrocyte
cell surface. In this aspect, the specific binding to
glycophorin provides important advantages because it
is present in very high copy number on the erythrocyte
surface (600,000 to 1 million per red blood cell),
which allows highly effective binding of the RGD
peptide-linker. Furthermore, since glycophorin has no
definable role in erythrocyte physiology, it
presumably can have the RGD-peptide linker bound to it
without distortion of the erythrocyte's physiological
or rheological properties.
In another embodiment of the invention, targeted
thrombo-erythrocytes and targeted erythrocytes are
provided, by conjugation of a targeting molecule such
as an antibody or physiological ligand to the thrombo-
erythrocytes or erythxocytes. In a preferred aspect,
2G the targeted erythrocytes are carrier erythrocytes,
which have been treated to release their contents
(form erythrocyte "ghosts") and then incorporate an
agent before resealing of their membrane, so that they
can be used as ln vivo delivery vehicles for their
internalized agent. As used herein, the term
"targeting molecule" refers to a molecule that can be
conjugated to an erythrocyte (or thrombo-erythrocyte)
and that binds specifically to a molecule found in
vivo, such as a receptor or other recognition molecule
or a molecule specific to a cell or cells, etc. In a
specific embodiment, the targeting molecule is a
peptide, e.g., a peptide containing the sequence Arg-
51y-Asp (R-G-D). In an embodiment where such a
peptide is as described in Section 5.1.1 and has been
conjugated to an erythrocyte as described in Section
,: . . ................. ,. , ~ : - :
,
~ 092/~#80~ 2~.JSt92~ 18 PCT/US91/0~30
5.1, resulting in the cell's retention of rheological
properties and specificity for activated platelets,
the cell is a targeted thrombo-erythrocyte. Such a
targeted thrombo-erythrocyte will react with activated
5 platelets in a thrombus, allowing imaging of the
thrombus or the delivery of therapeutic agents to the
thrombus. However, a targeted erythrocyte need not be
a thrombo-erythrocyte. In another embodiment, the
targeting molecule is an antibody, or fragment of an
antibody, a lectin, a steroid or a carbohydrate. More
than one targeting molecule can be used, for example,
by using two different molecules to target an
erythrocyte to the same ln vivo location.
5.1. PREPARATION OF THE THROMBO-ERYTHROCYTES
In order to produce thrombo-erythrocytes, a
polypeptide according to the present invention is
prepared and covalently conjugated to an erythrocyte
through a polyfunctional molecule according to methods
described nfra. However, it should be noted that
upon completion of the conjugation reaction, the
thrombo-erythrocytes should be tested for their
unusual ability to retain both normal rheological ~ -
properties and the platelet's specificity for forming
thrombi at sites of vascular injury, i.e., for their
ability to interact selectively with activated
platelets. The lack of significant difference in the
rheological properties displayed by the thrombo-
erythrocytes of the invention and those of untreated
erythrocytes can be o~served by detecting a lack of
significant difference between the thrombo-
erythrocytes and untreated red blood cells in one or
more of the foilowing characteristics: surface/volume
ratio, internal cell water, and/or membrane shear
rigidity, as tested by laser diffraction ektacytometry
. .-. .ri, '. . ........... . .
' '. : -' ' ' ' ' , . '; ' '"1'`~-'~`' "' ' ' '
:. ' ~; ,' ' .
.
~092/0~0~ 2 0 9 ~ 9 2 ~ PCT/~sgl/o~3n
~. -- 19 --
as described in Example 8, infra, or by other methods
known in the art. Examples of ln vitro assays that
can be used to demonstrate the ability of the thrombo-
erythrocytes to bind selectively to activated
5 platelets are described in Sections 8.1.5. and 8.1.8.
infra). In a preferred aspect, the thrombo-
erythrocytes of the invention are mainly conjugated
via glycophorin A and glycophorin B on the cell
surface, and have the ability, via their conjugated
RGD peptides to interact with the GPIIb/IIIa receptor
on activated platelets.
Certain mammals may provide the erythrocytes for
preparation of thrombo-erythrocytes. Human and monkey
erythrocytes are preferred for use, while baboon, dog,
and rat erythrocytes do not appear to be useful.
Erythrocytes may be purified and concentrated by
methods that are known in the art. Typically, by way
of example but not limitation, blood is removed from a
patient and added to an anti-coagulant such as
citrate. The blood is then centrifuged, and the
plasma supernatant is removed with a pipet, leaving
the erythrocytes. Buffer of about pH 6 to about pH 8
and about 0.15 N ionic strength, preferably phosphate
buffered saline (PBS), may be added and the mixture
re-centrifuged to wash the erythrocytes. This process
is repeated several times until the erythrocytes are
sufficiently pure. However, to avoid damaging the
erythrocytes, the washing steps are kept to a minimum.
The polypeptide and the erythrocytes are then
each covalently bonded to a polyfunctional molecule.
All operations are preferably performed in aqueous
solution in order to avoid lysing the erythrocytes,
which are sensitive to organic solvents. The pH
should be between 6 and 8, preferably between 6.5 and
7.5.
- ~ : : . , :
2D95925
W092/08804 PCT/US91/OX430
;~
It is preferable to use a heterobifunctional
cross-linking reagent that reacts directly with each
type of group. A heterobifunctional reagent that
works well is Mal-Sac-HNSA (N-maleimido-6-aminocaproyl
ester of l-hydroxy-2-nitrobenzene-4-sulfonic acid
sodium salt), which may be obtained from Bachem
Biosciences, Inc., Philadelphia, PA. Other cross-
linking agents known in the art can be used, and are
described in Section 5.l.2 infra, as long as the
resultant cell is tested for the retention of
rheological properties and specificity of binding to
activated platelets associated with the thrombo-
erythrocytes of the invention.
It was initially believed that the order of
adding the RGD polypeptide and the erythrocyte to the
polyfunctional molecule is not critical. Accordingly,
initially the three components were simply combined in
one reaction mixture, as illustrated in Section 6.
The dynamics of the one-step reaction described
in Section 6 showed that the thrombo-erythrocytes of
the present invention can be prepared and that they
bind to activated platelets. However, the dynamics of
the one-step reaction indicate that this one-step
method is unpredictable. First, a sulfhydryl (thiol)
group of the erythrocyte could react with the Mal-Sac-
HNSA linker, rather than the desired reaction between
the sulfhydryl (thiol) groups of the peptide and the
Nal-Sac-HNSA linker. Cross-linking of erythrocyte
cell-surface proteins would result. This potentially
competing and undesirable reaction may damage the
erythrocytes, and would make less linker available for
binding to the peptide.
Accordingly, a more preferred two-step method was
devised and tested, as shown in Scheme 3 (Figure 16)
and described in Section 7. In the preferred two-step
~o g~ g80~ 5 ~ 2 ~ PCT/~S91/0~130
- 21 -
method, the RGD polypeptide-linkers are prepared flrst
separately, and then subsequently reacted with
proteins on the erthrocyte. For example, this can be
carried out as follows: the erythrocytes are
5 maintained in a ~uffer solution at about pH 7.4. This
prevents any osmotic damage to the erythrocytes. The
polypeptide-linker is prepared separately at a pH of
about 6Ø After conjugating the peptide sulfhydryl
of the linker, the pH of the reaction solution is
raised to a pH of about 7.4. The peptide-linker
complex in solution at pH 7.4 can be added to an
erythrocyte suspension, thus allowing free amino
groups on the erythrocyte proteins to react with the
second reactive group on the linker. The peptide
linker complex can be lyophilized and stored for later
use.
Alternatively, a peptide-linker complex is
chemically synthesized by attaching the cross-linking
group as a subsequent step after peptide synthesis, by
standard chemical methods. This complex is suitable
in the present invention as long as the linker which
is attached to the peptide has an attachment point
that is available for linking to reactive functional
groups of the erythrocyte. In a specific aspect, the
RGD peptide-linker intermediates can be stored for
later use in conjugation to erythrocytes.
To ensure that the density of RGD peptides on the
erythrocytes is high enough to support the reaction
with platelets, the peptide must be added in great
molar excess to the erythrocytes. For example, the
molar excess of RGD peptides added to the erythrocytes
should be approximately 0.5 x lO8 to approximately 20 x
lO8, preferably approximately l x lO8 to approximately
lO x lO8, and more preferably approximately 3 x 108 to
approximately 7 x lO8. Preferably, the number of
- -. - - . : .. -.- : : . : :: :: ~ : . -.-
.: , . ; , ' ' . . '
- -: ~; : . : . ::
. '; . - . ~ .' ' ' '' : ~ ' :
. . :, , :
,
~'09~/0X8~ 2 09 ~9 2 S - 2~ - PCr/l591/08~30
polypeptides attached to each erythrocyte should be
approxi~ately o.oS x l0~, preferably approximately l x
l06, to approximately 20 x l06, although it is possible
that as few as 0.0l x 106 attached polypeptides will
yield a functional thrombo-erythrocyte.
Preferably, after the conjugation reaction is
- complete, excess cross-linker is removed by thorough
washing. Additionally, albumin or autologous serum
can be added during the ~ashing procedure to react
with any remaining reactive sites, and then be removed
in the wash step.
The erythrocytes have both amino and sulfhydryl
groups exposed on their surfaces. Either of these
groups may be used to form the covalent bond to one of
l~ the functional groups of the polyfunctional molecule.
Alternatively a carboxylic acid group can be used to
form a covalent bond to one functional group of the
polyfunctional molecule, e.g., via carbodiimide
activation. Another functional group of the
- 20 polyfunctional molecule is covalently bonded to the
RGD peptide. Preferably, an amino group will usually
form the bond to the polyfunctional molecule. In a
specific aspect, when the site of attachment on the
RGD peptide is cysteine, either the amino group or the
sulfhydryl group may be bonded to the polyfunctional
molecule. Where bonding is to the sulfhydryl group,
the amino group should be protected, e.g., by
acetylation.
5.l.l. THE RGD PEPTIDES OF THE INVENTION
The RGD peptide for conjugation to erythrocytes
in accordance with the present invention includes a
sequence of a~ino acids, prefera~ly naturally
occurring L-amino acids and glycine, having the
following formula (I):
. . . .. . , , ,, :
. - , : . . ~ ,
., , , . , ~. . : :
- ~' ' . :
~ 09~0~80~ 2 Q 9 5 9 2 5 PCT/~iS91/0~30
- 23 -
R~-Arg-Gly-Asp-R2
I
in which Rl represents an amino acid or a
sequence of more than one amino acid;
in a specific embodiment, Rl represents
XY(Z)n~ in which X, Y and Z independently
represent an amino acid; and n represents O
or l; R~ represents OH or NH2; or any amino
acid; or a sequence of more than one amino
acid. In a specific embodiment, R2
represents an amino acid other than serine,
threonine or cysteine or the amide thereof;
in another specfic embodiment, R2 is more
than one amino acid, the first amino acid in
the sequence, which is attached to asp,
being other than serine, threonine or
cysteine, or the amide of any free carboxyl
groups.
In Formula I, R~ and R2 may be any amino acid or
- 20 sequence thereof. The amino acids are preferably
naturally occurring. The most common naturally-
occurring amino acids are shown in Table I:
;~: :. : : . .. : :. :... , . ,.:: :: . ~ .
~ 092/0~80~ . PCT/~S91tO8430
2 ~ 9 ~ 24 -
TABLE I.
NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS
Name 3-Letter l-Letter
Abbreviation Abbreviation
(+)-Alanine Ala A
(+)-Arginine Arg R
(-)-Asparagine Asn N
10 (+~-Aspartic acid Asp D .
(-)-Cysteine Cys C
(+)-Glutamic acid Glu E
(+)-Glutamine Gln Q _
15 Glycine Gly G
(-)-Histidine His H
(+)-Isoleucine Ile I
(-)-Leucine Leu L
20 (+)-Lysine Lys K
(-)-Methionine Met M
(-)-Phenylalanine Phe F
(-)-Proline Pro P
(-)-Serine _ Ser S
25 (-)-Threonine Thr T _
(-)-Tryptophan Try W
(-)-Tyrosine Tyr Y
(-)-Valine Val V
However, R~ and R2 in Formula I are not limited to
the 20 natural amino acids. In other embodiments, R~
and R2 can be non-classical amino acids or cyclic
peptides or peptidomimetics (chemical peptide
analogs). Non-classical amino acids include but are
not limited to the D-isomers of the common amino
., ... ... . : ~
. .... .. . . .
. .
,: . . . ..
., : , ..... ..
~'092/0880~ ~2 ~0 ~ ~.9 2 ~ PCT/~S91/08430
- 25 -
acids, ~-amino isobutyric acid, 4-aminobutyric acid,
hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, ~-alanine, designer amino acids
such as ~-methyl amino acids, C~-methyl amino acids,
N~-methyl amino acids, and amino acid analogs in
general.
Furthermore, the Arg, and/or Asp in the RGD
sequence can be the D (dextrarotary) or L (levorotary)
amino acid.
In a specific embodiment, where Rl is XY(Z)D~ X
can be any amino acid, and specifically need not be
valine. Preferably, X represents a naturally
occurring amino acid, and most preferably cysteine or
glycine. In a specific embodiment, Y can represent
any amino acid, and specifically ~eed not be
threonine. Preferably Y represents a naturally
occurring amino acid, and most preferably glycine. In
a specific embodiment, Z can represent any amino acid,
~0 preferably a naturally occurring amino acid. Although
Z preferably represents glycine, Z need not represent
glycine when X represents valine and/or Y represents
threonine.
As discussed above, R2 can represent OH or NH2.
2~ In another embodiment, R2 may represent an amino acid,
preferably a naturally occurring L-amino acid or
glycine; in a specific embodiment, R2 does not
represent serine, threonine or cysteine or the amide
thereof. In a preferred embodiment, R2 represents
phenylalanine or the amide of phenylalanine. In yet a
further embodiment, R2 can represent a sequence of more
than one amino acid, in particular, the first amino
acid in the sequence, which is attached to the
carboxyl functional group of Asp, being other than
::, : . : :, . :
.. . . . . . . . . . .. . ..
092/08X01 ~ ~9~ ~ 2 ~ P CT~S91/0~430
- 26 ~
serine, threonine or cysteine, or the amide of any
free carboxyl groups in the sequence.
When R2 is a sequence of amino acids, there is no
necessary limitation on the number of amino acids in
the sequence. Accordingly, the polypeptide for
conjugation to erythrocytes can be any size, and
encompasses what might otherwise be called an
oligopeptide or a protein. Preferably, the
polypeptide will have no more than about l,000 amino
acids.
When combined, Rl and R2 may represent a sequence
of the amino acids discussed above. In a specific
embodiment, R2 is not serine, threonine or cysteineO
For example, in a preferred embodiment, where R~ is
XY(Z)n~ X represents cysteine or glycine, Y represents
glycine, and Z represents glycine. In a more
` preferred embodiment, X represents cysteine or
glycine, Y represents glycine, Z represents glycine,
- and R2 represents phenylalanine or the amide of
phenylalanine. In the most preferred embodiment, X
represents cysteine, Y represents glycine, Z
represents glycine, and R2 represents the amide of
phenylalanine.
When R~ is XY(Z)0, X, Y, and Z may represent any
tripeptide sequence. The tripeptide need not be Val-
Tyr-Gly.
The polypeptide may be prepared by methods that
are known in the art. For example, in brief, solid
phase peptide synthesis consists of coupling the
carboxyl group of the C-terminal amino acid to a resin
and successively adding N-alpha protected amino acids.
The protecting groups may be any known in the art or
those described in Section 5.l.2 infra. Before each
new amino acid is added to the growing chain, the
protecting group of the previous amino acid added to
, ,~ :' ,
.: ' . : ~ . '
:, ' ,.......... ., : ' . , : :
- . . .: . :
~o 92/n~804 2 ~ 9 ~ 9 ~ ~ PCT/~S91/08~0
- 27 -
the chain is removed. The coupling of amlno acids to
appropriate resins is described by Rivier et al., U.S.
Patent No. 4,244,946. Such solid phase syntheses have
been described, for example, by Merrifield, 1964, J.
5 Am. Chem. Soc. 85:2149; Vale et al., 1981, Science
213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc.
103:3178 and in U.S. Patent Nos. 4,305,872 and
4,316,891.
5.1.2. CROSSLINKING AGENTS
The polypeptide is conjugated to the erythrocytes
through a polyfunctional molecule, i.e., a
polyfunctional crosslinker. As used herein, the term
"polyfunctional molecule" encompasses molecules having
one functional group that can react more than one time
in succession, such as formaldehyde (although
formaldehyde is not indicated for use due to its
potential carcinogenicity), as well as molecules with
more than one reactive group. As used herein, the
term "reactive group" refers to a functional group on
the crosslinker that reacts with a functional group on
a peptide, protein, or carbohydrate so as to form a
covalent bond between the cross-linker and peptide or
protein. The term "functional group" retains its
standard meaning in organic chemistry. The
polyfunctional molecules which can be used are
biocompatible linkers, i.e., they are noncarcinogenic,
nontoxic, and substantially non-immunogenic in ivo.
Polyfunctional cross-linkers such as those known in
the art and described herein can be readily tested in
animal models to determine their biocompatibility.
The polyfunctional molecule is preferably
bifunctional. As used herein, the term "bifunctional
molecule" refers to a molecule with two reactive
groups. The bifunctional molecule may be
~ ~.
