Note: Descriptions are shown in the official language in which they were submitted.
05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 09
2176874
FIBRIN D-DOMAIN MULTIMER COATED PROSTI-IRlSESI AND METHODS
FOR TIiEIR PRODUCTION
T~ni~it~!si
The invention relates to methods for coating prosthetic surfacea with unti-
thrombogenic proteina, and to prosthetic devices prepared by those methods.
Racitgrot!nd of the Invention
A major limitation in developing prosthetic implants for medical uso is
that most prosthetic materials thus far developed tend to be excessively
thrornboQenie, i.e.
they trigger excessive blood clot formation, or thrombosis, at surl'aces of
the implant
exposed to the patient's blood. This problern can lead to severe
complications,
t s particularly in the case of prosthetic vascular grafts used to corndct
coranary artety
disease, lower limb ischemia, arterial aneurysms and othercirculatory
problems. About
20% to 30% of patients undergoing vascular bypass or replacement surgery
require
prosthetic grafts, because autologous veins are unavailable due to previous
surgery or
other roasons. In these patients, there is a high rate of graft failure due to
the high
tlirombogenicity of synthetic materials used to produce the prosthetic graits.
One multi-
center study demonstrated that the cumulative 4-year patency of prosthetic
grafts used for
distal arterial reconstruction was only 12% for polytetrafluoroethylene (PTFE)
$rafls; far
lower than the long-term patency obscrved for autologous gratts. Vieth et al.
(J_ Vasc.
h.1tCg. 3; 104-114, 1996).
The mechanism by which thrombosis occurs in prosthetic vascular grafts is
generally well u.nderstood. Shortly affter implantation of a synthatic graft,
adsorption and
accumulation of blood proteins on the lumenal surface of the graft begins. ]n
particular, a
surface coating of polymorized flbrin, referred to as the pseudointims,
appears first,
followed by an accumulation of thrombin bound to the fibrin. The thrombin
contributes
to platelet activation and fortnation of a platelet rich thrombus. Bound
thrombin may also
contribute to further fibrin accretion that in turn leads to distal embolism.
_, _ _ _ .
05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 10
217 6 8 7 4~-
2
An important diPPettimce between prosthetic and autologous vascular grafts
is that prosthetic grafts lack a lining of endothelirl cells which cover the
lumenal surface
of natural vessels. It is widely believed that the endothclial lining provides
an anti-
thrombogenic effect and is among the most important factors in tneintaining
long term
patency in autologous grafts. Accordingly, numerous atternpts have been made
to
artiticially seed endothelial cells onto surfaces of prosthetic gratts,
(reviewcd by
Mosquera and Goldman, Br. J. Surg= 71: 656-660, 1991). Most of these efforts
involved
coating the prosthetic surface with a "biological glue," comprised of adhesive
proteins
and other materials which enhance endothclial cell adhesion and growth. These
to investigations have used a variety of extracellular matrix proteins and
other matcrials,
including fibrin, fibrin gels cross-linked with factor XiII,
fibronectin,laminin, various
forms of collagen, aibumen and blood. Although endothelial seeding of gratb
has shown
benef3cial results in experimental settings, the technology has not progressed
sufficiently
that seeded grafts suitable for routine use can be produced.
ts Another primary focus of research concerning prosthetic vascular grafts
has involved efforts to develop non-cellular, anti-thrombogenic eoating:i,
such as protein
coatings, which may reducc the thrombogenicity of implanted grafts. One such
study
involved coating prosthetic surfaces with the anticoagulant protein heparin.
Nagaoka et
al. (A,rLifici Organs 17: 598-601, 1983). Other studies have used natural,
extracellular
2o matrix materials to coat vascular grafts. b'or example, European Patent No.
0 366 564
issued to Sawamoto et al. discloses polymeric materials coated with a
hydrolyzed fibrin
layer optionally cross-linked with factor XIII. In addition, U.S. Patent No.
5,324,647
issued to Rubens et al. discloses polymeric rnaterials coated with either a
layer of
ribrinogen, thermally denaturcd fwbrinogen or factor XIII croas-linked fibrin.
While
2s various of these natural coatings are reported to have anti-thrombogenic
eflf'ects, prosthetio
materials treated with such methods have not yct been widely employed in a
clinical
setting.
In view of the above, there is a clear need in the art for prosthetic
materials
having improved biological compatibility over currently availabie materials.
