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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratory for Thrombosis Research,
Interdisciplinary Research Center, K U Leuven Campus Kortrijk,
Kortrijk, Belgium; and the Department of Haematology and Cell Biology,
University of the Orange Free State, Bloemfontein, South Africa.
The interaction between collagen, von Willebrand factor (VWF), and
glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of
the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a
new kind of antithrombotic strategy and determine for the first time
experimental in vivo data concerning the importance of the collagen-VWF
interaction. We used a modified Folts model to study the antithrombotic
efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured
in the femoral artery. Administering a dose of 100, 300, and 600 µg/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs,
respectively. When 100 µg/kg 82D6A3 was infused into the baboons,
80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex
vivo inhibition of VWF binding to collagen, with no prolongation of the
bleeding time. The bleeding time was also not significantly prolonged
when the CFRs were abolished at doses of 300 µg/kg and 600 µg/kg.
At these doses 100% of VWF was occupied by the antibody and 100% ex
vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a
high affinity for VWF; after 48 hours still 68% VWF (300µg/kg) was
occupied with a pharmacologic effect up to 5 hours after administration
(80%-100% occupancy). In conclusion, these results clearly indicate
that the VWF-collagen interaction is important in vivo in thrombosis
under high shear conditions and thus might be a new target for
preventing arterial thrombosis.
(Blood. 2002;99:3623-3628) In normal hemostasis and thrombosis, platelets
adhere to the subendothelium of damaged blood vessels through an
interaction with von Willebrand factor (VWF), which forms a bridge
between collagen within the damaged vessel wall and the platelet
receptor glycoprotein Ib (GPIb/V/IX), an interaction especially
important under high shear conditions.1 This reversible
adhesion allows platelets to roll over the damaged area, which is then
followed by a firm adhesion through the collagen receptors (GPIa-IIa,
GPVI, GPIV, p65, TIIICBP) 2-4 resulting in
platelet activation. This leads to the conformational activation of the
platelet GPIIb/IIIa receptor, fibrinogen binding, and finally to
platelet aggregation.5
The VWF subunit is composed of several homologous domains each covering
different functions; VWF interacts through its A1 domain mainly with
the GPIb/V/IX complex,6 whereas its A3 domain predominantly interacts with fibrillar collagen fibers.7
Under normal conditions platelets and VWF do not interact. However, when VWF is bound to collagen at high shear rate, it is believed to
undergo a conformational change allowing its binding with the platelet
receptor GPIb/IX/V.8
One line of search for antiplatelet drugs in the prevention of
thrombosis is focusing on the inhibition of the VWF-GPIb axis. Compounds that interact with GPIb Specific blockade of the VWF-collagen interaction in vivo has not yet
been demonstrated but based on in vitro data could be a novel strategy
for the prevention of thrombus formation in stenosed arteries. We here
describe for the first time the antithrombotic effect of a murine
antihuman VWF mAb 82D6A3, known to bind to the A3 domain and to inhibit
VWF binding to fibrillar collagens type I and III but not to collagen
VI.17
The antithrombotic efficacy of 82D6A3 was demonstrated in baboons
by using a modified Folts model, where cyclic flow reductions (CFRs)
due to thrombus formation and its dislodgment are measured in
mechanically damaged, severely stenosed femoral
arteries.18
Purification of 82D6A3
Surgical preparation
After a 60-minute control period of reproducible CFRs (t = Platelet count, coagulation, and bleeding time All blood samples were collected on 0.32% (final concentration) trisodium citrate. The platelet count was determined using a Technicon H2 blood cell analyzer (Bayer Diagnostics, Tarrytown, NY). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured at 37°C using a coagulometer (Clotex II, Hyland, South Africa). The template bleeding time was measured using the Simplate II device (Organon Teknika, Durham, NC). The volar surface of the forearm was shaved and a pressure cuff was applied and inflated at 40 mm Hg. The wound was carefully dabbed every 15 seconds with filter paper. Baseline bleeding time measured in 13 baboons was 2.04 ± 0.59 minutes, with an interanimal coefficient of variation of 29%.All measurements were performed once at each time point. Plasma concentration of 82D6A3 Microtiter plates (96-well, Greiner, Frickenhausen, Germany) were coated overnight at 4 °C with 5 µg/mL (in phosphate-buffered saline [PBS], 100 µL/well) goat antimouse IgG whole molecule (Sigma, St Louis, MO). Plates were blocked with 3% milk powder for 2 hours at room temperature (RT). Next dilution series (1:2 in PBS) of the plasma samples (prewarmed for 5 minutes at 37°C) were added for 2 hours at RT and goat antimouse IgG labeled with horseradish peroxidase (HRP) was added for 1 hour at RT. Visualization was obtained with H2O2 and ortho-phenylenediamine (OPD, Sigma) and the coloring reaction was stopped with 4 moL/L H2SO4. The absorbance was determined at 490 nm. After each incubation step, plates were washed with PBS, 0.1% Tween-20, 3 times after coating and blocking steps and 12 times elsewhere. The plasma concentration of 82D6A3 in each sample was calculated from a standard curve where known amounts of 82D6A3 were added to baboon plasma.VWF-antigen levels Determination of the VWF-antigen (Ag) levels was performed