~\O 92/0~s81)~ 2 09~;9~ S PCI/I~S91/08~30
- 28 --
heterobifunctional or homobifunctional. Preferably,
the bifunctional molecule is heterobifunctional,
allowing for vectorial conjugation of the RGD peptide
and erythrocyte. It is particularly preferred for the
5 polyfunctional molecule to be sufficiently soluble in
water for reactions with the polypeptide and with the
substrate to occur in aqueous solutions such as in
aqueous solutions buffered at pH 6 to 8. Typically,
the polyfunctional molecule covalently bonds with an
amino or a sulfhydryl group on X of the polypeptide
and on the surface of the erythrocytes. However,
polyfunctional molecules reactive with other
functional groups, such as carboxylic acids or
hydroxyl groups, are contemplated in the present
invention.
The homobifunctional molecules have at least two
reactive functional groups, which are the same. The
reactive functional grou~s on a homobifunctional
molecule include, for example, aldehyde groups and
active ester groups. Homobifunctional molecules
having aldehyde groups include, for example,
glutaraldehyde and subaraldehyde. The use of
glutaraldehyde as a cross-linking agent was disclosed
by Poznansky et al., Science 223, 1304-1306 (1984).
2~ Hom~bifunctional molecules having at least two
active ester units include esters of dicarboxylic
acids and N-hydroxysuccinimide. Some examples of such
N-succinimidyl esters include disuccinimidyl suberate
and dithio-bis-(succinimidyl propionate), and their
soluble bis-sulfonic acid and bis-sulfonate salts such
as their sodium and potassium salts. These
homobifunctional reagents are available from Pierce,
Rockford, Illinois.
The heterobifunctional molecules have at least
two different reactive groups. The reactive groups
.
2`~
~092/08801 PCT/~S91/08~30
! - 29 -
react with different functional groups on the peptide
and on a protein on the surface of the erythrocyte.
These two different functional groups of the peptide
and of the erythrocyte protein that react with the
reactive group are usually an amino group, e.g., the
epsilon amino group of lysine, and a sulfhydryl group,
i.e., the thiol group of cysteine. However, the
carboxylic acid and hydroxyl functional groups on the
peptide and the erythrocyte protein can also react
with the crosslinker.
When a reactive group of a heterobifunctional
molecule forms a covalent bond with an amino group,
the covalent bond will usually be an amido or imido
bond. The reactive group that forms a covalent bond
with amino groups may, for example, be an activated
carboxylate group, a halocarbonyl group, or an ester
group. The preferred halocarbonyl group is a
chlorocarbonyl group. The ester groups are preferably
reactive ester groups such as, for example, an N-
hydroxy-succinimide ester group or that of Mal-Sac-
HNSA.
The other functional group typically is either a
thiol group, a group capable of being converted into a
thiol group, or a group that forms a covalent bond
with a thiol group. The covalent bond will usually be
a thioether bond or a disulfide.
The reactive group that forms a covalent bond -
with a thiol group may, for example, be a double bond
that reacts with thiol groups or an activated
disulfide. A reactive group containing a double bond
capable of reacting with a thiol group is the
maleimido group, although others, such as
acrylonitrile, are also possible. A reactive
disulfide group may, for example, be a 2-pyridyldithio
,
~., '~ ' ~ . `
u~o~/n~ol PCT/-'S91/08~30
- 30 -
20~2~
group or a 5,5'-dlthio-bis-(2-nitrobenzoic acid)
group.
Some examples of heterobifunctional reagents
containing reactive disulfide bonds include N-
succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson,
et al., 1978, Biochem J., 173:723-737), sodium S-4-
succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate,
and 4-succinimidyloxycarbonyl-alpha-methyl-(2-
pyridyldithio)toluene. N-succinimidyl 3-(2-
pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive
groups having a double bond that reacts with a thiol
group include succinimidyl 4-(N-
maleimidomethyl)cyclohexahe-1-carboxylate and
succinimidyl m-maleimidobenzoate.
Other heterobifunctional molecules include
succinimidyl 3-(maleimido)propionate,
sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate,
sulfosuccinimidyl 4-(N-maleimidomethyl-
cyclohexane)-l-carboxylate, maleimidobenzoyl-N-
hydroxy-succinimide ester. The sodium sulfonate salt
of succinimidyl m-maleimidobenzoate is preferred.
Many of the above-mentioned heterobi~unctional
reagents and their sulfonate salts are available from
Pierce.
Additional information regarding how to make and
use these as well as other polyfunctional reagents may
be obtained from the following publications or others
available in the art:
Carlsson, J. et al., 1978, Biochem. J. 173:723-737.
Cumber, J.A. et al., 1985, Methods in Enzymoloqy
112:207-224.
Jue, R. et al., 1978, Biochem 17:5399-5405.
Sun, T.T. et al., 1974, Biochem. 13:2334-2340.
Blattler, W.A. et al., 1985, Biochem. 24:1517-152.
.. . ...
:, ,,. . ~ . ,:
: - ~ . -: , . .. . .
.: ' : , :'. ' . :
~ ' .: . ' ' ' :
\~9'/0880~ 2 0 ~ 5 9 2 5 PCT/~'S91/08~0
. ~
- 31 -
Liu, F.T. et al., 1979, Biochem. 18:690-697.
Youle, R.J. and Neville, D.M. Jr., 1980, Proc. Natl.
Acad. Sci. U.S.A. 77:5483-5486.
Lerner, R.A. et al., 1981, Proc. Natl. Acad. Sci.
5 U.S.A. 78:3403-3407.
Jung, S.M. and Moroi, M., 1983, Biochem. Biophys.
Acta 761:162.
Caulfield, M.P. et al., 1984, Biochem. 81:7772-7776.
Staros, J.V., 1982, Biochem. 21:3950-3955.
Yoshitake, S. et al., 1979, Eur. J. Biochem. 101:395-
399.
Yoshitake, S. et al., 1982, J. Biochem. 92:1413-1424.
Pilch, P.F. and Czech, M.P., 1979, J. Biol. Chem.
254:3375-3381.
Novick, D. et al., 1987, J. Biol. Chem. 262:8483-8487.
Lomant, A.J. and Fairbanks, G., 1976, J. Mol. Biol.
104:243-261.
Hamada, H. and Tsuruo, T., 1987, Anal. Biochem.
160:483-488.
Hashida, S. et al., 1984, J. AP~lied Biochem. 6:56-63.
` Additionally, methods of cross-linking are
reviewed by Means and Feeney, 1990, Biocon~uqate Chem.
1:2-12.
Exemplary strategies for conjugating the peptide
to the substrate through a heterobifunctional reagent
are shown in schemes 1 and 2 (Figures 14 and 15,
respectively). In scheme 1, N-succinimidyl 3-(2-
pyridyldithio)-propionate (SPDP) is treated with a RGD
peptide. A free amino group on X of the polypeptide
rep: -es the succinimidyloxy group and forms the
corresponding 3-2-pyridyldithio)propionyl (PDP) amide.
The 2-pyridyldithio group may be cleaved, for example,
with dithiothreitol, forming the corresponding
thiopropionyl (TP) amide (II). SPDP is then treated
with an erythrocyte having at least one amino group,
~O9'/08~1 2~`959~ 32 - PC~/~S91/0~30
forming the corresponding PDP amide (III). Treat~ent
of II, which has a free sulfhydryl group, with III,
which has a group which forms a covalent bond with a
sulfhydryl group, namely a pyridyldithio group, yields
a compound wherein the peptide is covalently bonded to
the substrate (erythrocyte-protein) through two
polyfunctional molecules (IV).
In scheme 2, a polypeptide in accordance with the
invention is treated with succinimidyl 4-(N-maleimido-
methyl)cyclohexane-l-carboxylate (SMCC), forming the
corresponding N-maleimidomethylcyclohexane-l-
carboxylate amide (V). Reaction of v with the free
sulfhydryl groups of an erythrocyte surface protein
results in compound VI, wherein the peptide and the
erythrocyte are covalently bonded through a
polyfunctional molecule.
In a preferred aspect, Mal-Sac-HNSA is used,
instead of SMCC.
; It is also possible to bind a sulfhydryl group
from the polypeptide of the invention to the
polyfunctional reagent. This would occur when X
represents cysteine. In such a càse, the free amino
group of the cysteine residue is protected if the
polyfunctional molecule is a heterobifunctional
molecule that reacts with amino groups as well as with
sulfhydryl groups. The protecting group can be any of
the large number of protecting groups known in the
art. For example, an acetyl group can be added to the
free amino group by treating the polypeptide with
3~ acetic anhydride. Alternatively, a carbobenzoxy group
can be added by treating the polypeptide with
carbobenzoxy chloride. Other N-protecting groups that
are useful include the formyl, L-butoxycarbonyl-,
trifluoroacetyl-, tosyl-, p-nitrocarbobenzoxy-,
cyclopentyloxycarbonyl-, and phenoxycarbonyl- groups.
:' ': ' ' . -
. .... . ..... : : ~ '
- . ~
, . : , . ~ ~ ., .: , . :.
W092/0X8~ 2~ PCT/~is91/08430
- 33 -
5.2. USE OF THROMBO-ERYTHROCYTES TO CONTROL BLEEDING
The thrombo-erythrocytes of the invention may be
used to control bleeding ln vlvo. In particular, the
thrombo-erythrocytes may be used to control bleeding
from small wounds in thrombocytopenic mammals,
including humans. In a preferred aspect, the thrombo-
erythrocytes are administered autologously to control
bleeding. In a less preferred aspect, the
administration is allogeneic.
In a specific embodiment, approximately 0.286-
3.57 ml of blood per kg of the mammal is removed. The
erythrocytes are then washed and concentrated. The
washed erythrocytes are then covalently bonded to a
RGD peptide through a polyfunctional molecule as
described in Section 5.1, supra. The resulting
thrombo-erythrocyte is then introduced into the mammal
by means of standard transfusion techniques.
In one embodiment, the thrombo-erythrocytes of
the invention are used in the treatment of
thrombocytopenia, ie., to augment a deficiency in
platelet levels in a patient. In another embodiment,
the thrombo-erythrocytes of the invention are
introduced into a mammal, including a human, to help
control bleeding, e.g., after trauma or during -
surgery-
The thrombo-erythrocytes for administration to a
mammal are prefera~ly formulated in a pharmaceutical
composition, as described in Section 5O4~ infra. The
thrombo-erythrocytes can be administered to a mammal
by intravenous or intra-arterial bolus injection or by
intravenous drip. The number of thrombo-erythrocytes
to be administered, i.e., the dose, depends upon the
degree of thrombocytopenia in the mammal, and can be
determined on a case-by-case basis by one skilled in
the art. Preferably, the number of thrombo-
. , ,, ~ ...... . . . .... . .... . . .. .....
.. :: ,: -: . :: . .: , . ... ... : , ~. . - : -~.
:- . .: , . , :.: . , .... . ' . ~ ' ' : ,
. . . . :
- . . - - - : , : : , . . .: . .
\~o s2/o~n~ Pcr/~;ss1/og~3()
~5~ 34 -
erythrocytes augments the number of pla~elets in
proportion to the amount absent from the
thrombocytopenic individual relative to a normal
individual.
5.3. TARGETED CARRIER ERYTHR~CYTES
According to the instant invention, erythrocytes,
or in particular thrombo-erythrocytes, prepared in
accordance with the present invention can be modified
for delivery, to various target tissues, of labels or
biologically active agents that have been incorporated
into the erythrocytes ti.e. taken up by erythrocyte
ghosts) to form carrier erythrocytes. In one
embodiment, the carrier erythrocyte is a carrier
thrombo-erythrocyte (i.e., a thrombo-erythrocyte whose
intracellular contents have been replaced by a
composition comprising a label or agent, and then
whose membrane is resealed).
The carrier erythrocytes have advantages over
liposomes by virtue of their larger size, which a~oids
the problem of non-specific endocytosis of liposomes
by scavenging cells such as macrophages, and because
of the presence of an extensive cytoskeleton, that,
for example, protects the erythrocytes from complete
osmotic lysis under hypotonic conditions. Moreover,
the cell surface integral membrane proteins of
erythrocytes provide a convenient scaffold for cross-
linking targeting molecules. Most importantly, since
erythrocytes are inherently biocompatible, the carrier
erythrocytes are more likely also to be biocompatible.
5.3.l. MATERIALS FOR TARGETED DELIVERY
A large number of different molecules,
macromolecules or macromolecular material can be ~-
.: . ., . .~ .. .
, . . '.: ', - ' :, . :.. ~
2 ~ 2.~
~092/0~804 PCT/~iS91/08430
- 35 -
incorporated in the carrier erythrocytes of the
invention for delivery to a specific target.
In one embodiment, imaging agents can be
incorporated in the carrier erythrocyte. Imaging
agents include but are not limited to heavy metal
contrast agents for x-ray imaging, magnetic resonance
imaging agents, and radioactive nuclides (i.e.,
isotopes) for radio-imaging.
In another embodiment, the carrier erythrocyte
can be loaded with one or more therapeutic agents.
For example, and not by way of limitation, the
therapeutic agent can be a chemotherapeutic, an
enzyme, a neurotoxin, a growth factor, a neurotrophic
factor, a hormone, a thrombolytic agent, or any drug.
Generally, specific targeting of a drug to the site
where it is needed results in more effective therapy
because a larger therapeutic dose can be delivered
than could be tolerated systemically. For example,
larger doses of a chemotherapeutic can be delivered
locally to a tumor than can be tolerated systemically
by an organism, e.g., a human. In another example, a
thrombolytic agent can be administered to the site of
thrombosis in a concentration that would lead to
uncontrollable bleeding if administered systemically.
In a further embodiment, the carrier erythrocyte
can be loaded with nucleic acid se~uences. For
example, and not by way of limitation, the nucleic
acids can be anti-sense RNA or DNA for delivery to a
target cell. In another embodiment, the nucleic acids
can be genetic in~ormation, such as a gene for gene
therapy or an entire genome for fertilization. The
carrier erythrocyte can be loaded with sperm, or fused
with sperm to obtain the sperm haploid genome. In yet
another embodiment, the carrier erythrocyte can
..
: .
~O92/0~8n1 PCT/~iS91/08~30
~`9592~ - 36 -
contain plasmids, or modified virus or viral nucleic
acids targeted for delivery.
5.3.2. TARGETING MOLECULES
The instant invention provides for conjugating
targeting molecules to the erythrocytes or erythrocyte
ghosts. "Targeting molecule" as used herein shall
mean a molecule which, when administered ln vlvo,
localizes to desired location(s). The crosslinkers
for the conjugation of peptides to erythrocytes
described in Section 5.1.2, supra can be used to
conjugate the targeting molecule to the erythrocyte;
furthermore, carrier erythrocytes and carrier thrombo-
erythrocytes need not retain the rheological
15 properties of control red blood cells, in contrast to
non-carrier thrombo-erythrocytes. The targeting
molecule can be conjugated to the erythrocyte either
prior to or subsequent to the introduction of a
material into the carrier erythrocyte.
In various embodiments, the targeting molecule
can be a peptide or protein, antibody, lectin,
carbohydrate, or steroid. In one embodiment, the
targeting molecule is a peptide ligand of a receptor
on the target cell. In a specific embodiment, the
targeting molecule is a peptide sequence described in
Section 5.1.1, supra, or variants thereof that bind
RGD receptors on the surface of cells such as
endothelial cells, cancer cells, or ova, e.g., human
ova that have receptors that recognize the RGD
sequence. In a specific embodiment directed to th~
use of carrier thrombo-erythrocytes, the targeting
molecule is the peptide Rl-RGD-R2 attached as described
supra, and the erythrocyte targeting agent is loaded
with a thrombolytic agent. Such a thrombo-erythrocyte
is useful for the treatment of thrombosis,
U092/OX8n~ 2 ~ 9 5 9 2 5 PCT/~S91/08~30
37 -
particularly since it is targeted to activated
platelets.
In another embodiment, the targeting molecule is
an antibody. Preferably, the targeting molecule is a
5 monoclonal antibody. In one embodiment, to facilitate
crosslinking to the erythrocyte, the antibody can be
reduced to two heavy and light chain heterodimers, or
the F(ab' )2 fragment can be reduced, and crosslinked to
the erythrocy~e via the reduced sulfhydryl. In
another embodiment, the carbohydrate portion of the
antibody can be directly, or via a derivative,
utilized for attachment to the erythrocyte or thrombo-
erythrocyte.
Antibodies for use as targeting molecule are
~S specific for cell surface antigen. In one embodiment,
the antigen is a receptor. For example, an antibody
specific for a receptor on cancer cells, such as
melanoma cells, can be used. In another embodiment,
antibodies specific for leukocyte surface antigens,
such as lymphocyte antigens, CD (clusters of
differentiation) antigens, and receptors (e.g., T cell
antigen receptors) can be conjugated to the
erythrocyte ghosts. Any antibody known in the art
that is specific for a cell antigen can be used as a
2~ targeting molecule.
In another embodiment, where a desired antibody
is not available, the antibody can be prepared.
Various procedures known in the art can be used for
the production of antibodies specific for a target
antigen that can be used to modify erythrocytes to
prepare targeted erythrocytes. Such antibodies
include but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments and an Fab
expression library, although monoclonal antibodies or
a fragment thereof are preferred. For the production
.. . . . ..
.
: ~ . : .. .,. . :
~; '; ' ~., ' , , .
~ . , .. : . : .