In
3o particular, there is a need for prosthetic materials which hava reduced
thrombogenicity
compared to available synthetic materials used for prostheses. Such materiala
should be
05/16/1996 18:06 2066920446 STRATTONBALLEW-PALEV PAGE 11
2176874
3
resistant to the deposition of blood proteins and to platelet adherence. These
materials
would be useful for producing vascular grafts, synthetic heart valves,
a.rtificial organs and
in any other application where a surface of the prosthetic material will be
exposed to a
patient's blood, so as to create a potential for thrombogenesis at the exposed
prosthetic
S surface. The preserit invention provides such materials, as well as methods
for preparing
those materials. Materials prepared in accordance with the methods of the
invention are
useful for providing biocompatible implants, medical treatment methods
employing such
implants, as well as useful materials for experimental modeling of
thrombogenic and
fibrinolytic proccsses.
D'~1osq,re o the Inventinn
The present Invention provides methods for coating prosthetic surfaces
with anti-thrombogenic proteins, and prosthetic devices prepared according to
those
methods. The methods of the invention generally comprise contacting a
prosthetic
f s surface with a composition containing multimers of fibrin dcgradation
products which
have cross-linked D-domains, to produce an anti-thrombogenic coating of the
multimers
on the prosthetic surface. Fibrin degradation products useftil within the
invention are
limited to fibrin degradation products posscssing fibrin D-domains, preferably
in the form
of D-monomers or D-multimers generated by plasmin lysis. The multimers are
also
2o preferably formed by cross-linking the D-domains with Factor Xi1I, before
or after the
fibrin degradation products arc generated.
It is preferred within the invention to contact tha prosthetic surface with a
composition that consists essentially of the multimers of fibrin degradation
products in a
suitable carrier. It is also preferred to use a liquid carrier to form a
solution that is
25 substantially free of intact fibrin, and of fibrin degradation products
laeking D-domains.
In alternate ernbodimcnts of the invention, one or more additional agents
are added to the composition of multimers to enhanca endothelial cell growth
on the anti-
thrombogenic coating after the prosthetic surface has been contacted with the
composition. Useful proteins for this purpose include a variety of
extra.ceilular matrix
30 materials as well as chemotactic and/or cell growth factors. Specific
agents contemplated
for use in this manner include basic fibroblast growth factor, endothelial
cell growth
CA 02176874 2005-08-23
4
factor, u2 maeroglobulin, vittoneetin, Qbronectin, and oell-bindiag
fra:gtnenta of
fibronectin. In a related embodiment, coated prosthetic surfhce.a treated
according to one
of the above methods are seeded with endothelial cells to Ruther enhance
biocompatibility of the coated eurface.
With1n Another aspect of the invention, the composition of multimps Is a
solution, and the prosthetic surfaca Is incubated In the solution within a
aelected
temperature nnge. Within one embodimient, the incubation is oartied out
between
approximately 20 C and 25 C. Within another embodiment, tbo incubation I.
conducted
at a tcmperature aumcicnt to thermally denature the multimers, between about
56 C and
1000 C, preferably at about 70 C. in order to enhance adheronco of the
multimers onto
the prosthetic surfacx.
Preferred prosthetic materials for use withln the invention include a broad
range of biologically compatible polymers known to have rnedically useful
structures and
prosthetic surface characteristics. Useful polymera witbin this context
Include
polyethyieneterephthaiate, polytetrafluoroethylene, cxpanded
polytdratluoroethyiene,
polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethanes,
polypropylenes
and polyesters. Other usetW materials with suitnble p[osthetic surfaces
include stainless
9teel, titanium. cobalt chrome alloys and silivon-beaod materials.
Within another aspect of the invention, a variety of prosthetic devicea are
provided, which are made according to the coating methods of the invention. In
alternate
embodiments, the prosthetic surface to be eoated is a surface of an artiflcial
vascular
implant, duct implant, heart valve, bone implant, patch or other artiticial
impluit.
wthin yet other aspects of the invention are prosthetic members comprising a
coated prosthetic surface of this invention as well as the use of such a
member for treatment of
a manunalian patient by implantation of the member.
These and other aspects of the invention will become apparent upon reference
to the following detailed description.
Detailed Description of the Invention
The present invention provides methods for coating prosthetic surfaces with
anti-thrombogenic proteins, and also provides prosthetic devices prepared
according
CA 02176874 2005-08-23
s
to those methods. The methods of the invention generally comprise contacting a
prosthetic aurface with a composition containing multGncts of fibrin
degradation products
which have cross-linked D-domains, to produce an anti-thrombogenic coating of
the
multimers on the prosthetic surface. As used hercin, "anti-ttuombogenic
costing" refers
s to a surface layer, preferably a continuous surface coatin& which confers a
medicaily
significant reduced thrombogenicity. or blood clot Inducing affict, when the
costed
prosthetic surface Is exposed to a platelet preparation, or to an actual
patient's blood after
itnplamtation, cornpared to thrombogenicity of an unc,oated surfaco. To
determine a
medieally significant reduction In thrombogenictty, uneoated and coated
prostheaes eire
to preferably implanted into a mammal fot a suHicient period of tUne for
adsorption and
accumulation of blood proteins on a lumcnal surface of the uncoated prosthetie
surface.
