Note: Descriptions are shown in the official language in which they were submitted.
S012878J.32
Use of an anticoagulant as a diagnostic agent
The present invention relates to agents,
particularly annexines, which are labelled with a
detectable substance and the use thereof for diagnostic
purposes.
Blood consists of special unbound cells dispersed
in a plasma medium. The contents of the cell is
separated from the surrounding plasma by so-called
plasma membranes. These membranes are made up of
phospholipids in the form of a double layer and
associated proteins which partially penetrate this
double layer or protrude from it.
The various phospholipids are not randomly
distributed over the outer and inner shell of the double
layer but are held by the cell in an asymmetric
configuration (7,8). Whereas phosphatidyl choline (PC)
and sphingomyelin (SPH) are the dominating species of
the outer shell, phosphatidyl serine (PS), phosphatidyl
ethanolamine (PE) and phosphatidyl inositol (PI) are
located predominantly in the inner coat, facing the
cytosol. This energy-consuming state of asymmetry is of
exceptional physiological importance. PC and SPH are
the most inert species of the phospholipid family and in
stark contrast to the other species show exceptionally
neutral behaviour in the presence of the plasma
components. This reactive inertia of the outer coat
relative to the plasma proteins is the absolute
prerequisite for ensuring that the blood remains liquid.
Special plasma proteins which belong to the coagulation
cascade, namely the so-called coagulation factors, are
in fact able to convert liquid blood into a solid state
when they are activated (9). These coagulation factors
can be activated by phospholipids such as phosphatidyl
serine.
In many cases, e.g. after injury to a blood vessel,
- 2 -
it is necessary, not to mention crucial to survival, for
the coagulation factors to be activated. In such a
situation, a special blood cell, the platelet, can give
up its membrane asymmetry by activation mechanisms which
transport phosphatidyl serine to the outer coat, where
it aids the activation of the coagulation factors {10).
This systematic change in the phospholipid
composition of the outer coat of the platelet plasma
membrane is of major physiological importance in
haemostasis, as indicated for example by Scott syndrome
(11) .
However, physiology also includes pathology; thus,
the homoeostasis of the blood may in some cases slip
into a pathological state, as occurs in arterial,
coronary and venous thrombosis.
These haemostatic disorders are usually iodiopathic
and make it impossible for doctors to predict their
occurrence and develop preventive treatment.
The aim of the present invention was to provide
agents which would help in the early recognition of
haemostatic disorders.
Unlike the development of the symptoms, the
development of the disorders usually occurs slowly.
During this phase of symptomless progression, the
prothrombotic state, continuous activation of the
coagulation system is occurring locally. Connected with
this local activation there is, in the periphery, an
occurrence of platelets which are at an early stage of
activation. These platelets have already begun to
change the phospholipid composition of their outer
plasma membrane coat. Phosphatidyl serine is present in
the outer coat. Agents which could diagnose this so-
called prothrombotic state would therefore be of
extremely great clinical importance.
A further object of the invention was to provide
adjuvants which are capable of specifically
distinguishing phosphatidyl serine from phosphatidyl
c
2~'~~~4~1
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choline.
In addition to the occurrence of weakly activated
platelets in the periphery, fully activated platelets
will accumulate anywhere where the wall of the blood
vessel is in a pathological condition. This location
can be regarded as the trigger for the activation of the
haemostatic system. Locating this pathological site and
the thrombus forming at this point would be of major
therapeutic value.
A further aim of the invention was therefore to
provide an agent by means of which the starting point of
the activation of the haemostatic system and/or a
thrombus could be located.
The blood coagulation mechanism constitutes a
cascade of enzymatic reactions at the end of which is
the formation of thrombin which finally converts
fibrinogen into fibrin. Various procoagulant reactions
such as, for example, the activation of prothrombin by
factors Xa and Va are catalysed by phospholipid surfaces
to which the clotting factors bind.
The proteins which bind to phospholipids and
interfere with processes dependent on phospholipid
surfaces constitute a family which are Ca2'-dependent in
their binding to phospholipids.
This family, also known as the annexines, includes,
in addition to lipocortin i, calpactin I, protein II,
lipocortin III, p67-calelectrin, the vascular
anticoagulant protein (VAC) and IBC, PAP, PAPI, PP4,
endonexin II and lipocortin V.