~092/OXX01 S 925 38 - PCT/~S91/0~130
of antibodies, various host animals, including but not
limited to rabbits, mice, rats etc., may be immunized
by injection wlth a target antigen marker. In one
embodiment, target antigen is conjugated to an
immunogenic carrier. In another embodiment, a target
antigen epitope, e.a., a hapten, is conjugated to a
carrier, such as keyhole limpet hemocyanin. As used
herein, an "epitope" is a fragment of an antigen
capable of specific immunoactivity, e.g., antibody
binding. Various adjuvants may be used to increase
the immunological response, depending on the host
species, including but not limited to Freund's
(complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides,
oil emulsions, dinitrophenol, and potentially useful
human adjuvants such as BCG (bacille Calmette-Guerin)
and Corynebacterium parvum.
Nonoclonal antibodies to a target antigen can be
prepared by using any technique that provides for the
production of antibody molecules by continuous cell
lines in culture. These include but are not limited
to the hybridoma technique originally described by
Kohler and Milstein, (1975, Nature 256: 495-497), the
more recent human B-cell hybridoma technique (Kozbor
et al., 1983, ImmunoloqY Today 4:72) and the EBV-
hybridoma technique (Cole et al., 1985, Monoclonal
; Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). In an additional embodiment of the invention,
~ monoclonal antibodies specific for a target antigen
can be produced in germ-free animals utilizing recent
technology (PCT/US90/02545). According to the
invention, human antibodies can be used and can be
obtained by using human hybridomas (Cote et al., 1983,
Proc. Natl. Acad. Sci.. U.S.A. 80:2026-2030) or by
' ~ ' : " :
: .' , , . ' :
WO92/0~80~ 2 ~ 9 ~ 9 2 5 PCT/~S91/08~0
39 -
transforming human B cells with EBV virus in vitro
(Cole et al., 1985, in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,
according to the invention, techniques developed for
5 the production of "chimeric antibodies" (Morrison et
al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855;
Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing the genes
from a mouse antibody molecule specific for target
1D antigen together with genes from a human antibody
molecule of appropriate biological activity can be
used; such antibodies are within the scope of this
invention.
According to the invention, techniques described
for the production of single chain antibodies (U.S.
Patent 4,946,778) can be adapted to produce target
antigen-specific single chain antibodies. An
additional embodiment of the invention utilizes the
techniques described for the construction of Fab
expression libraries (Huse et al., 1989, Science
, 246:1275-1281) to allow rapid and easy identification
of monoclonal Fab fragments with the desired
specificity for target antigen.
- Antibody fragments that contain sites specific
for target antigen can be generated by known
techniques. For example, such fragments include but
are not limited to: the F(ab' )2 fragments, which can be
produced by pepsin digestion of the antibody molecule
and the Fab' fragments, which ~an be generated by
reducing the disulfide brides of the F(ab' ~2 fragments.
Preferably reduced fragments are used, since these can
be conjugated to erythrocyte protein via their
sulfhydryl groups.
This invention further provides for the use of
other targeting molecules, such as lectins,
~: ; , .: ~..
:. , :,. .
, : ~ . :,
~'092/08~04 ` PCT/~S91/0~30
2 09 59 2~ ~ 40 -
carbohydrates, proteins and steroids, conjugated to
erythrocytes.
5.3.3. PREPARATION OF CARRIER ERYTHROCYTES
The key to the use of the present invention for
targeting is the ability of red cells to be made
temporarily "leaky". Making these modified
erythrocytes leaky allows them to release their
contents and take up the desired molecules, small
cells such as sperm, viruses, drugs or altered genetic
material; after which the erythrocytes may be
resealed. As is well known to those skilled in the
art, many modifications to and variations of this
method can be used, and therefore it is intended that
the present invention encompass all such variations.
Erythrocytes can be prepared as described in
Section 5.1, su~ra. Any mammal can be the source of
such erythrocytes. In order to incorporate molecules
into erythrocytes for transport to selected target
tissues, any procedure known in the art can be used to
make the erythrocytes leak their contents (i.e.,
prepare erythrocyte ghosts) and then take up new
molecules before being resealed. Such methods are
described in the following references: (editorial),
25 1988, Lancet pp.1437-1438; Brearley et al., 1990, J. ~ -
Pharm. Pharmacol. 42:297-301; Tonetti et al., 1990,
Biotech. Appl. Biochem. 12:261-269; Updike and
Rokania, 1983, J. Lab. Clin. Med. pp. 679-691; Ramsey
et al., 1986, Clin. Res. 34:468A.
In one embodiment, by way of example but not
limitation, the following procedure can be used: -
Resealed erythrocytes are prepared by a gel-
filtration method similar to that described by
Kaplan in "Sodium pump-mediated ATP:ADP exchange:
The sided effect of sodium and potassium ions",
.. : , : , .
~'0 92/()880~ 2 0 9 ~ 9 2 5 PCI`/I~S91/08~30
, i,. ~
- 41 -
Journal of General PhYsiology 80:915-937 (1982);
and Sachs in Volume-sensitive K influx in human
red cell ghosts", Journal of General Phvsioloqy,
92:~85-711 (19~8).
The ~odified cells are separated from plasma
and washed with a 150 mM choline-chloride
solution that contains 0.1 mM EDTA
(ethylenediamine tetraacetic acid) and lO mM
PIPES (piperazine-N,N'-bis (2-ethanesulfonic
acid) adjusted to pH 5.5 with Tris (Tris
(hydroxymethyl) aminomethane). The cells are
washed repeatedly until the pH of the cell
suspension is 6Ø The cells are then brought to -
50% hematocrit in the wash solution and stored on
ice until run into a column.
The column is 45 x 10 cm and is filled with
Bio Gel A50 beads (Bio Rad Inc., Rockville
Center, NY); the bed volume is 3.5 liters. The
column is enclosed in a water jacket and
maintained at about 1C. The gel is equilibrated
with a solution that contain~ 10 mM PIPES, 11.2
mM choline chloride, and 0.1 mM EDTA; the
solution is adjusted to pH 6.0 with Tris (buffer
A)-
2 To prepare thrombo-erythrocyte ghosts, 200
ml of solution identical to buffer A except that
the choline-chloride concentration is 150 mM
(buffer B) is run into the column followed by 75-
100 ml of cell suspension. The cells hemolyse
(leak their content of hemoglobin and other
materials) on the column and intracellular
contents are retained by the beads. Ghosts are
eluted with buffer B and collected on ice. They
are concentrated by centrifugation (40,000 g for
10 min) and aspiration of the supernatant,
:: ~ . : . . . - :
~092/08~0~ PC~/~S91/08430
~09~92~ - 42 - ~;
collected in one or two tubes, and resuspended in
buffer A. The ghosts are again centrifuged, the
supernatant removed, and the ghosts distributed
to resealing solutions. These contain 2~ by
volume (final volume including ghosts) of a 500-
mM Tris HEPES (4-[2-hydroxymethyl]-l-
piperazineethanesulfonic acid) solution (500 mM
HEPES adjusted to pH 8.0 at 37 C with Tris), 0.5
mM Tris EGTA (ethyleneglycol bis-[~-
aminoethylether] N,N'-tetraacetic acid), 50
mg/l00 ml albumin, and the molecules designed for
incorporation into the thrombo-erythrocytes.
Ghosts account for 10-40% of the volume of
the suspension. The ghost suspension is kept at
0 C for 5 min and then incubated at 37O C for 60
min. The resealed thrombo-erythrocyte ghosts
are separated from the suspension by washing 3
times in 0.15 M Nacl, 0.l M NaP04, l mg/ml human
albumin, pH 7.4, and are then ready for use in
vivo or in vitro.
Molecules to be introduced into carrier
erythrocytes are discussed in Section 5.3.I.
In one embodiment, where it is desired to
facilitate delivery of the molecule-incorporated
within the carrier erythrocyte, the lipid composition
of the red blood cell can be manipulated by known
methods to destabilize the cell membrane or treated by
other methods (e.g., heating or removal of surface
sialic acid residues) to reduce ln vivo half-life of
the carrier erythrocyte.
5.3.4. ADMINISTRATION OF TARGETED
CARRIER ERYTHROCYTES
The present invention provides for administering
the targeted carrier erythrocytes to a subject via any
route known in the art. For example, the erythrocyte
. . ., , , ~ .
. , ~ . . . .
~092/0880~ 2~ PCT/~1S91/08~0
- 43 -
targeting agents can be administered via any route
used to administer liposomes. In other embodiments,
erythrocyte targeting agents can be administered
intraventricularly, intraperitoneally,
intramuscularly, subcutaneously, intravenously, and
intraarterially, to mention but a few routes.
Preferably, the administration is intravenously or
intraarterially.
In a preferred embodiment, the targeted carrier
erythrocytes are administered in a pharmaceutical
composition comprising the targeted erythrocytes and a
pharmaceutically acceptable carrier or excipient (see
Section 5.4.l, infra).
5.4. PHARMACEUTICAL COMPOSITIONS COMPRISING
THROMBO-ERYTHROCYTES OR TARGETED CARRIER
ERYTHROCYTES
The present invention contemplates administering
thrombo-erythrocytes or targeted carrier erythrocytes
to a mammal, preferably in admixture with a
pharmaceutically acceptable carrier or excipient.
Such admixtures comprise a pharmaceutical composition
of the invention.
A pharmaceutically acceptable carrier or
2~ excipient for use in the invention should comprise an
aqueous solution having the following characteristics:
pH of between about pH 6 and about pH 8; ionic
strength of about 0.15 N to maintain the appropriate
osmotic environment for the modified erythrocytes; and
physiological compatibility. The pharmaceutically
acceptable carrier or excipient should not disrupt or
solubilize the modified erythrocytes, e.g., contain
oils, emulsifiers, detergents, or surfactants at
concentrations lytic to the cell membrane.
Within the above parameters, the pharmaceutically
acceptable carrier or excipient can comprise dextrose,
: ~ ....... .. . , . ~ ' : -
'.. . ,,, , '
~ .
~092/08804 2 0 9 S 9 2 5 - 44 - PCT/~S91/08430
glucose, starch, lacto~e and the like in aqueous
solution or suspension.
6. EXAMPLE: PREPARATION AND TESTING
OF THROMBO-ERYTHROCYTES
Blood (l0 ml) was drawn from a human by syringe
and a l9 gauge needle and placed into a polypropylene
tube containing 0.l ml 40% trisodium citrate. The
blood was centrifuged at approximately 2,000 X g for
l0 min at 22C and the supernatant plasma removed.
The erythrocyte pellet was washed three times with
buffer A (O.15 M NaCl, 0.05 M phosphate, 5 mM glucose,
2 mM KCl, pH 7.4) by repetitive centrifugation at
approximately 2,000 X g for l0 min at 22C. An
15 aliquot of 0.5 ml of the washed erythrocytes in the
same buffer at a concentration such that 60% of the
volume was comprised of erythrocytes (60% hematocrit)
was removed. The aliquot was mixed with 0.5 ml Buffer
; B (0.15 M NaCl, 0.0l M Na phosphate, pH 7.0)
20 containing 2.9 mg/ml solution of the polypeptide:
acetyl-Cys-Gly-Gly-Arg-Gly-Asp-Phe-amide
A 50 ~l aliquot of 10 mg/ml Mal-Sac-HNSA (N-
7 maleimido-6-aminocaproyl ester of l-hydroxy-2-
nitrobenzene-4-sulfonic acid sodium salt; Bachem
25 Biosciences, Inc.; Philadelphia, PA) in buffer D was
then added and the reaction allowed to proceed at 22~C
for 2 hr with rocking. After the incubation, 0.5 ml
was removed and washed three times in buffer A. The
resulting thrombo-erythrocytes were resuspended in
30 buffer A to a hematocrit of 10%. A control sample of
erythrocytes was treated identically but no peptide or
Mal-Sac-HNSA was added.
The assay contained 50 ~l of citrated platelet-
rich plasma (prepared by centrifuging whole blood
3 anticoagulated with 0.0l volume of trisodium citrate
at 700 X g for 3.5 min at 22C and adjusting the count
.: . . ................ . . ...... : . . .......... : : :
- . . . : : : .: : .. . . :: .~ - . ::
~ n 9~/08X0~ 2 0 9 ~i 9 2~ PCT/~S91/08430
- - 45 -
to 3.0 X loR platelets per ml with plasma free of
platelets) and 5 ~Ll of the thrombo-erythrocytes with,
or without, adding 5 ~l of adenosine diphosphate (ADP)
(lO0 ~M stock solution) to activate the platelets.
Agglutination of erythrocytes was graded from l-
4~ based on microscopic examination after rotating the
samples in a microtiter plate at 260 rpm at 22C for
various periods of time. At 2-3 min, the thrombo-
erythrocytes produced 0-l+ agglutination in the
absence of ADP and 4+ agglutination in the presence of
ADP. These values remained unchanged for the
remaining 6 mi~ of observation. The control
erythrocytes did not agglutinate.
7. EXAMPLE: TWO-STEP PROTOCOL FOR THE
PREPARATION OF THROMBO-ERYTHROCYTES
The dynamics of the one-step reaction described
in Example 6 illustrated that the thrombo-erythrocytes
of the present invention can be prepared and that they
bind to activated platelets. However, the dynamics of
the one-step reaction provide reason for believing
that this one-step method is at best unpredictable.
First, the sulfhydryl (thiol) groups of the
erythrocyte could react with the Mal-Sac-HNSA linker,
rather than the desired reaction between the
sulfhydryl (thiol) groups of the peptide and the Mal-
Sac-HNSA linker. This potentially competing and
undesirable reaction may damage the erythrocytes, and
would make less linker available for binding to the
peptide.
Accordingl~, a more preferred two-step method was
devised and tested, as shown in Scheme 3 (Figure 16)
ar.d described herein.
Pre~aration of erYthrocvtes - Blood (l0 ml) was
drawn from a human by syringe and a 19 gauge needle
and placed into a polypropylene tube containing 0.l ml
. , ", '
-. : ,
- ~ . , : . . . .
,~ , . .
. ~ . ', ~'. ' ' '
' '' , ~ ' :. ~ .: ' ',
2~9~2~ ~
~092/0~8~ PCT/~S91/08~30
- 46 -
40~ trisodium citrate. The blood was centrifuged at
approximately 2,000 X g for 10 min at 22C and the
supernatant plasma removed. The erythrocyte pellet
was washed three times with buffer A (0.15 M NaCl,
0.05 M phosphate, 5 mM glucose, 2 mM KCl, pH 7.4) by
repetitive centrifugation at approximately 700 X g for
5 min at 22C. An aliquot of 0.5 ml of the washed
erythrocytes in the same buffer at a density such that
60% of the volume was comprised of erythrocytes ~60%
hematocrit) was removed.
Preparation of peptide - linker - A 0.5 aliquot
of a solution of 2.0 mg/ml of the polypeptide with the -~
formula shown in Section 6, supra, was prepared in
buffer B (0.15 M Nacl, 0.01 M Na phosphate, pH 6.0).
To this solution was added a 50 ~1 aliquot of 10 mg/ml
Nal-Sac-HNSA (N-maleimido-6-aminocaproyl ester of 1-
hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt;
Bachem Biosciences, Inc.; Philadelphia, PA) in buffer
B. The reaction was allowed to proceed at 22C for 5
minutes at a pH of 6.0, thus forming the peptide-
linker complex. The peptide-linker complex could be
lyophilized and stored at approximately -70C for
future use.
The peptide-linker complex was adjusted with a
0.1 M solution of sodium hydroxide (NaOH) to a pH of
7.4. The 0.5 ml aliquot containing the linked peptide
was added and mixed with the 0.5 ml aliquot of
erythrocytes prepared as described above and the
mixture was rocked for 120 minutes at 22C and pH of
7.4.
After the incubation, the thrombo-erythrocytes
were washed three times in buffer A. The resulting
thrombo-erythrocytes were resuspended in buffer A to a
hematocrit of 10%. A control sample of erythrocytes
~09t0~80~ 2 0 9 ~ ~ 2 ~ PCT/~S91/0~0
- 47 -
were treated identically but no peptide or Mal-Sac-
HNSA was added.
The assay contained l00 ~l of citrated platelet-
rich plasma (prepared by centrifuging whole blood
5 anticoagulated with 0.0l volume of trisodium citrate
at 700 g for 3.5 min at 22C and adjusting the count
to 3.0 X loB platelets per ml with plasma free of
platelets) and l0 ~l of the thrombo-erythrocytes with,
or without, adding l0 ~l of adenosine diphosphate
(ADP) (l00 ~M stock solution) to activate the
platelets.
Agglutination of erythrocytes was graded from l-
4+ based on microscopic examination after rotating the
sample~ in a microtiter plate at 260 rpm at 22C for
various periods of time. At 2-3 min, the thrombo-
~ erythrocytes produced 0-l+ agglutination in the
; absence of ADP and 4~ agglutination in the presence of
ADP. These values remained unchanged for the
remaining 6 min of observation. The control
erythrocytes did not agglutinate.
8. EXAMPLE: DETAILED CH~RACTERIZATION OF
THROMB0-ERYTHROCYTES
The present example shows that erythrocytes
coated with RGD-containing peptides interact with
platelets, and critically, that the interactions are
selective for activated platelets, a prerequisite for
diminishing the risks of indiscriminate thrombus
formation.