Subsequently, the uncoated and coated proAeses arc rcmoved and comparatively
analyzed microscopically, or by other conventional methods, to deterroine
whetber the
coating of multirners on the prosthetic surface confers an anti-thrombogenic
etFect. i.e.
I s whether thrombus formation is significantly reduced due to the presence of
tha coating.
Altereatively, several in vitro models may be used to determine the anti-
thrombogenic
effect of the multimer coating. for example models which compare protein
adsflrption
andlor platelet adhasion on native fibrinogen coated surfficm to adsorption
and/or
adhesion on rnultimer coated surrtiaces.
20 A variety of fibrin degradstion products me usotltl within the invention,
but they are limited to degradation produets which include substantially
eomplete fibrin
U-domains. As used herein, "D-domains" of fibrin Qenerally refea to globular
portions
of the fibrin molecule disposed near opposite ends of the molecule. T'tK D-
domsin
includes roughly two-thirds of the carboxy termini of ttie fibrin and y
cbains, and a
25 small portion of ihc carboxy termimts of the fibrin a chain (see
iis~mta8i3~ud
Thrninhnsis, Ba:ic Prin ts and Clinical P ic= 3d edition, eds. R.W. Cobnan et
al.,
J.B. L.ippincott, Co., Philadelphia, !'994,
and sec in particular Chapter 14, Fibrinogen Siructwe and Physiology, p. 271).
The fibrin degradation products useful in the invention are preferably D-
30 monomers or D-multimers generated by plasmin lysif. Plaat-in digestion of
ctoas-linked
fibrin in the presence of calcium gives ri:e to a series of croa-tinked
interqAediate
CA 02176874 2005-08-23
6
fragments Including D dimers, trimers and tetramers, with D-0imen
predominating
(Slebeniist et al., J. iol. Chem. 2A2: 2841429419, 1994; Robson et al., $pi.l.
tlaemato1. M: 322-326, 1994). A
suitable method for preparing D-domain from purified human fibrinogen is
described in
Delvos et al. (bleClri0S18~s18: 99-103.19$0).
Briefly, Fibrinogen is incubated with plasrnin in the pnesence of CaCI= and,
after
the digestion has progressed an apprapriate amount of time to yield the
desired end
products, the reaction is terminated by the addition of aprotinin. The digest
is then passed
over a Sepharose-lysine column, as described in Haverlcate et al.
(T1upmbJRem.1II: 803-
to 812, 1977). The effiuent is then dialyzed against a sodium bicarbonato
buffer and eluted
fYom a DEAE-Sephadex column with a linear pH/salt gradient betvreea the
dialysis butyer
and bicarbonate buffer. Samples ftom each protein-containing fraction are then
asacsaed
by SDS-PAt3E. Fractions with the highest purity of D-domain arc eoncentrated
10-fold
by ultrahltration. To prepare D-dimers, buman fibrinogen is Incubated in the
preseme of
is rFXIII, as described in Haverkate et al. (Fur_ J. Clin. Invest. Q: 2S3-2SS,
1979). The
cross-linked fibrin clot is squeezed with filter paper to remove entrapped
liquid, frozen,
lyophilized, ground to a fine powder, and resuspended (approx. 10 mg/mL) in an
appropriate buffer containing plasmin. After plasmin digestion, aprotinin is
added and the
solution is chromatographed on Sepharose-Iysine. D-dimer Is Isolated by
passage of the
2o e#liuent through a Sephacryl S-300 HR column. Fractions identified as
containing D-
dimer are then concentrated by ultrnfiltmtion.
Although D-dimers are the preferred flbrin degradation products for usa in
the invention, other fibrin desradation products having substantially complete
D-domains
are also contemplated. Such alteraate degradation products can be generated by
a variety
23 of convcntional methods, auch as limited trypsin digestion.
In a prefcrred embodiment of the invention, the prosthetic surface is
contactcd with a composition that consists essentially of the muftimers of
fibrin
degradation products in a suitable earrier. A composition is defined as
"consisting
essentially of multimers of fibrin degradation products" if there aro no
additional
30 substances preaent In the composition that materially alter the anti-
thrombogenic effect of
the multimer coating. It is also prefeered to use a composition that Is
substantially free of
05/16l1996 18:0E 2066820446 STRATTONBALLEW-PALEV PAGE 15
2176874
7
intact fibrin, and of ftbrin degradation products lacking D-domains. A
solution is
considered substantially free of intact flbrin and of ilbrin degradation
products lacking D-
domains when it contains about 90% pure, m.ultirnerie fibrin dcgradation
products that
include D-domains. The composition which contains the multimcrs can be in the
form of
a liquid solution, a solid, such as a fine powder, a paste or a colloidal
suspension.