The structural features common to the annexines are
presumably the basis for their similar Caz' and
phospholipid-binding properties. Although this general
property applies to all annexines, there is clear
individuality with regard to their affinity for Ca2'" and
the various types of phospholipid.
The physiological functions of the annexines relate
to membrane-associated processes. The basic mechanism
L'
2a~0~~'~
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of the anti-coagulant effect of VAC was recognised as an
inhibition of the catalytic capacity of the
phospholipids by the binding of VAC to their surface,
thereby preventing the formation of the coagulation-
promoting complex on their surface.
Studies of binding have shown that VAC associates
reversibly with procoagulatory phospholipids in calcium-
dependent manner.
Other bivalent cations from the series Cd2+, Znz+,
Mn2+ and Co2+ also have a positive effect on association,
but not to the same extent as Caz+.
Furthermore, it has surprisingly been found that
VAC absorption on phospholipids is positively influenced
to an exceptional degree in the presence of CaZ+ and Zn2+
ions.
Surprisingly, it has been found that, at plasma-
calcium concentrations, VAC binds to phosphatidyl serine
but not to phosphatidyl choline and sphingomyelin. VAC
will therefore specifically recognise and bind
peripheral platelets with PS in their outer membrane
coat. Furthermore, VAC will also specifically recognise
those locations in the vascular system which are
presenting PS to the blood.
This differentiation among phospholipids makes VAC,
like the other annexines, an ideal reagent for achieving
early recognition of the prothrombotic state as
described above.
By means of the present invention it is,
surprisingly, possible for the first time to recognise
the prothrombotic state of the vascular system. This
diagnosis is made possible by the specificity of
substances, of agents which are capable of recognising
the prothrombotic state of the platelets, which is
different from the normal state. Since the
prothrombotic state differs from the normal state of the
platelets in that the outer coat of only the
prothrombotic platelet shows phosphatidyl serine, this
~~7o~4rr
- 5 -
principle can be exploited according to the invention by
any agent capable of specifically distinguishing
phosphatidyl serine from phosphatidyl choline. The
agents which may be used according to the invention are
characterised by their specificity for phosphatidyl
serine, which can be determined by the binding tests
described in the specification.
By making use of this specificity of the agents
according to the invention it is also possible to locate
the starting point for the activation of the haemostatic
system and/or the thrombus.
Consequently, the present invention provides, for
the first time, agents which make it possible, by early
diagnosis of a state which might possibly develop into a
health-threatening condition, to adopt suitable
therapeutic measures.
Preferred agents according to the invention are
anticoagulant polypeptides provided that they have the
necessary specificity for the phospholipid phosphatidyl
serene.
The family of the annexines, particularly VAC, is
particularly preferred.
In order to be able to use the agents according to
the invention, particularly VAC or the other annexines
as a diagnostic agent, they are labelled in a manner
known per se. Suitable labelling may be achieved, for
example, by labelling with fluorescent groups or by
radioactive labelling. A fluorescent marker which may
be used to advantage is fluorescein isothiocyanate,
whilst radioactive markers which may be used to
advantage are the radioisotopes of the halogens,
particularly those of iodine, for example '3'I or ~Z~I or
lead, mercury, thallium, technetium or indium (2o3Pb,
~9aHg~ 2o~Tl ~ ~r~,c~ ii~In) .
Fluorescein isothiocyanate (Serva) may be used for
labelling VAC in a manner known per se (13). Labelling
is also possible by means of a paramagnetic contrast
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CA 02070647 2000-07-31
27855-40
- 6 -
agent which is detectable in a MRI (magnetic resonance
imaging) system. It is possible to use gadolinium,
cobalt, nickel, manganese or iron complexes by means of
which conjugates may be provided as diagnostic agents
which are detectable in a MRI system. A strong magnetic
field is used in such systems in order to adjust the
nuclear spin vectors of the atoms in the organism. Then
the field is destroyed which causes the nuclei to return
to their initial state. This process is observed and
recorded.
The anticoagulant polypeptide thus provided with a
detectable marker is then administered by the
intraarterial or intravenous route. The quantity
applied has to be such that it suffices for the
subsequent measurement after a sufficient incubation
period.