8.l. MATERIALS AND METHODS
8.l.l. PEPTIDES
The peptide Ac-CGGRGDF-NH2 was made on an
automated peptide synthesizer (Applied Biosystems
430A; Foster City, CA) using t-boc chemistry and a 4-
methylbenzhydrylamine resin. In 4 of the 5 syntheses,
- . ' ,
- ~ : . ': , ' :. ' ,
~'092/OX~0~ PCT/~S91/08430
20959~ - 48 - ~ ~
the coupling solvent was dimethylformamide, whereas in
the fifth it was N-methyl-pyrrolidone. The protecting
groups were ~ benzyl ester for the aspartic acid,
tosyl for the arginine, and 4-methylbenzyl for the
5 cysteine. Double couplings were performed with the
phenylalanine in three of the syntheses and arginine
in all of the syntheses. The amino-terminus was
acetylated while the peptide was still on the resin by
reaction with acetic anhydride. Cleavage of the
peptide from the resin was accomplished with anhydrous
HF in the presence of dimethylsulfide, parathiocresol,
and anisole, starting at -10C. After HF cleavage,
the peptide-resin mixture was washed with ether alone
(first 2 syntheses) or ether and dichloromethane (last
3 syntheses), and then extracted into acetic acid
before lyophilization. HPLC analysis (C-8 column, 220
X 4.6 mm, Applied Biosystems 300 RP) demonstrated a
single dominant peak in each synthesis representing
45-57~ of the total absorbance at 220 nm. For some
experiments the peptide was purified by HPLC before
use. Fast atom bombardment mass spectrometry (xenon
gun parameters: 7 kV, 1 mA, 0.4 mA ion current; mass
spectrometer parameters: acceleration potential 6 kV,
mass range 132-1172, resolution 1,500, scan speed 10
sec/decade; lyophilized sample transferred to glycerin
or thioglycerin matrix) was performed on 2 of the 5
peptides and demonstrated that the peptides had the
expected mass (751).
The peptide concentration for the coupling
experiments was determined by titrating the free
sulfhydryl groups with 5,5'-dithio-bis-(2-nitrobenzoic
acid) (Ellman's reagent; Pierce Chemicals, Rockford,
IL) using cysteine as a standard. A radiolabeled
peptide was prepared by performing the peptide
acetylation reaction (0.3 mg of resin) with 0.05 mmol
:. : ' -
U'092/088~ 2 0 9 ~ 9 2 5 PCTI~;S91/OX430
( - .
(25 mCi) of 3H-acetic anhydride (Amersham Corp.,
Arlington Heights, IL) in a mixture of 4.75 ml
dichloromethane and 0.25 ml diisopropyl ethylamine for
120 min at 22C with stirring and then adding 0.5 ml
(5 mmol) unlabeled acetic anhydride for an additional
5 min. The resin was then allowed to float
undisturbed, the infranatant fluid was removed, and 5
ml of 10% acetic anhydride (~5 mmol) in dichloro-
methane was added to the resin and allowed to react
0 for another 5 min. The resin was then filtered,
washed first in dichloromethane and then in methanol,
and cleaved from the resin with HF in the presence of
scavengers. The 3H-peptide had a specific activity of
1.3 X 10ll dpm per mmol peptide. HPLC analysis
demonstrated that 83% of the radioactivity eluted with
the peptide peak.
8.1.2. PREPARATION OF THROMBO-ERYTHROCYTES
Preparation of thrombo-erythrocytes. The
crosslinking strategy was: 1) to join the peptide to
the heterobifunctional crosslinking agent N-maleimido-
6 aminocaproyl ester of 1-hydroxy-2-nitrobenzene-4-
sulfonic acid (mal-sac-HNSA; Bachem Bioscience,
Bubendorf, Switzerland) via a reaction between the
2~ free sulfhydryl on the peptide and the crosslinker's
maleimide moiety, and then, 2) to join the peptide-
crosslinker to the erythrocyte via a reaction between
the erythrocytes' amino groups and the aminocaproyl
ester, resulti~g in the release of the highly
absorbent 1-hydroxy-2-nitrobenzene-4-sulfo~ic acid
dianion from the mal-sac-HNSA. In order to minimize
hydrolysis of the ester during the maleimide-
sulfhydryl reaction, a pH of 6.0 was chosen for the
first reaction. To speed the reaction between the
ester and the erythrocyte amino groups and to insure a
. .
. : .
'. , ' ' -
`~' "; ' . '
. . .
~09~/0X~ PCT/~S91/08~30
?t~ 5 - 50 -
physiological pH for the erythrocytes, a pH of 7.4 was
chosen for the second reaction.
Whole blood was collected by syringe and placed
in a polypropylene test tube containing either 0.1 ml
40% trisodium citrate or 1.2 ml CPDA-1 anticoagulant
(89 mM trisodium citrate, 16 mM citric acid, 16 mM
NaH2PO4, 160 mM dextrose, 2 mM adenine) so that the
final volume was 10 ml. The blood was centrifuged at
700 X g for 3.5 min at 22C for platelet-rich plasma
(PRP). After removing the PRP, the blood was
recentrifuged at 1600 X g for 10 min at 22C and the
resulting platelet-poor plasma (PPP) was removed. The
buffy coat layer was then removed and discarded, and
the erythrocytes were brought up to 50 ml with buffer
A (140 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM Na
phosphate, pH 7.4). The erythrocytes were then washed
3 times in buffer A and resuspended to a hematocrit of
10% in the same buffer. A 3 ml sample was transferred
to a small polypropylene tube and centrifuged at 700 x
2C g for 5 min at 22C; 2.5 ml of the supernatant buffer
was then removed, leaving 0.5 ml of a 60% hematocrit
solution (3.3 X 109 erythrocytes). In some
experiments, a slightly different buffer was employed
(150 mM NaCl, 50 mM Na phosphate, 2 mM XCl, 5 mM
2c glucose, pH 7.4) and the results were the same.
The Ac-CGGRGDF-NH2 peptide was then dissolved in
buffer B (150 mM NaCl, 10 mM Na phosphate, pH 6.0) at
-2.0 mg/ml (2.6 mM) and the solution was readjusted to
pH 6.0 with 1 M NaO~. The mal-sac-HNSA was then
dissol~ed at 10 mg/ml in buffer B, and 0.5 ml of the
peptide solution (1.3 ~mol) and 0.05 of the mal-sac-
HNSA (1.1 ~mol) were incubated at room temperature for
10 min. The pH of the solution was then increased to
7.4 with 0.1 M NaOH, and the solution was immediately
added to the 0.5 ml of erythrocytes in buffer A. The
.
. ...................... :: ., . . ,. :
' ~ ' . ~' . . ' ' ,.,: '
\~O92/08X~4 2 ~ 9 5 ~ 2 ~ pCT/~S91/08~30
- 51 -
tube was then gently rocked at 22OC for various
periods of time, usually up to 2 hr, but in some cases
18 hr. In other experiments, the reaction took place
in one step, with the peptide, crosslinker, and
5 erythrocytes incubated together at pH 7.4~7.5. After
the reaction was complete, the thrombo-erythrocytes
were washed X 3 in buffer A. Thrombo-erythrocytes
were used immediately or stored at 4C.
Preliminary studies monitored with Ellman's
0 reagent (to assess the reaction between the maleimide
group of mal-sac-HNSA and the sulfhydryl group on the
peptide) indicated that when equimolar (2 mM)
concentrations of crosslinker and peptide were used at
pH 6.0, the maleimide-cysteine reaction was >95
complete within 5 min. These preliminary reactions
were also monitored at 405 nm for release of the
1-hydroxy-2-nitrobenzene-4-sulfonic acid dianion from
mal-sac-HNSA as an indicator of hydrolysis of the
amino-reactive moiety on the crosslinXer (Aldwin and
Nitecki, 1987 Anal. Biochem. 164:494-501). At the end
of these experiments, samples were treated with 0.05
volume of 5 N NaOH, which produces complete release of
the dianion (id.), to establish the percentage of
total dianion that had been released during the
reaction. The results indicated that less than -1% of
the amino-reactive groups on mal-sac-HNSA were
hydrolyzed during the 10 min maleimide-cysteine
reaction at pH 6Ø
8.1.3. OUANTIFICATION OF PEPTIDE BINDING
To determine the number of peptide molecules
crosslinked to each thrombo-erythrocyte, the
radiolabeled peptide was used in combination with
unlabeled peptide. At selected time intervals,
thrombo-erythrocytes were removed from the incubation
.:
:: . . ` ,:
- ' ', ` .
.:
'0 9~/OXXO~ PCI/~ S91/08~30 1~
2095~2~ - S~ - f
mixtures, washed X 3 in buffer A, and then subjected
to hypotonic lysis to produce erythrocyte ghosts.
This was accomplished by first incubating the
erythrocytes with 10% buffer A (i.e., buffer A diluted
5 to 10% of its original concentration) at 0C, then
centrifuging at 38,000 g for 20 min at ooc, removing
both the supernatant fluid and the hard red button of
cell debris, resuspending the remaining pink ghosts in
1% cold buffer A, and washing X 2 in cold 1% buffer A.
In some experiments, 0.5 mM EDTA was added to the wash
buffer to prevent resealing of erythrocyte ghosts.
Finally, the erythrocyte ghosts were solubilized in
0.1 - 0.4 ml 1% sodium dodecyl sulfate (SDS), and this
solution was added to 6 ml of scintillation fluid
(Ultima Gold; Packard) and counted in a liquid
scintillation counter (Packard 1900CA, Downers Grove,
- IL). The number of peptide molecules bound per
erythrocyte was then calculated from the
radioactivity specifically incorporated into the
; 20 thrombo-erythrocytes [i.e., radioactivity associated
- with the ghosts after reaction with the full thrombo-
erythrocyte incubation mixture (er~vthrocytes + peptide
+ crosslinker) minus the radioactivity associated with
the ghosts of the nonspecific control (erythrocytes +
peptide)]. In one experiment, the 10~ and 1% lysis
buffers contained the protease inhibitors PMSF (1 mM),
leupeptin (0.~ mM), and EDTA (0.5 mM). An extra,
final wash in the 1% lysis buffer with just 0.5 mM
EDTA was employed in this experiment because the
ghosts were not easy to resuspend, and solubilization
at 37C was achieved with a mixture of 200 ~1 10~ SDS
+ 20 ~1 0.1 M NaOH + 200 ~1 of a tissue solubilizer
(TS-2, 0.5 N; Research Products International, Mount
Prospect, IL). The solubili2ed ghosts were then added
to 18 ml of scintillation fluid and counted.
. '".. ~ ,'''' , ' ' .' ' '' '.. '" ''"' . ' ' ', . ''`"',' ",.. ,.i "' . ' ., ' ', . ', ' ~ ',
... ' ' ' ~ ".' ., ' ' ... ' ,' .. .. ' '.' ' ' .': . ", '' ,' '.. ' .. ' ' ' .', , ' "' . . , ' :
~092/0X~0~ 2 ~ 9 ~ 9 2 5 PCT/US91/08430 1
- 53 -
8.1.4. IDENTIFICATION OF ERYTHROCYTE PROTEINS
TO WHICH PEPTIDES ARE CROSSLINKED
Pure 3H-peptide (1.3 ~mol) was reacted for 120 min
with erythrocytes in a 2-step reaction as described
above. The thrombo-erythrocytes were then lysed using
the cold, hypotonic buffers containing EDTA (0.5 mM),
and the resulting ghosts were then dissolved in 100 ~1
1.7% SDS at 15C and frozen at -80c. A platelet-
thrombo-erythrocyte co-aggregation assay (see below)
on a sample removed before lysis demonstrated that the
thrombo-erythrocytes were active in the assay.
Subsequently, a 20 ~1 sample of the solubilized
thrombo-erythrocytes was thawed, mixed with 20 ~l of
sample mixer and 2 ~l of 2-mercaptoethanol, heated to
100C for 3 min, and electrophoresed in a
polyacrylamide gel (3% stacking, 12.5% resolving gels)
according to the method of Laemmli (1970, Nature,
227:680-682). The gel was then stained with the
periodic acid-Schiff method by fixing overnight in 25%
isopropanol-10% acetic acid, washing with 10% acetic
acid, incubating with 1% periodic acid in 3% acetic
acid for 60 min, washing with water X 4, reacting with
Schiff stain for 60 min in the dark, and washing with
Na2S205 in 0.1 M HCl X 3. The gel was then stored
overnight in 7% acetic acid at 4C, photographed,
stained with Coomassie blue, destained, and
rephotographe~d. Finally, the gel was prepared for
fluorography by fixing in 30% methanol, 10% acetic
3C acid for 30 min X 3, incubating in a precipitating
reagent (solution A) of a fluorography preparation kit
(Entensify, New England Nuclear Research Products,
Boston, MA) for 30 min, and incubating in an aqueous
fluorescent reagent (solution B) for 30 min. The gel
was then dried and placed in a cassette with XAR-5
:: .: . : . . . : .,
, : ...................... . . : .
W092/08~0~ ~ Y 5 9 25 54 PC~/~SYI/08430
film (Eastman Kodak, Rochester, NY) for 7 days at
-70C.
8.1.5. PLATELET-THROMBOERYTHROCYTE
CO-AGGREGATION ASSAY _ _
To assess the ability of thrombo-erythrocytes to
enter into developing platelet aggregates, a
microtiter assay was developed. Platelet-rich plasma
(PRP) was prepared from blood anticoagulated with 0.01
volume 40% sodium citrate and adjusted to a platelet
count of 3.5 X 108/ml with platelet-poor plasma.
Aliquots (50 or 100 ~1) of the PRP were added to ~-
microtiter wells and then 5 or 10 ~1 of ADP (100 ~M
stock solution) was added to selected wells, followed
by the addition of 5 or 10 ~1 of thrombo-erythrocytes
(10% hematocrit in buffer A). The microtiter plate
was then rotated at 270 rpm at 22C for variable
periods of time between 0.5 and 20 min and the extent
of platelet-thrombo-erythrocyte coaggregation was
assessed visually on a scale from 0-4+ with the aid of
a magnifying mirror. To assess the specificity of the
reaction, in some experiments the PRP was preincubated
with 10 mM EDTA, 300 ~g/ml of the peptide RGDF, or 20
~g/ml of an antibody directed against both the
2~ GPIIb/IIIa receptor and the ~V~3 vitronectin receptor
; that blocks fibrinogen binding to activated platelets
(7E3) (Coller, 1985, J. Clin. Invest. 76:101-108;
Coller et al., 1991, Blood 77 75-83?. Blood smears
were made from the samples in some experiments and
stained with a standard Wright stain (Hemastain,
-~ Geometric Data, Wayne, PA). -
To obtain more quantitative data on the
co-aggregation of thrombo-erythrocytes-with platelets,
the assay was adapted to the aggregometer. PRP was
3~ prepared from whole blood anticoagulated with ACD-A
(8.5:1.5) and gel-filtered over a column of Sepharose
~ 092/088~ 2 0 9 ~ 9 2 5 PCT/~iS91/084~(~
`.i`.
- 55 -
2B (Pharmacia) using a modified Tyrodes buffer (140 mM
NaCl, 3 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 10 mM
HEPES, 2 mM MgCl2, 0.2~ bovine serum albumin, 5 mM
glucose, pH 7.4). Samples consisted of 450 ~l of ~el-
5 filtered platelets + 20 ~l of thrombo-erythrocytes
(10% hematocrit) or control erythrocytes (i.e.,
erythrocytes incubated with peptide but no
crosslinker). Maximal transmission was set with 450
~l of buffer + 20 ~l of control erythrocytes.
Platelets were activated with ADP (4.3 ~M final
concentration) or epinephrine (10 ~M).
8.1.6. ASSESSMENT OF HEMOLYSIS
Hemolysis of erythrocytes during thrombo-
erythrocyte preparation was assayed by the reaction of
any free hemoglobin with leucomalachite green (Kodak;
0.1% p,p'-benzylidenebis-(N,N-dimethylaniline) in 3.3
M acetic acid). The resulting compound was detected
by absorbance at 617 nm, a wavelength that is not
interfered with by the mal-sac-HNSA dianion.
Standards were prepared by lysing known amounts of
erythrocytes in deionized water. The assay consisted
of 10 ~l of sample (the supernatant of the reaction
mixture after centrifuging to remove intact
erythrocytes), 1 ml of leucomalachite green, and 1 ml
of 0.1% H202. After 10 min the absorbance of each
sample was read at 617 nm.
8.1.7. OSMOTIC FRAGILITY
Thrombo-erythrocytes, control erythrocytes, and
untreated erythrocytes were added to NaCl solutions of
various concentrations. After 20 min at 22C the
samples were centrifuged and the optical density of
the supernatant fluid assessed at 540 nm. Results
were expressed as the percent hemolysis, with 100%
- , , ; : , . - , :, . . . . . .
, - ' : ' ' :, . ' : : . ; ~ ,
~ ;,'?^
U092,08804 PCT/~S91/0~430
- 56 -
hemolysis defined as the optical density of a sample
of erythrocytes added to water.
8.1.8. BINDING OF THROMBO-ERYTHROCYTES TO
PLATELETS ADHERENT TO COLLAGEN
The first stage of the assay, invol~ing the
adhesion of platelets to purified type 1 rat skin
collagen, was performed as described previously, but
without radiolabeling the platelets (Coller et al.,
1989, Blood 74:182-192). In brief, a sample of gel-
filtered platelets (100 ~1; 5.5 X 108/ml) in the
presence of 2 mM MgCl2 was added to microtiter plate
wells precoated with collagen and the platelets were
allowed to adhere for 1 hour at 22C. The wells were
then emptied and washed X 3 with buffer (0.15 M NaCl,
0.01 M Tris/HCl, 0.5~ bovine serum albumin, 5 mM
glucose, pH 7.4). Control erythrocytes or thrombo-
erythrocytes (50 ~1; 10% hematocrit) were then added
to the wells in the same buffer, which was now
supplemented with 2 mM MgC12. After 60 min, the wells
were emptied and washed X 3 as above. The wells were
then visually inspected at 400X magnification with the
aid of a microscope with Nomarski optics. The effect
of 20 ~g/ml of an antibody to GPIIb/IIIa that blocks
fibrinogen binding and platelet aggregation ~lOE5~
(Coller et al., 1983, J. Clin. Invest. 72:325-338) and
400 ~g/ml of the peptide RGDF on thrombo-erythrocyte
adhesion to platelets was tested by adding these
agents to the thrombo-erythrocytes immediately before
3~ the thrombo erythrocytes were added to the microtiter
wells.