Suitable carriers include non-corrosive, biocompatible, solid or liquid
carriers suitable for
application to prosthetic materiais, and in which the multimors remain intact
and cross-
linked.
Within a preferred embodiment of the invention, the composition of
to multimers is a solution, and the prosthetic surface is incubated in the
solution within a
selected temperaturc rangc. Preferred solutions include standard biological or
medical
buffers, such as low ionic strength aqueous buffers at near neutral pH, for
example tris
buffered saline (TBS). The incubation is carried out at a temperature at which
the
solution is a liquid and the prosthetic surface is not degraded. Within one
embodiment of
13 the invention, the incubation is carried out at less than 56 C, preferably
less than 37 C,
most preferabiy at about 20-25 C, so that the multimers remain in an
undenatured state.
tfowever, it may be preferable in certain applications to carry out the
incubation at a
tcmperature sufficient to thermally denature the multimers, betwcen about 56
C and 1000
C, and preferably at about 70 C, to enhance adsorption of the muitimers onto
the
20 prosthetic surfacc.
Whcn a solution of multimers is used to coat the prosthetic sttrfaee, it is
generally desired to provide a eoncentration of multimers in the solution of
between about
0.05 mg/mt and 100 mg/ml. Preferably, the solution has a multimer
concentration of
betwesn about 0.1 mg/ml and 10 mglml. Higher concentrations may be employed
25 however, depending on solubility considerations, which may Improve the rate
or extent of
adsorption of the multimers onto the prosthetic surface,
Preferred prosthetic materials for use within the invention include a broad
range of biologically compatible polymers known to have generally useful
structures and
surface characteristics for medical or experimetttal, prosthetic purposes.
Useful polymers
30 within this context include polyethyleneterephthalate (eg. Dacron ),
polytctrafluorocthylene, cxpandcd polytetrafluorocthylcne (cg. (3ore-Tcx%,
W.L. Gore,
05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 16
2176874
:
Flagstaff, Ariz.), polymers of lactide-glycolide, polyglactin, polydioxanone,
polyurethanes, polypropylenes and polyesters. Other useful materials with
suitable
prosthetic surfaces include stainless steel, titanium, cobalt chrome alloys
and silicon-
based materials.
The inethods of the present invention are usoful to produce a variety of
prosthetic devices, such as artificial vascular implants, duct implants,
urological implants,
internal organs, heart valves, bone implants, patches, webs, or other
artificial prosthetic
structures, including those that are exposed to blood flow after implantation.
More
specifically, artificial duct implanta which may be coated according to the
methods of the
lo invention include artificial urinary ducts, artificial kidney tubules,
artificial lymphatic
ducts, artificial bile ducts, artificial pancreatic ducts, indwelling
catheters, shunts and
drains. It is also contemplated that the methods of the invantion may be
effectively
employed for imparting an anti-thrombogenic coating to heterograft or
xenogratt tissue or
organ implants. Surfaces of such devices and implants are prepared as
disclosed herein,
i s and the devices or implants are implanted in a patient according to
standard surgical
techniques. In addition, the coated surfaces of the present invention provide
a usefut in
vitro model for studying tibrin accretion, platelet adhesion and other
phenomena related
to vascular graft failure, as well as for testing of potential anti-
thrombogenic agents.
Coated prosthetic surfaces prepared according to the present Invention may
2o be seeded with endothelial cells. Endothelial cell seeding of vascular
grafts and othar
surfacos exposed to the blood provides prosthetic materials that are actively
anti-
thrombogenic and exhibit increased resistance to occlusion. The stability of
the muttimcr
coating makes it an excellent substrate for endothelial call seeding. In
aiternate
embodiments of the invention, one or more additional agents arc added to the
25 composition of multimers, to enhance endothelial call growth on the
prosthetic aurface
after it has been coated. Useful proteins for this purpose include a variety
of extracellular
matrix materials, as well as chemotactic and/or cell growth factors. Specific
agents
contemplated for use in this manner include basic fibroblast growth factor
(basic FCih')
and endothelial cell growth factor (vascular endothelial cell growth factor),
ai
30 maeroglobulin, vitronectin, fibronectin, and fibronecdn fragments
containing binding
determinsnts for endothelial cells. Freparation ofthe4o proteins and protein
fragments is
CA 02176874 2005-08-23
9
within the level of ordinary skill in the art. See, for example,
C3ospodarowicz et al., U.S.