In order to diagnose a prothrombotic state, the
polypeptide or agent labelled according to the invention
is added extracorporally to the blood to be examined,
optionally in the presence of a further anticoagulant
which does not decrease the plasma calcium concentration
such as, for example, heparin, and then the labelling
associated with specific types of cells is analysed.
The polypeptide, labelled according to the
invention, may be used for the purpose of the invention
in blood-isotonic aqueous solution or with adjuvants.
Adjuvants may, for example, include TWEFN 80* arginine,
phosphate buffers and physiologically compatible
preservatives. Other substances are well known to the
skilled man and may also be used.
The radioactive labelling is carried out using, for
example, the known iodogen method (12) or the
conventional chloramine-T method. In view of its half-
life of 8 days, '3'I is recommended for in vivo
diagnosis. The radioactively labelled agent is taken up
in blood-isotonic aqueous solution. After sterile
filtration it is injected. The whole body scintigraphs
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are taken with a gamma camera, e.g. for '3'I, 1, 2, 4 and
7 days after the injection.
As well as the genuine forms of the annexines it is
also possible to use altered forms for the purposes of
the invention.
Reference should be made in particular to the
mutants described in EPA 0 293 567. Furthermore, the
fragments or chemically modified derivatives of the
annexines may also be used which are specific to the
phospholipids phosphatidyl serine/phosphatidyl choline
and are therefore capable of recognising the
prothrombotic state of the platelets involved.
The present invention relates specifically to:
- An anticoagulant polypeptide from the family of the
annexines, preferably VAC which has a detectable
label.
An anticoagulant polypeptide which, as the
detectable labelling, contains fluorescent
labelling, preferably fluorescein isothiocyanate, a
radioisotope of a halogen, technetium, lead,
mercury, thallium or indium, particularly
preferably '3'I or ~25I or a paramagnetic contrast
agent.
- An anticoagulant polypeptide as described above for
distinguishing phosphatidyl serine from
phosphatidyl choline.
- An anticoagulant polypeptide as described above for
use as a diagnostic agent.
- A process for locating the starting point for the
activation of the haemostatic system, in which
CA 02070647 2000-07-31
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_ g _
a) an anticoagulant polypeptide or agent, provided
with a detectable label, from the family of
annexines, preferably VAC, is administered to
the system, preferably by intraarterial or
intravenous route and
b) after an incubation period the distribution of
said polypeptide is observed, either
extracorporally with a gamma-scintillation
camera or by magnetic resonance measurement.
- A process for diagnosing the prothrombotic state,
in which
a) the blood to be examined is mixed
extracorporally with an anticoagulant
polypeptide or agent from the family of
annexines, preferably VAC which has a detectable
marker and
b) the labelling associated with specific cell
types is analysed.
An agent which besides an anticoagulant polypeptide
from the family of the annexines, preferably VAC
which has a detectable marker, contains
additionally an adjuvant such as, for example, a
physiological solution of sodium chloride, TWEEN
80* arginine and/or phosphate buffer, an
anticoagulant, preferably heparin, which does not
reduce the plasma calcium concentration optionally
being used.
- A kit for the diagnostic detection of the
prothrombotic state or the starting point of
activation of the haemostatic system and/or a
thrombus containing an agent or an anticoagulant
Trade-mark
2a'~~64'~
polypeptide of the invention which is capable of
distinguishing phosphatidyl serine from
phosphatidyl choline.
- Use of an anticoagulant polypeptide or agEnt of the
invention for distinguishing phosphatidyl serine
from phosphatidyl choline.
- Use of an anticoagulant polypeptide or agent of the
invention for the diagnosis of the prothrombotic
state or the starting point of disorder of the
haemostatic system or the thrombus.
Materials and Methods
VAC is produced analogously to either EPA 0 181 465
or EPA 0 293 567. The following experiments were
carried out with VACa, but the results can also be
applied to the other annexines, particularly VAC/3.
Lipids
Dioleoyl-phosphatidyl choline (DOPC, No. P-1013)
Dioleoyl-phosphatidyl ethanolamine (DOPE, No. P-0510),
Cardiolipin (CL, No. C-5646),
Dioleoyl-phosphatidyl glycerol (DOPG, No. P-9664),
Phosphatidyl inositol (PI, No. P-0639),
Dioleoyl-phosphatidic acid (DOPA, No. P-2767),
Stearylamine (SA, S-6755) and egg yolk sphingomyelin
(S-0756) were obtained from the firm Sigma Chemical Co.