.
8.1.9. ASSESSMENT OF THROMBO-ER-YTHROCYTE
VOLUME AND SURFACE PROPERTIES
Laser diffraction ektacytometry was performed on -
the thrombo-erythrocytes and control erythrocytes
- . - . ......... . , . . , . :
. . . ~.: . , , . : . . i, .
~92/0X80~ 2 ~ 9 ~ 9 2 ~ PCT/~IS91 /08130
- 57 -
essentially as previously described (Mohandas et al.,
1980, J. Clin. Invest. 66:563-579; Clarck et al.,
1983, Blood 61:899-910) using samples sent by
overnight mail on 4C cold packs from Stony Brook to
5 Berkeley. Control erythrocytes or thrombo-
erythrocytes (20 ~l of a ~33% suspension in 0.15 M
NaCl, 0.01 M Tris/HCl, 5 mM KCl, 10 mM glucose, 1%
bovine serum albumin, pH 7.4 buffer) were added to 3.5
ml of 4% (w/v) polyvinyl pyrrolidone in phosphate
buffered NaCl adjusted to 290 mOsm (viscosity = 22
cp). The samples were then placed in the instrument
and the deformability index (a measure of the change
in cell shape from circle to ellipse) was measured
continuously as the cells were subjected to increasing
shear rates (0 - 1,037 s~~).
8.2. RESULTS
8.2.1. CHARACTERIZATION OF THROMBO-ERYTHROCYTES
The supernatant fluid after performing the
thrombo-erythrocyte reaction had 0.40 + 0.09% (mean +
SD; n=6) erythrocyte hemolysis compared with 0.13 +
0.04% (n=6) in the control reaction. Studies of
osmotic fragility showed only minor differences
between thrombo-erythrocytes and control erythrocytes
(Figure 1 contains data on 3 separate experiments),
and the control erythrocytes did not differ from
untreated erythrocytes. Laser ektacytometry
demonstrated that the thrombo-erythrocytes had the
same deformability properties as did control
erythrocytes and untreated erythrocytes (Figure 2).
Both control erythrocytes and thrombo-erythrocytes had
plateau values that were within the normal range
determined from studies on more than 200 individuals
(0.6 + 0.02; mean + SD).
- . : , . . . .. - , , , .. , ~ . :-
:' , :. ,: .
: '' . . . ' : . ' ~ ': . : ,.
.. . . ~ . . :
.
~'092/0~80~ 2 0 9 5~ 2 ~ 58 - PCT/~;S91/08~30
8.2.2. STUDIES WITH THE 3H-PEPTIDE
The results of 5 separate experiments to
determine the number of peptide molecules that bound
per thrombo-erythrocyte are shown in Table II.
S .
TABLEII
Bindtngof~H-CGGRGDF Pep~ide lo E~throcyles
Exlxrimenl I Slep ~H- Mal-Sac- Number of Peptide Molecules I -
or Peptide HNSA Bound Per Thr~mbo-E~ythrocyte l
_ Step (llmol)' (llmol)' Time (min) I ..
120
.
l l 0.9 ~.l 570,000
" l I .3I . I780,000 I ,100,000 I ,400,000 I ,400,000
3 l ~.3 1.1 750.000 l
11
t 0 .60.55 480.000570,000
. _ .
,~ 1.3 1.1540,000 750,000870.0001,100,000
11
4 ~ 1.3 1.1 1,000,~00
11
,, 1.3 1.1 500,000
. . ~
;~0 The number of llmol of the 'H-CGGRGDFpeptide and lhe crosslinking reagent (mal-sac-HNSA)
employed in the reactions are indicated. E~ch reaction muture conlained - 3.3 X 109 er~nhrocytes.
There was a progressive increase in 3H-peptides bound
per thrombo-erythrocyte as a function of time, with the
reaction slowing down or stopping at the 9o-120 min
25 time point. Maximum specific incorporation using 1.3
~mol of peptide and 1.1 ~mol of crosslinker per 3.3 X
109 erythrocytes was 0.5 - 1.4 X 1o6 peptide molecules
per thrombo-erythrocyte, representing ~0.3 - 0.7% of
the added peptide. Nonspecific association of peptide
with control erythrocytes was <3% of the specific
incorporation as judged by control samples in which the
crosslinker was omitted. In the 3 experiments in which
the reaction was conducted in a single step, the
35 results were comparable to those achieved using the 2-
step reaction. In studies where radiolabeled thrombo-
- . . . . . . . . .
- . :
2 ~ 5
~O9~/088~4 PCT/~S91/0~30
- 59 -
erythrocytes were solubilized in SDS and subjected to
polyacrylamide gel electrophoresis, there were 3
identifiable radioactive bands (Figure 3). The
strongest was at Mr 87 kD, corresponding to the major
5 periodic acid-Schiff (PAS)-staining band (PAS-1)
(Thompson and Maddy, 1982, in Red Cell Membranes - A
Methodological Approach, Ellory and Young, eds.
Academic Press, NY, pp. 67-93). The second was at Mr
42 kD and corresponded to the second PAS-positive band
l0 (PAS-2), and the third was a weak band at Mr 22 kD
corresponding to the third PAS-positive band (PAS-3).
8.2.3. PLATELET-THROMBO-ERYTHROCYTE INTERACTIONS
In more than 20 separate experiments, thrombo-
15 erythrocytes prepared by incubating the peptide-
crosslinker with erythrocytes for 120 min gave a
positive response in the platelet-thrombo-erythrocyte
co-aggregation assay using ADP; epinephrine and
thrombin were tested in a smaller number of experiments
~0 and also found to be effective in stimulating platelet-
thrombo-erythrocyte interactions, visible as
macroscopic, red co-aggregates (Figure 4). Microscopic
examination of stained smears confirmed the intimate
association between platelets and thrombo-erythrocytes
~5 (Figure 5), with the platelet aggregates acting as
bridges between thrombo-erythrocytes. In contrast, the
thrombo-erythrocytes did not interact with platelets
when no agonist was added, demonstrating the
selectivity of the thrombo-erythrocytes for activated
30 platelets. Control erythrocytes, which had been
reacted with the peptide but not the crosslinker, did
not interact with either unactivated or activated
platelets (Figure 4). When the platelets in these
samples were activated, pure platelet aggregates could
be identified microscopically (Figure 5);
' '' ~ . , ~ ' ' .'
- ~ ~ ' ' '
. .
~ ~'092/08~04 PCT/~S91/08430_
2 n g j ~9 ~ 5 60 ~
macroscopically these appeared as small white clumps
(Figure 4). In time course experiments, crosslinking
incubation times as short as 15 min were found
sufficient to produce thrombo-erythrocytes that gave
5 positive reacti~ns in this assay, although the
reactions tended to be less strong.
To exclude any confounding effects of the citrate
anticoagulant used in these studies, the assay was also
performed with PRP anticoagulated with heparin (4 U/ml)
lO or hirudin (10 U/ml; Sigma) and similar results were
obtained, although as expected, thrombin-induced
activation did not occur with these anticoagulants.
It is important to note that since these assays contain
normal plasma, fibrinogen is available for binding to
15 activated GPIIb/IIIa receptors; thus, the thrombo-
erythrocytes were able to compete effectively with
fibrinogen for the GPIIb/IIIa receptors.
Several inhibitors were used to assess whether the
thrombo-erythrocytes were actually binding to the RGD
?0 binding site on the activated platelets' GPIIb/IIIa
receptors. In fact, the co-aggregation was inhibited
by a fluid-phase RGD peptide, a monoclonal antibody to
GPIIb/IIIa and the ~V~3 vitronectin receptor that blocks
both the binding of fibrinogen to platelets and the
25 interactions between RGD-coated beads and platelets
(Coller, 1985, J. Clin. Invest. 76: 101-108; Coller et
al., 1991, ~lood 77:75-83), and EDTA, a strong divalent
cation chelator that inhibits the interactions of all
ligands with integrin receptors (Figure 4).
To obtain more quantitative data, an assay was
developed using gel-filtered platelets and thrombo-
erythrocytes in an aggregometer. Figure 6 depicts the
results of an experiment demonstrating that thrombo-
erythrocytes, but not control erythrocytes, interact
with ADP-activated platelets. The thrombo-erythrocytes
, . . ~,. , . , - ~
.: , : ~ .. . .:
... . . . . . .
: ~ .
. ~
. .
~092/~880~ 2 0 9 ~ 9 2 5 PCT/~S91/08S30
- 61 -
did not interact with unactivated platelets despite the
stirring and 37C temperature. lOE5, a monoclonal
antibody to GPIIb/IIIa that blocks the binding of
fibrinogen to platelets and partially blocks the
5 interaction of platelets with RGD-coated beads (Coller
et al., 1983, J. Clin. Invest. 72:325-338) inhibited
the interaction between platelets and thrombo-
erythrocytes. In this assay, plasma proteins were
removed in the gel-filtration step and thus there was
little or no exogenous fibrinogen to compete with the
thrombo-erythrocytes for binding to the activated
platelets.
8.2.4. BINDING OF THROMBO-ERYTHROCYTES TO
PLATELETS ADHERENT TO COLLAGEN
Hemostasis in vivo is thought to be initiated by
adhesion of platelets to subendothelial proteins, in
particular collagen, when blood vessels are damaged
(Coller et al., 1989, Blood, 74:182-192). Platelets
20 then aggregate on top of the adherent platelets,
presumably as a result of the GPIIb/IIIa receptors on
the lumenal surface of the adherent platelets
undergoing the transformation that allows them to bind
adhesive glycoproteins such as fibrinogen and von
25 Willebrand factor with high affinity (Plow and
Ginsberg, 1989, Proq. Hem. Thromb. 10:117-156). We
therefore tested the ability of thrombo-erythrocytes to
bind to platelets that had adhered to collagen. As in
our previous studies (Coller et al., supra), a dense
30 lawn of platelets adhered to the collagen in the
presence of 2 mM MgCl2. When control erythrocytes were
then added, virtually none of the erythrocytes bound to
the platelets (Figure 7). In sharp contrast, the
thrombo-erythrocytes formed a dense lawn on top of the
35 platelet lawn and this reaction could be virtually
.. , , . . ~. . . .
-, .
~'092~0~0~ PCT/~IS91/08430
2 n~ s9 ~ ~ 62 _
completely inhibited by antibody 10E5 or the peptide
RGDF (Figure 7).
8.3. DISCUSSION
In an attempt to succeed where previous approaches
have failed, we have covalently attached the peptide
Ac-CGGRGDF-NH2 to erythrocytes via surface amino groups
with the aid of a heterobifunctional crosslinking
reagent. Approximately 0.5.-1. 5 X 106 peptide molecules
were cross-linked per erythrocyte after 120 min. The
peptide appeared to be ~electively crosslinked to-
glycoproteins that are present in the PAS-1, PAS-2, and
PAS-3 regions, making it most likely that it is
crosslinked to glycophorin A (whose dimeric form is
15 largely responsible for PAS-1 and whose monomeric form
is largely responsible for PAS-2), and the related
glycoprotein, glycophorin B (which is largely
responsible for PAS-3) (Anstee, 1990, Vox Sang. 58:1-
20). It is interesting that there are an estimated 0.2
~ - 10 X 106 glycophorin A molecules per erythrocyte and
~0.25 X 106 glycophorin B molecules per erythrocyte
(Anstee, 1990, VoxSanq. 58:1-20.), raising the
possibility that there is 1:1 stoichiometry between the
number of crosslinked peptide molecules and the number
25 of glycophorin A + glycophorin B molecules.
Thrombo-erythrocytes were analyzed in several
ways. The crosslinking reaction itself produced only
slightly more hemolysis than simply washing the
erythrocytes. In addition, there were only minimal
changes in osmotic fragility. Laser diffraction
ektacytometry, a technique that is sensitive to changes
in the erythrocyte membrane and the hydration state of
the cytoplasm of the erythrocyte, has been a useful
35 tool in analyzing erythrocytes altered in vitro and
erythrocytes from patients with a variety of disorders
, . . . , . , , . . . , . . . .............. . , ,. ~ .. . : . ~
.: : - .,. . ,,. . -., . ~ ., : ,
- , - . : , . : .
......
~ 2 ~
~09~/0880~ PCT/~S91/084~0
- 63 -
(Mohandas et al., 1980, J. Clin. Invest. 66:563-573;
Clarck et al., 1983, Blood 61:899-910; Pasvol et al.,
1989, Blood 74:1~36-1843). Abnormal values are
obtained in many disorders associated with shortened in
5 vivo erythrocyte survival (Clarck et al., 1983, Blood
61:899-910). It is notable, therefore that thrombo-
erythrocytes were indistinguishable from untreated
erythrocytes in this assay.
Thrombo-erythrocytes are able to selectively
10 interact with platelets activated with ADP,
epinephrine, or thrombin to produce large aggregates
containing mixtures of platelets and erythrocytes.
Studies wich monoclonal antibodies to GPIIb/IIIa and
fluid phase RGD peptides indicate that the RGD peptides
15 on the erythrocytes bind to the activated GPIIb/IIIa
receptors on the platelets. The interactions occur
even in the presence of normal amounts of plasma
- fibrinogen, indicating that the thrombo-erythrocytes
can compete effectively with fibrinogen for binding to
20 activated GPIIb/IIIa receptors. In addition, the
interactions are not limited to platelets in citrated --
PRP since platelets in PRP prepared from blood
anticoagulated with either heparin or hirudin are also
able to interact with the thrombo-erythrocytes.
To simulate better the likely in vivo situation at
a site of vascular injury, where platelets first adhere
to adhesive proteins in the blood vessel wall, we also
tested the ability of the thrombo-erythrocytes to bind
to platelets that had adhered to collagen. The
30 thrombo-erythrocytes, but not control erythrocytes,
bound readily to the adherent platelets, and studies
with a monoclonal antibody to GPIIb/IIIa and RGD
peptides again supported a mechanism involving the
interaction of the RGD peptides with the activated
GPIIb/IIIa receptors.
.. ..
~092/0~ PCT/~S91/0~30
2 0 9 5 9 2 ~ 64 ~!
These in vitro studies are positive indicators of
the utility of the thrombo-erythrocyte as a potential
alternative to fresh platelets. Since there are 20
- times as many erythrocytes as platelets in the -
5 circulation of normal individuals, conversion of the
erythrocytes contained in 50 ml of blood into thrombo-
erythrocytes would produce as many thrombo-erythrocytes
as there are platelets in 1 liter of blood, or
approximately 2 conventional units of platelets.
10 Moreover, since erythrocytes are 9 times as large as
platelets, the 50 ml of blood would yield the
equivalent of 18 conventional units of platelets by
mass. The technique of erythrocyte washing is already
standard practice in blood banks and the cross-linking
~5 reaction can be carried out within 1-2 hours, depending
upon the density of peptides selected. Thus, thrombo-
erythrocytes can function as an autologous, semi-
artificial platelet alternative.
In addition to their functions in platelet
20 adhesion and aggregation, platelets make other
contributions to enhancing hemostasis and so it is
appropriate to question whether thrombo-erythrocytes
might also serve to enhance the hemostatic response.
One of the functions platelets serve is to act as a
~5 surface on which coagulation reactions ta~e place
(Walsh and Schmaier, 1987, In Homeostasis and
Thrombosis: Basic Princi~les and Clinical Practice,
Colman et al., eds., Lip~incott, Philadelphia, pp. 689-
703). Both unique platelet receptors and the
30 platelets' non-specific phospholipid membrane have been
implicated in this function and it is unclear how much
each contributes (Walsh and Schmaier, 1987, In -
Homeostasis and Thrombosis: Basic Princi~les and
Clinical Practice, Colman et al., eds., Lippincott,
Philadelphia, pp. 689-703). The erythrocyte membrane
- ` , ~ ` '. ,, ' ~ ~" :
.
. .
~09~/0~80~ 2 0 9 ~ 9 2 5 PCT/~S91/08~30
- 65 - -
can also serve to accel~rate coagulation reactions
under certain circumstances and so it is possible that
thrombo-erythrocytes may also be able to facilitate
thrombin formation (Zwaal et al., 1989, Molec. Cell
5 Biochem. 91:23-31). The recent discovery that
erythrocytes can enhance platelet activation via
cooperative biochemical interactions with platelets
involving eicosanoid metabolism (Santos et al., 1991,
J. Clin. Invest. 87:571-591) provides another potential
0 mechanism by which thrombo-erythrocytes may enhance the
function of residual platelets. Platelets release ADP
from their dense granules when stimulated, leading to
ADP-induced platelet activation; erythrocytes are rich
in ADP and so it is possible that ADP may leak from
~5 thrombo-erythrocytes that become enmeshed in hemostatic
plugs. Finally, the identification of nitric oxide
produced by cells in the blood vessel wall as a potent
inhibitor of platelet activation suggests another
potential mechanism by which thrombo-erythrocytes may
20 enhance platelet function since free hemoglobin and
hemoglobin in erythrocytes have been demonstrated to
neutralize the effect of nitric oxide (Houston et al.,
1990, Blood 76:953-958).