Pat. Nos. 4,785,079 and 4,902,782; Obam et al.. VEBSjatC 2U: 261-264, 1987;
Dufour
et al., PMROt, I: 2661-2671,1988; Tischer et al., WO 91/02058; Boel et al.,
?,Q: 4081-4087.1990; Ruoslabti et a1., U.S. Pat. No. 4,614,317;
s Pierschbacher et al.; U.S. Pat. No. 4,589,881; and Suzuki ct al., EMBO d.4;
2519-2524,
1985. These proteins will
generally be Included in the multimer composition at concentrations between
about 0.5
and 10 g/mL.
E.ndotbelial cells are obtained by standard proeedures ftom umbilical vein,
to saphenous vein or other souroca. See, for example, Balconi et al., bdid,
BigL 54 231-
243, i986; Ryan et al., Tisna Cell jZ: 171-176, 1985: Budd et al.,M
I..$uCg.26: 1259-
1261, 1989. Cells can be harvratted by mechanical or, prcferably, enzymatic
methods.
Briefly, veins are flushed to remove blood and tilled with a coliagenase
solution to
dislodge the endothelial cells. The cells are collected and cultured in a
conventional
13 medium, generally at about 370 C. in a 3 /. CO2 atmosphere. Satisfactory
attachment of
the cells to the coated prosthetic surface is generally obtained within one to
two hours. In
the alternative, cultuR+ed endothelial cells are added to the composition
containing the
muitimers, thus trapping them vrithin the coating. Ths coated prosthetic
material is then
incubated to allow the cells to reproducz.
20 Factor XIII for use within the present invention may be prepared from
piasma according to known methods. such as those disclosed by Cooke and
Holbrook
(HWdl=,y ,14,1: 799-84, 1974) and Curtis and Lorand (MethndaEazXID41. !a: 177-
191,
1976). The aj dinur form of factor X1I1 may be
prepared from placenta as disclosed in U.S. Pat. Nos. 3,904,751; 3.931,399;
4,597,899
Zs and 4,283,933. It is preferred, however, to ust
recombinant factor X1lI so as to avoid to the use of blood- or tissue-derived
products that
carry a risk of disease tranamission and avoid contamination with other
protcins.
Methods for preparing recombinant factor XIII are known in the art. See,
for example. Davie et al., EP 268,772 and (3rundmann et al., AU-A-69896/87.
30 Within a profcrred cmbodiment, the
factor XIII a2 diiner is prapared cytoplasmieally in the ye" Saccharmnyces
cerev-siae.
CA 02176874 2005-08-23
The celia are harvested and lysed. and a cleared lysate is prepared. The
lysate I.
fractionated by anion exchange chromatography at neutral to slightly alkaline
pH using a
column of derivatized agat+osc, such as DEAE FAST-FLOW SEPHAROSETm
(Pharmacia) or the like. Factor XIII is then prccipitated from the eolumn
eluate by
5 concentrating the eluate and adjusting the pH to 5.2-5.5. such as by
diafiltration against
ammonium succinate buffer. The preeipitate is then disaolval and further
purified using
conventional chromatographic teehaiques, such as gel liltrstion and
hydrophobic
interaction chrornatography.
Although it Is prefenrcd to use the factor XIII &2 dimer within the present
io invention, other xymogen forrtms of factor XIII (e.g. e7bz tetramer) may be
used. Zymogen
factor XIII is activated by thrombin present on the tibrin nutbce. However,
activated
factor XIII (factor Xilla) may also be used.
Proteins for use within the present invention (inoluding thrombin, plasmin,
fibrinogen and factor XIII) can be obtained from a variety of mammalian
sources, such as
ts human. bovine, porcine and ovine. As will be appreciated by those skilled
in the art, in
prosthetic applications it is prefenmd to use proteins syngenesious with the
patient in
order to reduce the risk of inducing an immtme rqponse. Non-human proteins are
particularly useful in the preparation of materials for veterinary use or for
use in
experimental models.
The following examples are offered by way of 9llustration, not limitation.