The purity of DOPC and DOPE was tested by thin
layer chromatography. Dioleoyl-phosphatidyl serine
(DOPS) was produced by conversion of DOPC according to
(1). ~4C-labelled DOPS (specific activity
100,000 dpm/~.g) was obtained from Amersham.
c°
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Preparation of the phospholipid double layers on silicon
plates
Phospholipid double layers were applied using a
"Langmuir-film balance" (Lauda type FW-1) as described
in Corsel et al. (2). Hydrophilic silicon plates were
treated for 24 hours in 30% chromosulphuric acid and
water and stored in 50% ethanol/water. Before use they
were thoroughly washed with detergent and water. The
film balance was filled with demineralised water and
50 ~M CaCl2. 20 ~cl of a solution containing about 2 g/1
of phospholipid in chloroform were applied to this
substrate. The DOPS fractions in the double layers were
tested with '4C-labelled DOPS mixed with DOPC. The
double layers filled up were removed from the silicon
plates with the scintillation detergent (Du Pont Formula
989) and the total radioactivity was measured in a
scintillation counter.
Measurement of binding by ellipsometry
The adsorption of VAC on the phospholipid double
layers was measured by means of an automatic
ellipsometer as described (2,3).
The binding tests were carried out in a hydrophilic
cuvette containing 5 ml of a stirred buffer (0.05 M
Tris/HC1; 0.1 M NaCl; pH=7.5; T=20°C). The divalent
cations were added stepwise as chlorides.
At VAC-concentrations of <0.1 ~,g/ml, the buffer
which contained the specific VAC concentration was added
continuously in order to create an adequate buffer
capacity for VAC.
The refractive index and the thickness d of the
adsorbed film were determined from the combined
polarising and analysing data (4). The quantity t of
the adsorbed protein layer was determined from the
refractive index and the thickness using a modified
Lorentz-Lorenz equation [1] (3,5):
c
- 11 -
[ 1 ] r=3 d ( nZ-nbz ) / [ ( nz+2 ) ( r ( nb2+2 ) -v ( nb2-1 ) ) ] ;
nb is the refractive index of the buffer. The values
r=0.254 and v=0.71 were used for the specific molar
refractivity and the partial specific volume (3).
Results
The effect of divalent cations on the binding of the VAC
to ~hosphol ipids
VAC binds to phospholipid membranes consisting of
20% DOPS/80% DOPC, depending on the calcium
concentration. The subsequent addition of EDTA resulted
in immediate and total desorption (Fig. 1). By varying
the free Ca2+ concentration it was possible to initiate
adsorption several times, without any noticeable change
in the quantity adsorbed or the rate of adsorption.
Irreversible changes to the VAC molecule or the
phospholipid double layers caused by adsorption or
desorption are therefore improbable. The binding was
also totally reversible when the cuvette was rinsed out
with Ca2+-free buffer.
The Ca2+ dependency of the VAC binding to
phospholipids is shown in Fig. 2. The Ca2'-dosage-
activity curve shows quite clearly a CaZ+ concentration
at which half the maximum VAC adsorption is achieved:
[Caz+] i~Z. The [Ca2+] »z value depends on the composition
of the phospholipid surface. With phospholipid surfaces
containing 100%, 20%, 5% and to DOPS, [CaZ+]»Z values of
36 ~,M, 220 ~,M, 1.5 mM and 8.6 mM were measured,
respectively (Table 1). These results accord
particularly well with the [Caz+]»Z value of 53 ~M, which
was measured for the endonexin II (=VAC) binding to
equimolar mixtures of PS/PC vesicles (6). The maximum
quantity of protein adsorbed (rmax) was independent of
the DOPS fraction of the membrane and amounted to
~o~os~~
- 12 -
approximately 0.217 ~,g/cmZ. No adsorption was detected
with pure DOPC double layers up to a Ca2+ concentration
of 3 mM.
The adsorption of VAC to the phospholipid double
layers of different concentrations is shown in Figure 5.