9. A BEAD MOD~L FOR RGD BINDING TO PLATELETS
In the series of studies described herein on
peptides of the general structure (G)D-RGDF (where n
equals the number of glycine residues) that were
covalently attached to polyacrylonitrile beads via
30 their amino termini, it was discovered that the length
of the peptide profoundly affected the ability of the
beads to interact with platelets. Thus, with beads
coated with peptides with n=1, very little interaction
occurred between the beads and either unactivated or
activated platelets, whereas when n=9, strong
.- - .......... . ........... . ~ . . - :
. : , :.,, , . . - : . . :
.. : :., . ~ . :
U'09 /0~0~ PCT/~S9~/0843~
2~9592~- 66 - ~
interactions occurred with both unactivated and
activated platelets. When the peptide had n=3, the
interactions were highly dependent on the state of
platelet activation, with platelets treated with PGE~
5 reacting poorly if at all, and platelets treated with
ADP reacting bris~ly.
Specifically, to gain additional information on
the RGD binding domains of the platelet integrins (see
Table III), a series of RGD peptides containing
10 variable numbers of glycine residues as spacers [(G)n~
RGDF] were immobilized on polyacrylonitrile beads via
their amino-terminal glycine residues and the ability
of these beads to interact with platelets was then
evaluated. The differential platelet agglutinating
l5 effects of these beads as a function of the number of
glycine residues permitted the exploration of the ~GD
binding site(s) under basal conditions and in the
presence of platelet agonists and inhibitors. In
addition, we were able to analyze a series of
20 monoclonal antibodies directed at the different
integrin receptors for their ability to inhibit the
interactions.
.
..
.
.. . .
- .. ~ ,.. .
,; - : . .
U'092t08804 ~ PCT/~S9ltO8~30 - 67 -
TABLE III
PLATELET INTEGRIN RECEPTORS
Mean Distance
5 ¦ CommonPlalelel Prolein Chain Number Belween
NameComposition Composilion Ligands of Surface Receptors
o~ 1~ Reccplor, (Angstroms)'
¦ Collagcn .
¦ Receptorla/lla (VLA-2) ~2 1~ Collagen _ 1,0001,490
10 l , . ~
Fibroneclin
ReceptorIc'/lla (VLA-5) ~s ~1 Fibronectin _ 1,000 1.490
Laminin
Recq~torlc/lla (VLA-6) C4 ~1 Laminin _ 1,0001,490
Vitroneclin Vitronectin,
l 5 Reccptor~/Illa ~v ~3 Fibrinogen - ~ 00 4 ,700
fa illerbrand
Throm!oo-
spondin .
_
Fibrinogen GPllbtllla ~ 33 Fibr~nogen, 50,000 214
Receptor Fibronectin,
FWailltebrand
Vitronectin,
Thrombo-
= spondin . -
25 + Assumes a platelet surface area of ~,2~,2 and equal spacing between receptors
9.1. MATERIALS AND METHODS
9.1.1. PL~TELET PREPARATION
Blood was drawn by syringe and placed in
3~ polypropylene test tubes containing 0.01 volume 40%
trisodium citrate. Platelet-rich plasma (PRP) was
prepared by centrifugation at -700 X g for 3.5 min at
22C and adjusted to 3 X l0ll/l with platelet-poor
plasma (PPP, prepared by centrifuging for 10 min at
3~ 1600 X g at 22C). Gel-filtered platelets (GFP) were
prepared as pre~iously described (Coller et al., 1989,
~.
~092tOX804 ~ PCTtUS9ltO843~
~ J ~ t; `~ 68 - ~ ;
Blood 74:182) by layering the PRP onto a column of
Sepharose 2B and eluting with a modified Tyrode's
solution containing no added CaCl2 and 2 mmol/l MgCl2
(138 mmol/l NaCl, 2.7 mmol/l KCl, 0.4 mmol/l NaH2PO4, 12
5 mmol/l NaHCO3, 2 mmol/l MgCl2, 0.2% bovine serum albumin
(BSA), 0.1% glucose, 10 mmol/l HEPES, pH 7.4).
9.1.2. PEPTIDES
The peptides (G) n-RGDF were synthesized on two
10 different occasions on a solid phase synthesizer
(Applied Biosystems, Foster City, CA, Model 430A) using
t-Boc chemistry; a 4-methylbenzhydrylamine (4-MBHA)
resin was used so that the peptides contained carboxy-
terminal amides rather than free carboxyl groups after
l5 cleavage. Benzyl-ester and tosyl side chain protection
groups were used, respectively, for aspartic acid and
arginine. In the first synthesis, the peptide Gl RGDF
was prepared and 20% of the resin was removed and
subjected to cleavage with HF. Two glycines were then
20 added to the peptide on the resin and another 20% was
removed for HF cleavage. The process was continued
until the entire series of peptides (G" G3, G5, G7 and G9
RGDF) were synthesized. The second synthesis followed
the same general scheme. but consisted of four
25 different syntheses: G, and G3 RGDF; Gs and G7 RGDF; Gg,
G~ and G,3 RGDF; and Gl5, Gl7 and Gl9 RGDF. Double
couplings were used for arginine, phenylalanine, the
fifth and all subsequent glycine residues in the first
synthesis, and the fourth and all subsequent glycine
residues in the second synthesis. The peptides were
cleaved from the resin with HF in the presence of
anisol and dimethylsulfide (10:1:1 by volume); the
starting temperature was -10C and the temperature was
35 maintained below -2C throughout the cleavage by adding
ice to the ice-salt mixture. The peptides were washed
.. , . .. . .. - .
~;`i~ !
~'092/0880~ PCT/~iS91/08430
(` ~O~S925
with ethyl ether and then extracted twice with 30% HAc
and twice with 10% HAc. The pooled extracts were then
diluted to a final concentration of ~10% HAc and
lyophilized. Peptide homogeneity was assessed by HPLC
5 using a C8 reverse phase column (Aquapore RP-300,
Applied Biosystems, 300 ~ pore size, 7 ~ spherical
silica, 4.6 X 220 mm) with an acetonitrile gradient of
0.60% in 0.1% trifluoroacetic acid that was programmed
to run over 40 min. Average purity was 72 + 5% (mean +
10 SEM) in the first synthesis and 80 + 3% in the second.
Since Glg RGDF exhibited the poorest homogeneity as
judged by HPLC (56~), it was purified to >95%
homogeneity with preparative HPLC on a larger column of
the same material (lO X 250 mm). The functional
t5 activities of the crude and purified G~g RGDF peptides
in the bead agglutination assay were the same.
fibrinogen ~-chain dodecapeptide (amino acids 400-411)
containing an added amino-terminal tyrosine (Y-
HHLGGAKQAGDV) was a gift from Dr. Ellinor Peerschke,
20 State University of New York at S~ony Brook, NY. The
snake venom peptide trigramin, which contains an RGD
sequence and inhibits fibrinogen binding to GPIIb/IIIa,
(Huang et al., 1987, J. Biol. Chem. 262:16157) was a
gift of Dr. Stephan Niewiarowski, Temple University,
25 Philadelphia, PA.
Fast atom bombardment mass spectrometry was
performed on peptides prepared in the second synthesis
by collecting the HPLC elution peaks, drying the
samples under vacuum (Speed Vac Concentrator, Savant
30 Instruments Inc., Farmingdale, NY), and redissolving
them either in methanol (G~7 RGDF) or 2S% HCl (G919
RGDF). For the G~l RGDF peptides, the mass spectrometry
probe was precoated with 1 ~1 of 50% glycerine/50%
35 thioglycerine matrix and then l ~1 of the peptide
solution was added; for the Gl3~9 RGDF peptides, the ~-
- - ~ .. .
.
UO92/Og80~ PCT/~;S91/0~430
209592~ 70 - ~'
probe was precoated with 1 ~1 of thioglycerine matrix
and then 1 ~1 of the peptide solution was added. The
fast atom bombardment mass spectra were generated on a
Kratos MS890/DS9O mass spectrometry system (Ramsey,
5 NJ). A saddle field ion source (Ion Tech, Middlesex,
England) was used as a source of fast xenon atoms; it
produced 1 mA of ion current when operated at 7 kV.
The mass spectrometer was operated at 6.8 kV and the
mass range was calibrated with cesium iodide in the
0 positive ion mode using a 10 sec/decade scan speed
after adjusting for the 1 amu added to the molecule by
protonation. The observed molecular weights of all
peptides prepared in the second synthesis (G~9 RGDF)
matched precisely the predicted molecular weights. To
15 verify the identity of the peptides prepared in the
first synthesis, 1:1 mixtures of the corresponding
peptides in the first and second syntheses were
analyzed by HPLC and all of them showed only a single
peak.
For studies to assess the effect of RGD-containing
peptides on the binding of radiolabeled monoclonal
antibodies to platelets, the peptide RGDS was obtained
from Peninsula Laboratories (Belmont, CA) or
synthesized as described above. A longer peptide that
25 is a composite of the ~-chain decapeptide (402- 411)
and one of the polyarginine RGDV peptides described by
Ruggeri et al. (Ruggeri et al., 1986, Proc Natl. Acad.
Sci. U.S.A. 83:5708) (LGGAKQAGDV(R)8RGDV) was also
tested. This peptide was synthesized as above; HPLC
3~ demonstrated a single major peak containing more than
67~ of the absorbance.
The BIOGRAPH molecular modeling computer program
(Bio Design, Inc., Pasadena, CA; version 1.34) was used
to determine the peptide lengths. Calculations were
made for the peptides in both the extended form and in
. .
~092/08~04 2 0 9 ~ 9 2 ~ PCT/~91/08~30
- 71 -
t~e alp~a-helical conformation so as to span these two
extreme possibilities. We do not know the exact
conformation of the peptides in solution, but we
suspect that the glycines attached to the RGDF assume a
5 multitude of random conformations because of the
rotational freedom of glycine residues. It is
possible, however, that immobilization of the peptides
on the beads at high density limits this conformational
; freedom.
9.1.3. FIBRINOGEN BEAD ASSAY AND FUNCTIONAL
ASSESSMENT OF G~-RGDF PEPTIDES
The fibrinogen bead agglutination assay was
performed as previously described (Coller, 1980, Blood
15 55:169) Briefly, fibrinogen (lot number PR2548, Cutter
Laboratories, Berkeley, CA) purified according to the
method of Mosesson (Mosesson, 1962, Biochim. Biophys.
Acta 57:204) was coupled to 1.3 ~ polyacrylonitrile
beads containing N-hydroxysuccinimide groups at a ratio
20 of 3 mg fibrinogen per 1 ml bead slurry (containing 67
mg beads) (Matrex 102; Amicon, Danvers, MA). After
coupling was completed, the beads were washed
extensively with 0.15 M NaCl, 10 mM Tris/HCl, pH 7.4,
containing 0.05~ sodium azide (TSA), resuspended in TSA
25 and kept at 4C. To perform the assay, thirty-five ~
of citrated PRP (3 X 10ll/1) was incubated with 35 ~1 of
peptide in TSA in a round bottom 96-well microtiter
plate for at least 10 min at 22C. Then 5 ~1 of beads
containing -0.4 ~g of bound fibrinogen (5 X 105
30 molecules per bead) was added and the plate was rotated
at 260 rpm. The degree of agglutination was graded
visually on a scale between 0 and 100~ as a function of
time. Antibody 10E5, which binds to GP~Ib/IIIa and
blocks fibrinogen binding (Coller et al., 1983, Blood
35 61:99), produces complete inhibition of bead
agglutination in this assay when used at 10-20 ~g/ml,
~: . : : .: ., : '. , .: .
,
.- . . - ,. : .:
, . . , : .
- -
092/0880~ PCT/~S9l/08~30
2 09 ~92 ~~ 72 - ~,-
and so a sample containing 10E5 instead of peptide was
included in each assay as a positive control. A
negative control consisting of TSA buffer instead of
peptide was also included in each assay; this control
5 consistently reached maximal agglutination (100%) after
4 minutes and so the inhibitory effects of the peptides
Gl9-RGDF in solution were also assessed after 4 minutes.
The longer peptides, G~9 RGDF, were poorly soluble
under these conditions and so could not be tested in
this manner (see below).
9.1.4. COVALENT COUPLING OF PEPTIDES TO BEADS
One ml of the polyacrylonitrile beads (~7 mg)
(Matrex 102, Lot nos. JC 1236 and 1239) in dry dioxane
15 was centrifuged at 10,000 X g for 1 min at 22C. The
supernatant dioxane was removed, and the beads were
rapidly washed twice with 0.05 M Na acetate, pH 5.5.
The pelleted beads were then resuspended in 1 ml of a
4.05 mM solution of peptides Gl, G3, G5, G7 or G9 RGDF in
20 0.05 M Na acetate buffer, pH 6.5, and allowed to rock
overnight at 4C. Any remaining reactive N-hydroxy- -
succinimide groups were blocked by adding 0.1 volume of
1 H glycine ethyl ester, pH 8 for 30 min at 22~C. The
beads were then washed extensively in TSA, pH 7.4,
2~ resuspended in a volume of 1.5 ml of TSA and stored at
4C. The G~l, G,3, Gl5, Gl7 and G,9-RGDF peptides were only
partially soluble in the acetate buffer, but they were
fully soluble in 0.1~ trifluoroacetic acid (TFA), pH
2.5, at 22C or 37C. The G~, G~3, G,5, G" and G,9-RGDF
peptides therefore, were coupled to the beads in 0.1%
TFA, pH 2.5 for 72 hrs. For comparison purposes, the
G~, G3, G5, G, and G9 RGDF peptides were also coupled to
beads under these circumstances and these beads were
35 similar in coupling efficiency and platelet
agglutinating activity to those coupled in the acetate
- ~ ~ , . .
' ; ' ~ ' ~ .
~:
:
,
~'O 92/O~R01 2 0 9 5 9 2 ~ PCT/l!S9l/08430
-- 73 --
buffer (see below). Bovine serum albumin (BSA,
essentially globulin free, Sigma) at 30 ~M was coupled
to l ml of the beads as a control. In another series
of experiments to assess the impact of decreasing the
5 density of peptides on the beads, the G3 RGDF and G9
RGDF peptides were diluted to concentrations between
0.4 ~M and 4.05 mM before coupling.
Coùpling efficiency was assessed by HPLC by
comparison of the areas under the peptide peaks in the
10 pre- and post-coupling samples; the results are shown
in Table IV for the standard method using 4.05 mM
peptide. The Gl, G3, Gs~ G7 and G9 RGDF peptides at pH
6.5 and the Gll, G,3, G,5, G,7 and G,9 RGDF peptides at pH
2.5 were coupled at -74% and -91% efficiency,
15 respectively.
2C
, . :. . .. ~ : .
.. : .. '. :' :. : . ~ - . :. .. : .. : ,, .. : , .. .. . . ...
:: . . :. . . : ,. : ,: ,.. , .: . .. . . .- - , .
: : . . .. . . :- . . .. : . . . .
,.~.` ~ 1
~092/0880~ PCTJ~;S91/08430
2 09~92~ - 74 - ~
~ABLE IV
EFFICIENCY OF PEPTIDE COUPLINGS TO POLYACRYLONITRILE BEADS
Concentration Coupling
Pe~tides (mM) B~ffer EfficiencY l%)
G~9-RGDF 4.05 Acetate, pH 6.5 73+1~(n+5) and
75+4(n=5)
G~ -RGDF 4.05 TFA- 9l+5 (n=5)
G~-RGDF 2 ~cetate, pH 6.5 94
G~-RGDF 1 Acetate, pH 6.5 9/
0 G~-RGDF 0.5 Acetate, pH 6.5 98
G~-RGDF 0.0004-0.5 Acetate, pH 6.5 >98
Go~RGDF 2 Acetate, pH 6.5 88
Go~RGDF l Acetate, pH 6.5 92
G~-RGDF 0.0004-0.5 Acetate, pH 6.5 98
G9-RGDF <0.5 Acetate, pH 6.5 >98
Albumin 0.03 Acetate, pH 6.5 98
*Mean + SEM
+Trifluoroacetic acid
Assuming an average coupling efficiency of 80% for
these peptides, then ~l.9 X 10'~ peptide molecules were
bound to each 67 mg of beads ((4.05 ~moles/ml) (6.02 X
1017 molecules/~mole) (0.8~); this number of molecules
25 represents ~40% of the N-hydroxysuccinimide groups on
the beads, indicating the high efficiency of coupling.
Since the estimated surface area of the beads is 6
m2/gram, the peptides are ~4.6 ~ apart on the beads ((6
m2/gram) x (0.067 gram) = 0.4 m7; ~0.4 m7 = 0.63 m or 6.3
30 x lo9 A; ~1.9 x 10l8 molecules = 1.38 X 10~ molecules;
(6.3 X 109 ~) j(1.38 X 109 molecules) = 4.6 ~ per
molecule) (Table V). The extraordinarily high peptide
density this represents is best appreciated by
translating the results into an equivalent molar
35 concentration assuming that the molecules are 4.6 ~
apart in fluid phase; the result is a 17 M solution.