EXAMWU
MaterimLis
For use in the cxperimmts pmeated below, Hepes, Tris, and porcine
Zs heparin wwe obtained front Sigma Chemical Co., St. Louis, MO; D-
phenylalanyl- L-
prolyl-L-arginyl chloromethylketone (FPRCH= Cl) from Glbiochem, San Diego, CA;
the chromogenic substrate 5-2238 from Helena Laboratories, Mississauga, Ont.,
Canada;
Nat29 I from ICN Canada Ltd., Montreal, Canada; the Enzymobead reagent from
BIo-Rad
Laboratories, Mississauga, Ont. Canada; Na="CrOil (200-500 Ci/g) from New
England
Nuclear, Dorval, Que., Canada; SP SephadexTM, Sephadex G-25T"", DEAE-
Cellulose, DEAE
SephadexTM, Sephacryl S-300 HRT"" and SepharoseTM-lysine from Pharmacia Fine
Chemicals,
CA 02176874 2005-08-23
11
Piscataway, NJ. Polyethylene tubing (tntramedic PE 240, internal diartuter
0.17 cm) was
obtained from Clay,Adamy, Parsippany, NJ. This tubing was rinsed with methanol
and
incubated overnight in Tris buffered saline (TBS, 0.1 M NaCI, 0.05 M Tris, pH
7.4)
befort It was exposed to protein eolutions.
Hunian plasmin was obtained #irom Helena Laboratories,.Bcaunaont, TX;
bovine albumin from Bochringer Mannhcim Canada, Dorval, Que, Canada; aprotinin
from Mobay Chemical Co., New York, NY; human a= thrombin was a Qenerous gift
of
Dr. J. Fenton II, New York State Department of Health, Albany, NY; apyraa was
prepared
from potatoes as described previously (Kinlough-Rathbone et al., ln; Methods
3u
HSn1.a,tQlAip-, Measirentents of Pl~tetet Ft!~tian L.A. Harker et al., eds.
Churchill
Livingstone, Edinburgh, 1983; pp 64-91). Recombineutt human platelet factor
XiII
(rFXIII) was provided by Zymogenctics, Seattle, WA. 'ilte characteristics of
this pratein
have been weli-defined (Bishop et al., $i0Ch01rtixtqc 24, 1861-1869, I990).
and under
reducing conditions it migrated as a single band on SDS-PA(3E. Human
fibrinogen was
is purified from citrated human plasma by sequential P- al,tnine precipitetion
(Straughn et
al. Thrombos. D'athes. l semerrh. (StuttQl 16: 198-206, 1966), and was 93-94%
clottable.
This fibr'tnogen was further puritied to remove factor XIII, fibrin
degradation products,
and plasminogen using DEAE-cellulose chromatography according to the method of
Lawric ot ai. (Bi he . Soc. Trans. _: 693-694, 1979).
D-domain was prepared Gom purifie4 hwnan tibrinogen according to the
method of Delvos et al. (HumajjasjA1$: 99-105, 1988). Fibrinogen (100 mg in S
mL
TBS) was incubated with plasmin (I S casein units/mL) for 18 h at 37'C In the
presence of
zs 2 mM CaCI2 . The reaction was teaainated by the addition of aprotinin (100
[UU/mL),
and the digest was passed over a SepharoseT""-lysine column. Haverkate et al.
(Thromb.
Res. 10:803-812, 1977). The effluent was dialyzed against 10 mM sodium
bicarbonate buffer (pH
8.9) and eluted from a DEAE-SephadexTM column with a linear pH/salt gradient
between the dialysis buffer and a 10 mM sodium bicarbonate buffer, pH B.O.
eontaining
0.3 M NaCI. Samples from each of the protein-cotttatninS flractions were
assessed by
SDS-PAGE. Fractions with the highest purity of D-domain, veritjed as mom than
906A
CA 02176874 2005-08-23
12
pure by SDS-PAGE, were concmitntod 10-fold using an ultn-tiltration eeil
(Cantriprep
Concentrator, Amicon Division, W.R. Orace & Co., Danvers, MA) and stored at -
70=C.
D-dimers wac preparod from htunat tibrinogen (200 mg in l0 mi. of
TBS) incubated at 37'C in the presence of rFXIII (S g/mL) and 30 U/mL huznan
ac-
s thrombin. Haverkate et al. (F>=r_ 1_ clin_ In.reat_ S: 233-255, 1979). The
crose-linked
fibrin clot was removed, squeezed with filter paper to reanove entrapped
liquid, frozen at -
70'C, lyophilixed, ground to a tine powder, and resuipended (approx. 10 mg/mL)
in a
buffer at pH 7.8 containing 0. 15 M NaCI. 0.05 M 1'ris. 5 mM CsC12. ard 0.13
camin
units/mG of plasmin. Brenna at ai. U. Lah. Clia.Med-1.13: 682-688,1989). After
ir.cubation at 37'C for 18 h, aprotinin (100 K1Y/mL) waa added and the
solution was
chromatographed on SepharoseT""-lysine. D-dimer was isolated by passage of the
effluent
through a Sephacryl S-300 HR column cluted with TBS coetaining 0.02$ M sodium
citrate, pli 7.4. The column had been calibrated with Bio-RadT"" gel
filtration molecular
weight standards. Fractions identified as containinQ D-dimer (195 kDa),
verified as more
than 90% pure by SDS-PAGE, were concentrated by ultraflltration as dawcribed
tbove
and stored at -701C.