It is shown quite clearly here that virtually no
adsorption of VAC is found with pure DOPC double layers.
The adsorption of VAC to stearylamide (SA) is also weak.
In experiments with cations other than Ca2+, it was
found that the binding of VAC to the phospholipids is
strongly Ca2+-specific (Fig. 3) . Cd2+, Znz+, Mnz+ and Co2'
showed little promotion of binding; Baz+ and Mg2+ had no
influence. This property of the cations can to some
extent be correlated with the ion radii thereof.
Zinc svneraism
High concentrations of zinc ions (1 mM) promote
VAC-adsorption only to a small extent (Fig. 3); 50 ~,M
have no effect whatsoever on adsorption. Surprisingly,
this concentration does influence binding in the
presence of Caz+; there is some synergism. The [Caz+) ~~Z
value fell from 8.6 to 2.7 mM for double layers having
only 1% DOPS ( [Zn2+]=50 ~.M) (Fig. 4) . 50 ~M [Zn2'J is
within the normal range of the plasma zinc
concentrations.
Diagnostic methods
1. In vitro diagnosis
a) VAC is labelled by methods known per se with
the fluorescein group, e.g. with fluorescein
isothiocyanate; in this way VAC-FITC is
obtained.
b) A patient's blood is placed in a plastic test
tube containing an anticoagulant which does not
- 13 - 2~"~0~4'~
reduce the plasma-calcium concentration (e. g.
heparin) and VAC-FITC.
c) After mixing, the blood cells are analysed
using an FACS (fluorescence activated cell
sorter). This analysis determines the
intensity of fluorescence, which is associated
with specific types of cell.
d) The analysis profiles show the quantity of
platelets with bound VAC-FITC, i.e. platelets
with exposed PS, and therefore the presence of
a prothrombotic condition. This health-
threatening condition can be recognised early
by this method and consequently treated at an
early stage.
2. In vivo diagnosis
a) VAC is labelled with a short-lived isotope, for
example '3'I using methods known per se; '3'I-VAC
is obtained.
b) '3'I-VAC is administered intravenously to a
patient.
c) After a certain incubation period the patient
is exposed to whole or partial body
scintigraphy. The distribution of
radioactivity can be observed using a large-
field-of-view gamma camera.
d) Intravascular sites with ~3~I-VAC accumulation
mark the point where the thrombosis is
progressing. Suitable thrombosis-preventing or
thrombosis-alleviating measures can be taken
early.
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CA 02070647 2000-07-31
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1. Radioactive labellinct
1.1 Preparations
1.1.1. Preparation of a 500 mmol/1 sodium phosphate
buffer
24.5 g of sodium dihydrogen phosphate monohydrate
were dissolved in 1 litre of twice distilled water
and added to a solution of 35.5 g of disodium
hydrogen phosphate in 1 litre of twice distilled
water until a pH of 7.5 was achieved.
1.1.2. Preparation of a 20 mmoljl sodium phosphate
buffer + 150 mmol NaCl lelution bufferl
2.76 g of sodium dihydrogen phosphate monohydrate
were dissolved in 1 litre of twice distilled water
and added to a solution of 2.84 g of disodium
hydrogen phosphate in 1 litre of twice distilled
water until a pH of 7.2 was obtained. The elution
buffer was prepared by adding 8.77 g NaCl
(150 mmol) to 1 litre of the buffer.
1.1.3. Eguilibration of the purification column
A PD-10 column (Sephadex G25, Messrs. Pharmacia)
was equilibrated with about 30 ml of the elution
buffer.
1.1.4. Preparation of the reaction vessel
2 mg of IODO-GEN (molecular mass: 432.09 g/mol)
were dissolved in 50 ml of highly pure
dichloromethane. 200 ~.1 of this solution were
pipetted into a 1.5 ml Eppendorf*container and then
the solvent was evaporated off at 37'C
(thermostatic block). In this way 8 ~.g (1.85 x
10'2 mmol) of IODO-GEN were finely distributed over
the wall of the reaction vessel.
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1.1.5. VAC-a used for labellinct
The starting material used was a solution of 50 mg
VAC-a in 4 ml of 20 mmol/1 sodium phosphate buffer
+ 150 mmol/1 NaCl, pH 7.2, diluted with 1 ml of
twice distilled water. Molecular mass VAC-a:
34000 g/mol.