,., , , j ., . , . .. . ~ - . .- ... , ... .. . ~... . , . .: . .:
t~
U092/08801 2 0 9 ~ 9 2 5 PCT/US91/08~30
.. _ 7~ _
For comparison, the mean distances between fluid phase
peptides at various concentrations are also given, as
are comparable values for fibrinogen molecules on
beads, fibrinogen molecules in plasma, albumin
5 molecules on beads and albumin molecules in plasma.
TABLE V
LIGAND DENSI~IES AND CONCENTRATIONS
Amount Cou- Mean
Added to plingDistanceMolar
67 mg Efficien-Betweenco~cen-
Beads cy~,oleculestration
Liaand ~moles)(~O) (A) Ecuivalent
Immobilized 4.05 -80 4.6 1.7 X 10
G,~9-RGDF 4.05 X 10l98 13 7.5 X 10l
Peptides 4.05 X 10-98 41 2.4 X 10-
4.05 X 10198 130 7.6 X 104
~.05 X 1oJ 98 4102.4 X 10'5
Soluble 550 l X 10'5
G~,g-RGDF 260 1 X 10'4
Peptides 120 1 X 10
94 2 X 10~
4 X 10~ .
Immobilized
Fibrinogen 5.9 X 10'3~88~ 113 1.2 X 10'3 : .-
Fibrinogen
In Plasma 570 8.8 X 10~ .
IITUTobilized
Albumin3.0 X 10-98 47 1.6 X 10-
Albumin I~
Plasma 140 6 X 10
9.1.5. MONOCLONAL ANTIBODIES
Table VI lists the antibodies, their specificities
and the concentrations used. They have all been --
30 characterized previously: antibodies 10.E5 (Coller et
al., 1983, J. Clin. Invest. 72:325), 7E~ (Coller B.S.,
1985, J. Clin. Invest. 76:101), 6D1 (Coller et al.,
1983, Blood 61:99), and 6F1 (Coller et al., 1989, Blood
74:182) are from the laboratory; antibody A~A4 (Bennet
35 et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2417)
was a gift of Dr. Joel Bennett, University of
' ':
Sl.5~S~; 7 U I E S'~_E~ ~ -
~'O 92/0880~ PCT/~S91/08430
2 ~ 9 ~ ~ 2 5 - 76 - (
Pennsylvania; antibody PAC-1 (Shattil et al., 1985, J.
Biol. Chem. 260:1107) was a gift of Dr. Sanford
Shattil, University of Pensylvania; antibody LM 609
(Cheresh et al., 1987, J. Biol. Chem. 262:17703) was a
5 gift of Dr. David Cheresh, Scripps Research Institute;
antibody GoH3 (Sonnenberg et al., 1988, Nature 336:487)
was a gift of Dr. A. Sonnenburg of the Netherlands Red
Cross; and antibody mAbl6 (Akiyama et al., 1989, J.
Cell. Biol. 109:863) was a gift of Dr. Kenneth Yamada,
National Institutes of Health, Bethesda, MD. The
platelets wère incubated with the antibody
concentrations indicated for 30-60 min before the
addition of the beads. In the case of PAC-1, which
binds only to activated platelets (Shattil et al. 1985,
15 J. Biol. Chem. 260:1107), the platelets were activated
with ADP (6.7 ~M final concentration) without stirring
for 5 min before adding the antibody.
-
.. . . . . . . ... .. . .
- . . .
:
,, . . : , .
: ,
"
-
, . - . , ,. . , , ~.
.: . :, . . . . .. . .
~O9~/08801 2 0 g ~ 9 2 5 PCT/~S91/08~30
.:
77 -
TA8LE VI
MONOCLONAL ANTIBODIES AND PEPTIDES TESTED
IN G9-RGDF BEAD ASSAY
Inhibition of
Gg-RGDF Bead
Aq~lutination ~%)
Desiqnation SPecificity Concentration -ADP+ADP
Antibodies
10E5 GPIIb/IIIa20 ~g/ml 40 21
~E3 GPIlb/IlIa+a/IIIa20 yg/ml 100 100
A~4 GPIIb/IIIa20 ~g/ml 62 45
PAC-l GPIIb/IIIa60 ~g/ml - 8
LM609 ~v/IIIa 100 ~g/ml 0 0
GoH3 GPIc/IIa100 ~g/ml 0 0
mA~16 GPI~*t~Ia20 ~g/ml 0 0
6Fl GPIa/IIa20 ~g/ml 0 0
6Dl GPIb 20 ~g/ml 0 0 .
Peptides
RGDF <4.2 X 10~M 0
1-4 X 103M50-100
Fibrinogen <4.5 X 10~M 0
Dodecapeptide (+Tyr) 1.8 X 103M 13
(Y-HHLGAKQAGDV) 3.6 X 103M 67
Trigramin 4.5 ~g/ml 0
9.1.6. AGGLUTINATION ASSAY WITH PEPTIDE-COATED BEADS
To 70 ~1 of PRP or GFP at a count of 3 X 101l
platelets/l in a 96 well round bottom polystyrene
microtiter plate, 5 ~1 of a well mixed bead suspension
(0.22 mg beads) was added and the plate was rotated at
~5 260 rpm (Orbit shaker, Lab Line Instruments, Melrose
Park, IL) for various times between 0 and 30 min (0.5,
1, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30 min). At the
indicated times the wells were observed from the bottom
with the aid of a magnifying mirror apparatus (Cooke
30 Microtiter System, Dynatech Laboratories, Inc.
Alexandria, VA) and the degree of the bead- :
agglutination was rated visually from o% (no visible :
ayg~utination, indistinguishable from the control,
albumin-coated beads) to 100~ (full agglutination)
(Figure 8A). Platelet c~unts in the supernatant plasma
~09_/()8X0~ PCT/~S91/08~30
2 ~ . 78 ~
or buffer were measured by removing 5 ~l from the
supernatant after allowing the bead agg~utinates to
settle for 3 min. The platelet counts decreased during
the agglutination, indicating that increasing nu~bers
5 of platelets were incorporated into the agglutinates
(Figure 8). In some experiments, platelets were
activated with 6.7 ~M ADP 30 sec prior to the addition
of the beads; in other experiments the platelets were
preincubated with 0.14 ~M PGE, (Sigma, St. Louis) for 30
10 min. Control experiments indicated that there was no
agglutination at all in the presence of EDTA (l0 mM).
There was only a minimal increase in the supernatant
lactate dehydrogenase level during the agglutination,
indicating that the platelet-bead interaction did not
15 produce significant platelet lysis.
9.1.7. RELEASE OF 14C~SEROTONIN FROM PLATELETS
Citrated PRP (3 X l011/l) was incubated with '4c-s-
serotonin (l0 ~Ci/ml stock solution, 20-25 nCi/ml final
20 concentration, New England Nuclear, Boston, MA) for 30
min at 22C with gentle stirring (~l00 rpm) using a
teflon coated stir bar. In order to maintain the PRP
pH constant at ~7.60, the PRP was overlayed with 5% C02,
95% air just before the incubation began. Serotonin
25 uptake was terminated by adding 5 ~M imipramine (Sigma)
and stirring was continued for another 5 min. Total
uptake under these conditions was 70-80% of the added
serotonin. The agglutination assay was performed in
quadruplicate in the same way as described above except
30 that after 8 min of shaking, 70 ~l of cold 2%
paraformaldehyde in PBS, pH 7.4 was added to the wells
and the microtiter plate was centrifuged at l000 X g
for 5 min at 22C. The top 50 ~l of the supernatant
~total volume in each well was 145 ~l) was added to
scintillation fluid and counted in a scintillation
- :, . . ...
... . . . . . . . . . .
: ~ ~, , , , , : :
.....
I
~092/08804 2 0 9 S 9 2 5 PcT~;sgl/0843n
- 79 - i
spectrometer. In some experiments the PRP was
preincubated with acetylsalicylic acid (50 ~M) or PGE~
(0.14 ~M) during the serotonin uptake.
9.1.8. EFFECT OF RGD PEPTIDES
ON 7E3 AND lOE5 BINDING
To assess the effect of RGD-containing peptides on
the initial rate of binding of 7E3 and 10E5, studies
were conducted with l25I-7E3 and l25I-10E5 essentially as
described before (Coller, B.S., 1985, J. Clin. Invest.
76:101). The RGDS or LGGAKQAGDV(R)8RGDV peptide was
incubated at 22C with citrated PRP (-3 x 108/ml) for 1
to 5 min at vari~us concentrations and then non-
saturating concentrations of the antibodies (1.5 - 2.5
~g/ml) were added for 1 to 2 min. The bound antibody
was separated from the free by centrifugation of the
PRP through 20~ sucrose and both the platelet pellet
and the supernatant were counted. Results were
expressed as either an increase or decrease in antibody
20 binding as compared to a buffer control.
9.2. RESULTS - -:
9.2.1. EFFECTS OF SOLUBLE G~9-RGDF PEPTIDES
ON THE FIBRINOGEN BEAD ASSAY
The ability of the soluble G~9-RGDF peptides to
inhibit platelet-fibrinogen interactions was tested
with the fibrinogen ~ead assay (Tab~e ~II). -
~'O9~/08804 i , PCT/~S91/08430
20959~25- ~ ~ . (;,,:'
TABLE VII
MINIMAL INHIBITO~Y CONCEMTRATIONS OF FRE~ (G)n-RGDF
PEPTIDES IN SOLUTIO~ IN THE FI8RINOGEN-BEAD ASSAY
Pe~tide Minimal Concentration Required to
Prevent ComPlete A~alutination (~M~
RGDF 16, 32*
G~-RGDF 32, 56
-RGDF 32, 56
0 Gs-RGDF 63, 113
G7-RGDF 113, 127
G~-RGDP 227, 253
*Values are from 2 different experiments ~sing platelet-rich
plasma.
There was a distinct decrease in potency with
increasing numbers of glycine residues, with the RGDF
peptide approximately ten-fold more potent than the G9- : -
RGDF peptide. The decreased potency of the longer
peptides is unlikely to be accounted for solely on the
basis of decreased diffusion coefficients since the
molecular weight of the Gy~~GDF peptide (786) is less
than twice that of the RGDF peptide (435).
9.2.2. PEPTIDE BEAD AGGLUTINATION ASSAY
Al~umin coated beads were not agglutinated by the
platelets, e~en when ADP was added. This indicates
that the beads are not non-specifically incorporated
30 into the platelet aggregates that form when ADP is
added.
The data in Figure 8 and 9 show the extent of
agglutination of (G)A-RGDF beads by PRP as a function of
time and the number of glycine residues. Both the
35 total platelet agglutinating activity and the speed of
agglutination increased dramatically as the number of
tSTlTuT- S~IE- ~ -
- - . . . . ........... ~ , . . , ............... . . . .;
.. ~ .. ~. . , ,. ., . . . ` -, .
:,,
~\O 92/0880~ 2 ~ g ~ PCr/~S91/08~30
glycine residues increased from 1 to 13 and then the
activity decreased as the number of glycines increased
further to 19. The G~-RGDF beads gave values in between
those produced by the G5-~GDF and G9-RGDF beads, and the
5 G"-RGDF and G1s-RGDF beads gave values that were similar
to those of the G13-~GDF ~eads. Thus, even though the
shorter RGDF peptides were more potent than the G9-RGDF
peptide when tested in fluid phase in inhibiting the
fibrinogen bead assay, the immobilized Gg-RGDF peptide
o was much more potent in agglutinating platelets than
the immobilized smaller peptides. The decrease in
platelet agglutinating activity of the longest peptides
(G,7- and G~g RGDF) was notable and perhaps suggests that
the peptides have sufficient freedom to fold back on
~5 themselves or interact with each other.
The addition of ADP to the platelets prior to the
(G)D_RGDF beads increased both the speed and extent of
agglutination with all of the different beads (Figure
lOA). The G3-RGDF beads showed the greatest increase in
20 total extent of agglutination with ADP stimulation,
going from 44 ~ 6% (n=17) to 96 + 4% (n=7) at 30 min.
Thus preactivation of the platelets with ADP appears to
increase the affinity of the RGD-binding sites andlor
reduce their distance from the platelet surface.
Preincubation of the PRP with PGEI significantly
diminished the ability of the PRP to agglutinate the
(G)n-RGDF beads (Figure lOB). The agglutination of the
Gl-RGDF and G3-~GDF beads was completely abolished, but ~ -
agglutination with the longer beads was less affected;
in fact, the Gg-R~DF beads underwent 75 + ~6% (n=7)
agglutination at 30 min and the G~5-~GDF beads underwent
88 ~ 13~ (n=4) at the same time p~int. Thus, the
inhibition of platelet activation by PGEI was not able
35 to prevent significant interactions between platelets
and the longer (G)n-RGDF beads. The beads coated with
- .: . ;: . .
~ ~ ,
~092/0880~ PCT/~iS91/0843
2 ~9 59 2 5 ~ 82 -
the G3-RGDF peptide showed the most marked differences
in platelet interaction as a function of platelet
activation; PGEI totally inhibited the interactions,
whereas ADP dramatically increased both the speed and
5 extent of agglutination (Figure l~C). Thus, the G3-RGDF
peptide appears to have a critical length or
flexibility for reporting on the state of activation of
the RGD receptor site(s).
Decreasing the number of peptides on the bead
10 surface decreased the agglutination reaction (Figure
11), but full agglutination could eventually be
achieved even when the peptide number was decreased
nearly 10-fold. Some agglutination of PRP occurred
even when the peptide number was reduced approximately
1~ lOO-fold. With ADP preactivation, all of the bead
preparations showed enhanced agglutination and full
agglutination was even achieved with the beads
containing approximately lOO-fold fewer peptides.
Based on the density calculations (Table V), these data
20 indicate that a peptide-peptide distance on the beads
<13 ~ is re~uired for full agglutination of non-
activated platelets, whereas a peptide-peptide distance
<41 ~ is adequate for full agglutination of ADP-
activated platelets.
The inhibitory effects of free ~GDF and ~-chain
peptides ~n the bead agglutination were also
investigated. High RGDF-concentrations were required
to inhibit the agglutination of the beads containin~
the longer peptides (Table VI) and lower concentrations
~ were required with the shorter peptide beads. For
example, 41 ~M RGDF abolished agglutinati~n with Gl-RGDF
beads, whereas 400 ~M was required to abolish
agglutination with G3-~GDF beads, and 3-4 mM RGD~ was
35 needed to abolish agglutination with beads containing 7
or more glycine residues. Lower concentrations of RGD
, - ~ - , ~ , . -
~ 0 , ~ a ~
9_/0880~ PCT/US9~/08~30
.: - 83 -
peptides produced incomplete inhibition that was most
nota~le during the early phase of platelet
agglutination. The fibrinoqen ~-chain dodecapeptide
derivative also inhibited agglutination of the (G)n-RGDF
5 beads; on a molar basis, the potency of the inhibition
of the ~-chain peptide was slightly less than that of
the RGDF peptide (Table VI).
9.2.3. THE RELEASE OF SEROTONIN
10 The interaction of the platelets in PRP with the :
longer immobilized peptides resulted in the release of
serotonin, and the extent of release correlated with
the extent of agglutination (Table VIII).
TABLE VIII
Relea~e of Serot~nin From Platelet~ in Platelet-Rich
Plasma Interacting With (G)~-RGDF ~ead~
For 8 Minutes
Serontonin ~eleaqe Agglutination
20 Pe~tide on Bead(% of Maximal~~ of Maximal~
Gl-RGDF 1~ 0~
G3-RGDF 2 17
G5-RGDF 9 75
G7-RGDF 16 88
25 Gg-R~DF 19 l~o
*Mean value~ for 2 or 3 experimen~s
Preincubation of the platelets with
acetylsalicylic acid (50 ~M) did not change this
pattern. PGE~, however, abolished release for the 8 min
of agglutination with all the beads, but the
agglutination response with the longer beads still
reached 50%. Thus it can ~e inferred that the release
35 reaction is not a prerequisite for agglutination, but
it may amplify and accelerate the response.
. : . , ~ . . ~ .
.: ;
. . .. .
.: ,
wO 92/Og801 ~ ~ 9 a ~ ~ - PCT/~'S9l/08430
9.2.4. THE INTERACTION OF GFP WITH RGDF BEADS
Gel-filtered platelets showed a pattern of
interaction with the beads that was similar to that
observed with PRP in that the agglutination was
5 dependent on-the peptide length and was enhanced by
activating the platelets with ADP (Figure 12). These
data indicate that interaction of the beads with plasma
proteins is not required for agglutination. There
were, however, some minor differences in agglutination
10 with GFP compared to PRP. Thus, with the b~ads
containing the shorter peptides, the agglutination by
GFP was slower and less extensive than with PRP. In
contrast, with the beads containing the longer
peptides, the agglutination by GFP was somewhat brisker
15 than that produced by PRP, although the ultimate extent
of agglutination was maximal with both platelet
preparations. The effects of gel-filtration on
platelet functions, the presence of plasma proteins,
and/or the differences in pH ~PRP p~ -7.70 and GFP pH
20 7.40) probably account for these minor differences.
9.2.5. EFFECT OF MONOCLONAL ANTIBODIES
ON RGDF BEAD AGGLUTINATION
Monoclonal antibodies 10E5, A2~, 7E3, and PAC-1,
2S which interact with GPIIb/lIIa and inhibit fibrinogen
binding to platelets, all inhibited the interaction of
platelets in PRP with G~-RGDF, G3-RGDF, and G5-~GDF beads
hy >90%, even with ADP activation. With the longer
~eads, however, differences in the antibodies'
30 inhibitory potency became apparent. As illustrated in
Figure 13A, with the G9-RGDF beads 10E5 markedly delayed
the initial agglutination response but by 30 min it
reached -60~ of the control value. Since 10E5 b~ocks
platelet aggregation, this demonstrates that platelet
35 aggregation is not required for agglutination to occur.