Radlnl hel nar nf PrnteirL
Fibrinogen, D-domain, and D-dimer wera labeled with 1251 using
Enzymobeads according to the supplier's protocol. Enzymobeads and free Iodide
were
removed by filtration through Sephadex G-25. Specific nadioactivities were;
flbrinogen
2 x 10s cpm/ g; D-domain 3.0 x l0r cpm/pg; D-dimer 1.0 x 10s cpm/pg. SDS=PAGE
and autoradiography of the 13s1-labeled fibrin and fibrin degradation products
showed
them to migratc as single bands that were coincident with their non-
radioactive
counterparts.
P1+ennration of H Mn Pla elet- Red Blood uRus,eensie~
Blood was obtained from donors who, in the pt+evious 2 weeks, had not
reccived drugs that affect platelet function. Platelets were isalated, labeled
with "Cr (10
Ci/mL) in the fitst washing fluid, and resuspended In 'Cyrode-tlburnin
solution which
contained 2 mM CaCi,, 1 mM MgC12, 0.14 M NaC1. 2.7 mM KC1, 11.9 mM NaHCO3,
05/16/1996 18:06 2066820446 STRQTTONBALLEW-PALEV PAGE 21
2176874
13
0.42 mM NaH2PO4, I mg/mL glucosc, 5 mM Hepes, 0.35% albumin, and 50 pL/mL
apyrase (pl 17.35) (Kinlough-Rathbone et al., ln: 11+tet Qds in Hgmgl0loey.
Measurements
of Platelet Function L.A. Harker et al.. eds. Churchill Livingstone,
Edinburgh, 1983; pp
64-91; Mustard et al., In: MSthods in EtI27tmQ14gXa PlatClt'dSo BweRtors.
Adheaion_
SGrtSfipp, Vol_ 1ff'Part A., J.J. Hawiger, ed. Academic Press, New York, 1989,
pp 3-11;
Cazenave et al., J. Lgb.Cliq-m,pgL ,$,}: 60-70, 1979). Red blood cells were
Isolated as
described previously (Ca2enave et al., I, i ab,s'lin_Med. 23: 60-70, 1979) and
added to
the platelet suspension (40% hematocrit). The platelet count wss adjustaf to
500,0001 L.
lo Fxample I
Comnarative ThrombagoWilXof Fjbrin tiDg, od FgAgL IIj CXoss-linked Fibrin
Coating, of PolXgthyjgneTybipg
To assess the potential and-thrombogenic cffect of cross-linking of flbrin,
polyethylene tubes were incubated in a labeled fibrinogen solution, cither
with or without
the inclusion of 1 ug/mL of rFXIII in the solution. Segments of PE 240 tubing,
12 cm in
length, were coated with thermally-denatured fibrinogen (Rubens et al., j,
HiQmed.Mol.
$g,g. 2~: 1651-1663, 1992) by filling them with a I mg/mL solution of
fibrinogen, with or
without i}tg/mL of rFXIII, and heating the filled tube segments for 10 min. at
70'C. The
segments were perfused with 40 mL of THS and then filled with a 1 g/mL
solution of a-
thrombin in 0.025 M NaCI, 0.025 M Tris, pH 7.4. After 5 min. at room
temperature, the
tubes wcre washed with 4 mL of this buffer and then perfused with 4 mL of TBS
containing fibrinogen (I mg/mL), 11s1-fibrinogen (1 pg/mL), and 2 mM CaCI=.
After
incubation for 5 min. at room temperature, the tubes were perfused with 40 mL
of TBS
and, unless indicated otherwise, were filled with 10uM FPRCH2C I in TBS and
incubated
for 5 min. to neutralize surface-bound thrombin. (Treatment with FPRCH2C1 was
found
to completely neutralize surface amidolytic activity as measured by the
chromogenic
substrate S-223 8). The tubes were rinsed with 20 mL of TBS and platelet
accumulation
was measured immediately, Representative tubes (not perfused with the
platelet; red
blood cell suspension) were filled with I x reducing sample buffer (Bio-Rad)
and
3o incubated at 37'C for 24-48 h. This treatment removes protein from the PE
surface.
CA 02176874 2005-08-23
14
Aliquots of the solubilized tubc eontents, containing 20,000 cpm, wero
analyzed by SD3-
PAGE.