1.1.6. I-125 used for labelling
Na~~SI made by Dupont, NEN Products, with 67.3 MBq
(=1.82 mCi) total radioactivity on the date of
calibration. The specific activity was 15.9 Ci/mg
of iodine = 1.98 kCi/mmol 1/2 I2, corresponding to
0.115 ~.g of iodine (9.2 x 10-~ mmol 1/2 IZ) . The
active NaI was dissolved in 5.5 ~,1 of 0.1 mol/1
NaOH.
1.2. Iodisation
Al.l the work was carried out with removal of the
isotope behind leaded glass screens. 20 ~,1
(= 200 ~cg) of the solution of VAC-a described in
1.1.5. were transferred into the reaction vessel
pretreated with IODO-GEN. This vessel was sealed
and shaken for 20 minutes at ambient temperature.
Then the reaction solution was applied by means of
a pipette to the prepared PD-10 column (see
1.1.3.). The reaction vessel was rinsed again with
500 ~.l of the elution buffer (see 1.1.2.) and this
solution was also applied to the PD-10 column. The
eluate flowing out was discarded.
1.3. Purification
By the application of 0.5 ml aliquots of elution
buffer (see 1.1.2) at 2 minute intervals, the VAC-a
[ ~25I ] was separated from the free ~ZSI/Na ~ZSI . After
12 fractions, this purification step was complete.
2fl'~0~4'~
- 16 -
The relative activity content of the fraction was
measured using a laboratory monitor (GMZ) (Fig. 7).
Fractions 6 and 7 were combined, made up to
precisely 2.0 ml with elution buffer, divided into
100 ul portions and frozen at -20°C. The substance
was kept available in these portions for analysis
and developmental studies.
2. Analytical part
2.1. Measurement of content
In the chromogenic substrate assay a VAC-a content
of 71.8 ~,g/2.0 ml of solution was measured.
2.2. Measurement of radioactivity
2.2.1. Total radioactivity
After a 100 ~cl portion had been thawed, 50 ~,1 of
this [~zSI] VAC-a solution was added by pipette to
950 ~,1 of the inactive VAC-a solution (see 1.1.5.),
thoroughly mixed and 50 ~,1 thereof was placed in an
LSC-Counter (Beckman) for measurement. 24.5 MBq
(= 0.663 mCi)/2.0 ml of total solution.
2.2.2. Specific activity
Measurement of the content and total radioactivity
yielded a specific activity of
341.5 MBq/mg = 11.61 TBq/mmol
(9.23 mCi/mg = 313.8 Ci(mmol)
2.2.3. Measurement of the protein-bound
radioactivity by TCA precipitation
100 ~.1 of the VAC-a solution were combined with
50 ~.1 of 3% BSA solution and 150 ~,1 of 40% aqueous
trichloroacetic acid, shaken thoroughly and left to
stand for 60 minutes in a refrigerator. The
~o~~s~~
- - 17 -
precipitate formed was removed by centrifuging.
Aliquots of the supernatant were measured in an
LSC-counter.
Result: 99.3% of the radioactivity was
precipitable.
2.2.4. Radioactivity yield
Of the 67.3 MBq put in, 24.5 MBq were found in the
VAC-a. The activity yield was therefore 36.4%.
2.3 Degree of modification
From the specific activity and the quantity of
inactive VAC-a used, it was calculated that
statistically every 6th VAC-a molecule was labelled
with a ~zSI-atom.
3.4 Identity and purity
Using the SDS-PAGE (gradient gel 7 to 17%, non-
reducing conditions) and subsequent evaluation of
the gel by silver staining (Oakley method),
autoradiography and detection under the linear
analyser (made by Berthold LB 282, probe LB 2820)
(Fig. 8) the substance was investigated by
comparison with the VAC-a used. The substances
were identical, the proportion of dimeric product
was significantly below the limit of 8o tolerated
for inactive charges.
c
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Legend accompan~ing the drawings
Fiq. 1: Alternating adsorption and desorption of VAC on
a phospholipid surface, induced by increasing and
lowering the Ca2' concentration. The adsorption of VAC
(1 ~g/ml) on a 20% DOPS/80% DOPC phospholipid double
layer. Addition of Caz+ (3,4,6 mM) is indicated by t or
v.