Increasing the lOE5 c~ncentration 3-fold (to 60 ~g/ml)
- , - . - . . .
. . . - - : . . : ~ : -
~ ~ . - - . .. . .
- . - . . . .
- : .: . , . . : ,
uo 92/088n~ 2 0 9 5 ~ 2 5 PCT/~S9l/08130
. . ~ ..
- 85 -
did not change this pattern. A2~ produced greater, but
still incomplete, inhibition, with the agglutination
reaching only 38% by 30 min. 7E3 produced the most
extensive inhibition, with no agglutination detectable
5 at 30 min. When the assay was performed with platelets
that were stimulatèd with ADP, lOE5 and A2~ were
somewhat less inhibitory, especially at the earlier
time points, but the differences were still dramatic
compared to the control (Figure 13B). 7E3 was still
10 able to produce complete inhibition under these
circumstances. With Gll-, G~3-, and G~7-RGDF beads and
ADP activated PRP, however, even 7E3 could not
completely inhibit agglutination, with the values
reaching 13-25~ after 30 min (data not shown).
One possible explanation for the inhibitory
potency of 7E3 is that it reacts with av/IIIa VnR in
addition to GPIIb/IIIa (Coller et al., 1991, Blood
77:75; Charo et al., 1987, J. Biol. Chem. 262:9935).
To assess this, we studied the effect of antibody LM
20 609, which reacts with a~/IIIa but not GPIIb/IIIa, and
can block av/IIIa function (Coller et al., 1991, supra;
Cheresh et al., 1987, J. Biol. Chem. 262:17703~.
Antibody 7E3 also can inhibit av/IIIa function, and
preincubation of platelets with 7E3 decreases the
2~ binding of 125I-LM 609, suggesting that 7E3 and LM 609
may bind to nearby sites on av/IIIa. LM 609 alone had
no effect on bead agglutination and the combination of
lOE5 (anti-GPIIb/IIIa) and LM 6099 (anti-~/IIIa) was
not more inhibitory than IOE5 alone. Thus, these data
do not support the hypothesis that 7E3 is a more potent
inhibitor than other antibodies to GPIIb/IIIa because
it also reacts with av/IIIa.
PAC-l, which effectively inhibited the
3S agglutination of G~-RGDF and G3-RGDF beads, had much
less inhibitory acti~ity than the other 3 anti~odies
: - - . . , -- . .
.
. , , . . : :
.. .
~092/088n~ PCT/~S91/08430
2Q9~925 ~1
when tested with the longer beads With the G9-RGDF
beads, for example, it produced only 8~ inhibition at
30 min (Table VI). Thus, even though convincing
evidence indicates that PAC-l binds to the RGD-binding
5 site (Taub et al., 1989 J. Biol. Chem. 264:259), it was
much less inhibitory than the other antibodies.
Trigramin at 3-4.5 ~g/ml (~0.6 ~M), which is
approximately twice the concentration reported to
nearly saturate platelet GPIIb/IIIa receptors (Huang et
10 al., 1987, J. Biol. Chem. 262:16157), inhibited the
agglutination with the shorter beads, but with the
longer beads it inhibited only the early phase, such
that at 30 min no inhibition was observed (Table VI).
A similar inhibitory pattern was observed with the
15 soluble RGDF peptide at ~200 ~g/ml (~400 ~M),
consistent with data for Huang et al. (supra) showing
trigramin to be approximately 500-fold more potent than
RGDS peptides in inhibiting fibrinogen binding to
platelets.
All of the antibodies against the other integrin
receptors on platelets that may recognize RGD-
Gont~ining sequences within their ligands (GoH3 (anti-
Ic/Ila), mAbl6 (anti-Ic*/IIa), and 6F1 (anti-Ia/IIa)3
did not inhibit the bead agglutination (Table VI; 3
25 separate experiments with each antibody). 6Dl,
directed against the non-integrin receptor GPIb, also
had no effect on the agglutination. Alth~ugh these
data suggest that the interactions between these
receptors and the (G)n-~GDF peptides do not contribute
30 to the observed agglutination, this conclusion must be
tempered because it is possible that these antibodies
bind to the receptors in a way that inhibits the
binding of the macromolecular ligands to the receptors
without blocking access to the ~GD binding site .
, , - , , - : , : ~
- . . . . : . .............. - ....... . , , ~
- , ~ - - ::
09_/0880~2 ~ ~ 5 ~ ~ ~ PCT/~S9l/08i30
.
l - 87 -
.
9.2.6. EFFECT OF RGD PEPTIDES ON
7E~ AND 10E5 BINDING
Incubation of cltrated PRP with RGDS at
concentrations up to 5 mM did not inhibit the initial
rate of 7E3 binding; in fact, there was a consistent
increase in the rate (at 5 mM a 66% increase in rate
(n=2), at 2.5 mN a 63% increase in rate (n=l), and at
O.5-1 mM a 21~ increase (n=4). RGDS at similar
concentrations had little or no effect on 10E5 binding,
decreasing the rate by 5~ in one experiment and
increasing it by 2% and 24% in 2 other experiments.
The longer RGD peptide [LGGAKQAGDV(R)~RGDV], however,
consistently inhibited the initial rate of binding of
both 7E3 and 10E5. The effect on 7E3 was more
15 pronounced, with increasing concentrations of the
peptide showing increasing inhibition of the initial
rate of 7E3 binding up to a maximum of -73% inhibition
(n=2) at 20 ~M. Concentrations of peptide much higher
than this (80 ~M - 1.67 mM) failed to increase the
20 inhibition significantly (76%; n=8). Concentrations of
the peptide up to 20 ~M caused progressively greater
inhibition of the initial rate of 10E5 binding to
platelets, plateauing at 43% inhibition; as with 7E3,
further increases in peptide concentration failed t~
2L increase the inhibition (80 ~M - 1.67 mM = 46%; n=4).
9.3. DISCUSSION
The interaction of the RGD sequences contained in
adhesive glycoproteins with receptors on the platelet
30 surface is thought to ~e crucial to normal platelet
function. In the present study, we have use~
immobilized RGD peptides of varying lengths as
structural probes of the platelet receptor(s) t~at
mediate these interactions. We found that the shortest
35 immobilized peptide (Gl-~GDF) reacted minimally with
either unactivated or activated platelets in PRP,
~092/O~X0~ PCT/~S91/08~30
- 88 -
2095^925
whereas the long peptides (>G9-RGDF) interacted well
with both unactivated and activated platelets, although
the latter showed brisker interactions. In addition,
the interactions with these longer peptides could
5 induce the platelet release reaction, perhaps by
causing receptor clustering. Inhibition of platelet
activation with PGE1 abolished the interaction of
platelets with the beads containing the shorter
peptides, but only partially inhibited the interaction
10 of platelets with beads coated with the longer
peptides. Peptides of intermediate size (G3-RGDF)
demonstrated the greatest sensitivity to the platelet's
activation state, with virtually no interaction with
platelets pretreated with PGE~, slow and incomplete
15 interaction with native platelets, and brisk and
extensive interaction with ADP-activated platelets.
In contrast to the enhanced ability of the longer
immobilized peptides to interact with platelets, the
longer peptides in solution were less potent than the
2~ shorter ones in inhibiting the interaction between
platelets and fibrinogen-coated beads. This indicates
that the immobilized longer peptides do not show
enhanced interaction with platelets as a result of an
intrinsic affinity advantage conferred by the increased
25 number of glycine residues; in fact, they had to
overcome an intrinsic disad~antage with regard to
affinity. Differences in peptide density on the beads
also cannot account for the observed differences
~ecause the peptides were all immobiIized at
3~ approximately the same density and dilutional studies
indicated little effect of minor differences in peptide
density. It is most likely, therefore, that the longer
peptides were more effective because they could more
easily gain access to the RGD binding sites in the
receptors. Both the increased le~gth and increased
.... . . . - . . . .................... .. . .
- . .: . . . . . . . , . .~ : :
.: . .: , : . . , . .................... . ~ . . , . . :
. . .: . : : . . .. :.
~ 92/0880~ 2 ~ 9 5 9 2 ~ PCT/~;S91/08430
; - 89 -
flexibility of the longer peptides could contribute to
this enhanced ability to interact with the receptors.
The minimal ability of the shortest peptides to
interact with platelets under any conditions indicates
5 that the RGD binding sites may be recessed from the
surface of the receptors by at least several angstroms.
The ~radient of increased interactions as the peptide
length increased suggests that the RGD binding sites
may be arrayed at various depths, either because the
10 receptors themselves are at variable distances from the
platelet surface or because the RGD binding sites are
variably recessed in the receptors. Since the increase
in reactivity was most dramatic between the G3-RGDF and
G,-RGDF beads, and since there was very little increase
15 in platelet reactivity after the peptide length was
increased beyond 9 glycine residues, it is likely that
most of the receptors lie within reach of the G3-RGDF
and G9-RGDF peptides. To estimate the maximum lengths
this range of peptides represents, we assume that the
20 glycines adopt an extended conformation with 3.5 A per
glycine residue rather than an alpha-helical
conformation in which case the glycines would be only
l.55 ~ apart. We conclude, therefore, that the
majority of RGD binding sites can be reached by
25 peptides that extend out -11.32 A from the surface of
the bead. Since not even minor increases in
agglutination occurred with beads containing peptides
longer than Gl3-RGDF, we conclude that virtually all ~f
the receptors can be reached by peptides that extend
out -46 ~ from the surface of the bead.
The increased response after ADP-activation
indicates that activation either causes the RGD ~indin~
sites to move closer to the platelet surface or
35-increases the affinity of the receptor for the RGD
peptides, perhaps by decreasing steric hindrance. One
..
, - . ,: . . ; - : - . . . - :
UO92/08RW PCT/~S91/08~3n
- 90 -
~g~;~,2~ .
could visualize the latter process as either the
opening up of an overlying constricting region or the
straightening of an otherwise tortuous path to the
binding site. The reduced agglutination response
5 produced by PGEl suggests either that it causes the RGD
binding sites to become more recessed or to have lower
affinity, perhaps as a result of greater steric
hindrance. It is important to emphasize, however, that
even with PGEI pretreatment, the longer beads were able
lO to produce substantial agglutination. One possible
explanation is that even under maximal inhibition by an
agent such as PGE~ that increases platelet cAMP, the
receptors are in a dynamic equilibrium between
conformations that can interact with RGD-containing
75 ligands and others that cannot. The binding of a
platelet via a receptor in the proper conformation to
an RGD peptide on a bead would then allow the platelet
to linger at the bead surface while additional
receptors transiently adopt the proper conformation.
~O Since the density of RGD peptides is so high, it is
very likely that the receptor will find an RGD peptide
to interact with even if the length of time the
receptor stays in the proper conformation is brief. In
this way, each interaction makes the platelet linger
2~ longer, facilitating additional interactions and
encouraging the process to continue to full
agglutination. Alternatively, or additionally, the
length and flexibility of the longer peptides may allow
them, with sufficient time, to insinuate themselves -
30 into the RGD binding sites of otherwise inaccessible
receptors. The extraordinarily high density of
peptides on the beads would also fa~or even such low
affinity interactions.
- . ,~. - .. .. -. . -
. .
.
- , . , . ' . ., . ' ' . ' . ' -
.
~ 92/08804 2 0 9 5 9 2 5 pcr/~;s9l/og43o
9 1 --
10. EXAMPLE: A MONCLONAL ANTIBODY
COUPLED TO ERYTHROCYTES
As described supra in Section 5.3, various
targeting molecules can be coupled to erythrocytes, to
produce targeted erythrocytes, specifically targeted
carrier erythrocytes. The present Example demonstrates
targeting of erythrocytes to platelets by coupling a
monoclonal antibody to the erythrocytes.
10.1. MATERIAL AND METHODS
10.1.1. PREPARATION OF ERYTHROCYTES
Whole blood was collected into a tube containing
1.2 ml of CPD-A1 anticoagulant for a final volume la
15 ml. The blood was centrifuged at 700 x g for 3.5 min
at 22C. The platelet-rich plasma (PRP) was removed
and centrifuged at 1700 X g for 10 min at 22C. The
buffy coat and platelet-poor plasma (PPP) were removed,
leaving 2 ml of erythrocytes. The erythrocytes were
20 washed three times in Buffer C (140 mM NaCl, 5 mM KCL,
lOmM NaPO4, and 10 mM glucose, pH 7.4), and suspended in
buffer C at a 10% hematocrit.
10.1.2. MONOCLONAL ANTIBODY 10E5
A 1.1 mg/ml solutio~ of platelet glycoprotein
GPIIb/IIIa-specific monoclonal antibody 10E5 was
prepared (see Sections 8 and 9, supra). To
approximately 1 ml of the l~E5 solution was added 10 ~l -
of l25I-labeled 10E5 anti~ody (21 ~g/ml). ~he cold and
30 radiolabelled 10E5 antibody solution was dialyzed using
12,000-14,000 molecu~ar weight cut-off dialysis tubing.
Buffer C was degassed with N2 bu~bling and equilibrated
with 10 ~1 of 10-DG chromatography support (BioP~ad
Econopac). The 10E5 was reduced ( into a variety of
35 forms including a pair of heavy chain-light chain
univalent molecules) by adding 1 ~l (about 14 mM) of 2-
.
., : ; . ,; ., ~ ~ ~; , , : -
... ..
:~
~'092/~88~ PCTt~S91/08430
2 0 9 59 25 92 -
mercaptoethanol and incubating at 22c for 60 min. The
reduced lOE5 was chromatographed on the l0-DG column
eluted with Buffer C. Twenty fractions of 0.5 ml each
were collected. Fractions #5-8 (2.2 ml) were
5 radioactive and contained 0.S3 mg of antibody (1.77 x
l05 cpm). From these pooled fractions, 75 ~l was
removed for gel electrophoresis and Ellman's assay.
l0.l.3. COUPLING OF MONOCLONAL
0 ANTIBODY TO ERYTHROCYTES
Three ml of erythrocytes (l0~ hematocrit) were
centrifuged at 430 x g for 4 min at 22C. The pellet
was resuspended to 30% hematocrit (l ml) in Buffer C.
To the erythrocytes was added 0.5 mg of freshly
15 prepared l0 mg/ml mal-sac-HNSA in 50 ~l of Buffer C.
The reaction mixture was rocked for 120 min at 22C,
then washed four times in Buffer C and suspended to l
ml in Buffer C. A 250 ~l aliquot of the erythrocytes
was then reacted with 0.53 mg (2.l ml) of reduced l0E5
20 antibody for 30 min at 22C; the reaction mixture was
overlayed with N~
Control erythrocytes coupled with mal-sac-HNSA
were reacted with 2.l ml of buffer C (no antibody) for
30 min at 22~C.
After reacting with l0E5 or buffer alone, cells
were centrifuged 4 min at 430 x g at 22C. The
supernatant was removed and stored frozen. The cells
were washed three times with Buffer C, and the
radioacti~ity determined. Calculations based on the
30 specific activity of the l0E5 antibody indicated that
approximately l,360 antibody molecules bound per
erythrocyte.
.. . .
.. . . . ~ . . .. ~ ,. :
U'092/0880-1 2 0 9 ~ 9 2 ~ PCT/~Sgl/Og430
,,; ~.. `.
- 93 -
10.1.4. ACTIVITY ASSAYS
Platelet co-agglutination assays were
performed. One hundred ~l of erythrocytes (10%
5 hematocrit; lOE5 conjugated, mal-sac-HNSA controls and
unmodified controls) were mixed and incubated for 10
min. The mixed cells were rotated for 8 min and
microscopic cell association of the fresh samples was
immediately determined using a 400X phase contrast
10 microscope. In addition, blood smears were prepared,
stained, and viewed microscopically at 1000X using an
oil immersion lens.
Microscopic examination clearly differentiated
between the 10E5-conjugated erythrocytes, which
l5 interacted with platelets, and the mal-sac-conjugated
erythrocytes, which did not.
To demonstrate that the association of 10E5
conjugated erythrocytes with platelets was specific,
the co-aggregation assay was run in the presence of
20 soluble 10E5 antibody. Microscopic examination of the
samples revealed that thP platelets, when preincubated
with sol~ble 10E5, did not associate with the 10E5-
conjugated erythrocytes, whereas, in the absence of
soluble 10E5, platelets again showed the interaction
25 with the 10E5-conjugated erythr~cytes.
10.2. DISCUSSI~N
These results clearly indicate that erythrocytes
can be targeted to a specific cell by conjugation with
30 a targeting molecule. In this case, the targeting
molec~le was reduced univalent monoclonal antibody
10E5, which is specific for the glycoprotein GPIIb/IIIa
~n platelets.
,' ." . ,:
.: :. : , -
.,
. .. ~ .. . . . . . .
WO92/O~#n~ PCT/~S91/08430
209592S 94 ~ ~ ~.
The present invention is not to be limited in
scope by the specific embodiments described herein
since such embodiments are intended as but single
illustrations of one aspect of the invention and any
5 embodiments which are functionally equivalent are
within the scope of this invention. Indeed, various
modifications of the invention in addition to those
shown and described herein will become apparent to
those sXilled in the art from the foregoing description
10 and accompanying drawings. Such modifications are
intended to fall within the scope of the appended
claims.
Various references are cited herein, the
disclosures of which are incorporated by reference
15 hèrein in their entireties.
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