To evaiuate the eomparadve thrombogenicity attributable to the flbrin
coating and ct+oss-linked fibrin coating trcatments, platelet accumulation on
the differently
treated tube segmer;ts was compared. The coated tube tegnents were attached to
silicone
tubing qnd aasernbled on a peristaltic pump (Drake-WillockT"", Mode14504,
Portland, OR).
Suspensions of s'Cr-Labeled Platelets with unlabeled red blood cells at 37'C
wera
perfused through the tubing for 10 min. at 10 mUmin. and a wall shear rate of
764 aec
The segments were then perfused under ehe same conditions for 10 min. with the
moditied Tyrode solution desaribed above. The s'Cr remmalning in the segments
was
measured using a PackardT"" gamma counter. The number of platelets that
accumulated per
mm2 was calculated from the surtltce area of the Inside of the segments and
the specific
radioactivity of the platelets In the platelet: red blood cell suspension.
Data from these
studies were determined as a mean-t t+tandard deviation (S. D.), a d were
analyzed by
ts Student's t-test (two-tailed).
The above studics showcd that Including rFXIIl (1 Ng/mL) In the solution
of labeled fibrinogen did not diminish the amount of labeled fibrin on the PE
tubing, and
perfasion did not alter the amount of labeled fibrin on the surface. Analysis
by SDS-
PAGE of the surface of tubas coated with fibrin in the prewnee and absence of
rFXIII
9howcd the presence of an approximately 100 kDa band, correspondinS to the y-r
dimer.
in the fibrin formed in the presence of rFX1II, confimning that cross-linking
had occurred.
The inclusion of rFXIII (I g/mL) in the solution used to coat the tubing with
labeled
librin subatantially decreased the exaent of platelet socumulation from a
value of 46,9741
9702 platelets per mm1(no rF)III) to 36,111 8 17964 plateleb per mm= (with
rF3QII) (
n-12, p<0.01).
F.xim~lo2
Semnerrtive Thrambogeeeai ef D-Domwin C ng. and Fector ti Cren-linlked D-
I? jõcner ontina_ nf Polyet ene Tubinn
To as9css the potential anti-thrombogenic effect of cxoss-linkino of D-
domain portions of fibrits, polyethylene tubes were incubated in a solution of
either
CA 02176874 2005-08-23
ts
labeled fibrin D-domains or D-dimers, prepared aa desadbod above. Segmenb of
PE 240
tubing, 12 cm in length, were filled with solutions of D-domaln or D-dirner at
1 mg/mL in
T$3, containing tracer amounts of the correeponding "l-labeled fngment. Tho
segments were incubated for 1 h at 23'C annd rinsed with a modified Tyrode
solution
(without Ca2' or Me+ , but containing 0.01 M EDTA and 0.1% glucose). Platelet
accumulation was meaaured as above. BrieBy, the caded tube segments wero
attached to
a peristaltic pump with silicone tubing, and suspensions of s'Cr-Labeled
Platelds with
unlabclcd red blood ce11s at 37'C were perfimed througb the tubing for 10 min.
at 10
mUmin. and a wall shear rate of 764 see 't. The segrnrnts were then perfused
unckr the
io same conditions for 10 min. with the modified 1'yroda solutiop described
above. 'flte "Cr
rtmaining in the segments was measured using a PackardT"" gamma counter. The
number of
platelets that accumulated per mm3 was calculated as above.
These studies showed that platelet accumulation on D-dimer coated
pofyethylene surfaces was greatly reduced compared to piatelet actamtulatlon
on non
is cross-linked, D-domain coatod surfaces. The surface protein eoncentration
of the tubing
coated with D-domain was 0.70 t 0.02 g/cm3, and the number of platelets that
accumulated at high shear was 34.3$3 3905 per mm1(n-3). In contrast, the
surface
protein cottcentration of P4 tubing eoated with D-dimer wsu 0.13 # 0.05
g/cm=, and the
number of platelets that accumulated was 563 257 per mm= (n--4). Thus, the
20 thrombogenicity of the D-dimer coated surfaces nteasured 60-fold lass than
that of the D-
domain coated surfaces. Even when these data are approximately normalized to
account
for the i'ive-fold lower protein ads,orption of the D-dimers to the tubing,
eomparod to the
protein adsorption of the D-domains, it is nonethess evident that the D-dimer
coating ls at
least ten times more anti-thmmbogenic on polyethylaro tubing then the D-
dottfain
25 coating.
Although certain embodinents of the inventlon have been described In
detail for purposes of illusttation, It will be readily apQmnt to those
skilled in the art that
the methods aad producta described herein may be modified without deviating
frotn the
spirit and scope of tihe invention. Accordingly, the invention Is not limited
by the above
30 deycription but is to be detesmined in scope by the ciaima which follow.