Fig. 2: Influence of the phospholipid composition and
CaZ+ concentration on the adsorption of VAC on a
phospholipid surface.
0 100% DOPS; ~ 20% DOPS; 0 5% DOPS; D to DOPS% 100%
DOPC% all the mixtures were supplemented with DOPC.
[VAC] - 1 ~g/ml.
Fig. 3: Effect of bivalent ions on the adsorption of
VAC. VAC adsorption on double layers of 20% DOPS and
80% DOPC in the presence of the ions specified (1 or
3 mM). [VAC] - 1 ~,g/ml.
Fist. 4: Synergistic effect of ZnZ' on the Caz'-dependent
adsorption of VAC on the phospholipid surface. The
effect of Ca2' on the VAC adsorption on 1% DOPS and 99%
DOPC in the presence of 50 ~,M Znz+ was measured.
[VAC] - 1 ~.g/ml.
Fig'. 5: Adsorption of VAC on phospholipid double layers
of varying composition.
VAC adsorption on dioleoyl phosphatidyl serine (DOPS),
cardiolipin (CL) and dioleoyl phosphatidyl ethanolamine
(DOPE), either pure or mixed with 80% dioleoyl
phosphatidyl choline (DOPC), on dioleoyl phosphatidyl
glycerol (DOPG), phosphatidyl inositol (PI) and
stearylamine mixed with 80% DOPC or on pure DOPC. [VAC]
- 1 ug/ml % [ Caz+ ] - 3 mM .
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CA 02070647 2000-07-31
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Fig. 6: 1,3,4,6-Tetrachloro-3a-6a-diphenyl-glycoluril
(IODO-GEN)
Fiq_ 7: Distribution of radioactivity in the fractions
1-12.
Fig. 8: Distribution of radioactivity under the linear
analyser.
Table 1
Evaluation of the phospholipid double layers applied to
silicon plates
~4C-DOPS Quantity of Activity DOPS
on film phospholipids fraction
balance (by ellipsometry) measured
ug/cm2 DPM %
2 0.396 453 1.9
5 0.409 1133 4.5
20 0.401 5006 20
100 0.442 27174 99
The calculated mixture was placed on the film
balance. The quantity of double layer was measured by
ellipsometry and the activity of the '4C-labelled DOPS
was measured using a Beckmann 6S 3801 scintillation
counter (s. d. <2%) and corrected by the background
radiation (60 DPM). The DOPS fraction in the double
layer was calculated using equation 2:
Quantity(~g/cm2) x spec. activity (DPM.ug'~)
Fraction =
Activity (DPM) x area (cm2)
The specific activity of DOPS was 100,000 DPM.~,g'~,
the area occupied by phospholipids was 0.62 cm2.
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_ 20 _ 2U7~~~'~
Table 2
Half the maximum VAC-binding to various phospholipid
surfaces.
Lipid (mol%/molo) rmax ~ S.D. [Ca2']»2~S.D.
( I~g/ cm2 ) mM
DOPS(100) 0.195 0.025 0.036 0.013
DOPS/ DOPC (20/80) 0.222 0.014 0.22 0.06
DOPS/ DOPC (5/95) 0.229 0.004 1.5 0.5
DOPS/ DOPC (1/99) 0.234 0.007 8.6 2.5
Cardiolipin / DOPC (20/80) 0.209 0.011 0.039 0.022
DOPG/ DOPC (20/80) 0.212 0.003 0.155 0.027
PI / DOPC (20/80) 0.221 0.005 0.47 0.05
DOPA/ DOPC (20/80) 0.207 0.006 0.75 0.26
DOPE/ DOPC (20/80) 0.213 0.003 0.86 0.21
Sphingomyelin 0.225 0.014 7 3
/
DOPC
(20/80)
DOPC(100) n.d. >30
mM
The maximum VAC-adsorption (rmax) on the phospholipid
surfaces specified together with the calcium concentration
resulting in half the maximum VAC binding [Ca2+]»2 are
given as the averages of at least three different
experiments with the corresponding standard deviations.
n.d. - not determined.
z:
.~ ~o~o~~~
- 21 -
Biblioqraphy
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