essentially as described.20 Briefly, microtiter plates were coated with a polyclonal anti-VWF-Ig solution (Dako, Glostrup, Denmark), blocked with 3% milk powder and plasma samples, preincubated for 5 minutes at 37°C, and added to the wells at 1:40 to 1:2560 dilutions. Bound VWF was detected with rabbit antihuman VWF HRP antibodies (Dako). VWF-Ag levels were calculated from a standard curve obtained by adding 1:40 to 1:2560 dilutions to the coated wells of a human plasma pool, known to contain 10 µg/mL human VWF.VWF occupancy Microtiter plates (96-well) were coated overnight at 4°C with 125 µL/well of a polyclonal anti-VWF-Ig solution (Dako; 1:1000 in PBS). Plates were blocked with 3% milk powder solution for 2 hours at RT. A dilution series (1:2 in PBS) of the plasma samples (prewarmed for 5 minutes at 37°C) was added for 2 hours at RT. Samples containing 100% occupied VWF were obtained by adding a saturating amount of 82D6A3 (6 µg/mL) to each corresponding baboon plasma. Bound 82D6A3 was detected by addition of goat antimouse IgG-HRP (1 hour at RT). Visualization and wash steps were performed as described above. The VWF occupancy of each sample was calculated as follows: (A490-nm sample/A490-nm sample saturated with 82D6A3) · 100.Determination of the VWF-collagen binding activity The enzyme-linked immunosorbent assay (ELISA) was performed essentially as described.20 Briefly, microtiter plates were coated with human collagen type I (Sigma), blocked with 3% milk powder solution and 1/2 dilution series of baboon plasma (prewarmed for 5 minutes at 37°C) were added. Bound VWF was detected with rabbit antihuman VWF-HRP antibodies. Binding of baboon VWF to collagen in the different plasma samples was compared to the binding of VWF in the respective blood sample taken at time zero (presample), which was set as 100%.Inhibition of the VWF binding to collagen by 82D6A3 The ELISA was performed as described for the determination of the VWF-collagen binding activity except that serial dilutions of 82D6A3 were either preincubated with constant amounts of VWF in a preblocked plate for 30 minutes or were directly added to the plate containing collagen-bound VWF.Statistics Data are expressed as mean ± SD. Student t test (paired) or one-factor ANOVA followed by Fisher test was used for statistical evaluation. P < .05 was considered as statistically significant.
In vitro interaction of 82D6A3 with baboon VWF 82D6A3, known to inhibit human VWF binding to collagen,17 cross-reacts with baboon VWF. 82D6A3 has a comparable affinity for baboon VWF as for human VWF (median effective concentration [EC50] 200 ng/mL) and inhibited the binding of baboon VWF to collagen with an inhibitory concentration of 50% (IC50) of 3.5 µg/mL (Figure 1).
82D6A3 not only prevented binding of VWF to collagen, but also was able
to remove the bound VWF from a collagen surface with somewhat lower
efficacy (Figure 2).
Antithrombotic effect Using a Folts model, the antithrombotic efficacy of 82D6A3 was tested by administering different doses of 82D6A3 to baboons. In Figure 3, a representative tracing of CFR data is given.
Injection of saline did not affect CFRs (107% ± 7%). In a first
study, a dose of 600 µg/kg 82D6A3 resulted in a complete
disappearance of the CFRs (Figure 4).
This dose was expected to be an overdose because according to
theoretical calculations, a dose of 600 µg/kg would result in a
plasma concentration of 13 µg/mL (45 mL/kg plasma volume), which in
in vitro experiments results in a 100% inhibition of the VWF binding
to collagen (Figure 1). As a result, in all subsequent in vivo studies
lower doses of 82D6A3 were used. Administration of 100 µg/kg 82D6A3
resulted in a significant reduction of the CFRs by 58.3% ± 4.8%
(Figure 4). Administration of a total doses of 300 µg/kg completely
abolished the CFRs (Figure 4), which could not be restored by
increasing intimal damage or increasing stenosis.
Coagulation, platelet count, and bleeding time No significant changes in PT and aPTT were observed in any of the animals (data not shown). No major changes in platelet count were observed for the 100 µg/kg (not significant [NS]), 300 µg/kg (NS), and 600 µg/kg 82D6A3 doses (Tables 1 and 2). The bleeding time was not significantly prolonged after injection of 100 µg/kg. After injection of 300 µg/kg the bleeding time was increased 2.7 times at 60 minutes and 2.4 times at 150 minutes; however, these changes were not statistically significant. After injection of 600 µg/kg the bleeding time was increased 1.9, 3.0, 2.8, and 1.7 times after 30, 60, 150, and 300 minutes, respectively.
82D6A3 plasma concentration, VWF-Ag levels, VWF occupancy, and ex vivo VWF-collagen binding The 82D6A3 plasma levels remained relatively constant during the first 3 hours of the experiment. Then, they decreased to 69%, 23%, and 7.6% after 300 minutes, 24 hours, and 48 hours, respectively, when 300 µg/kg 82D6A3 was administered (Table 1).Thirty minutes after injection of the different doses of 82D6A3, plasma VWF-Ag levels decreased slightly and increased above baseline at 24 hours. None of these changes were significant (Tables 1 and 2). At 60 minutes after administration VWF occupancy was 80% for the 100-µg/kg dose and nearly 100% for the 300-µg/kg and 600-µg/kg doses. VWF remained occupied for an extended period; even 48 hours after the injection of 300 µg/kg 82D6A3, still 63% of the VWF-binding sites were occupied by 82D6A3 (Table 1). Injection of 100 µg/kg 82D6A3 resulted in an ex vivo inhibition of the VWF-collagen binding of 31% (blood sample taken after 1 hour; Table 1). At doses of 300 µg/kg and 600 µg/kg no interaction between baboon VWF and collagen was observed in samples taken up to 5 hours after the administration of the antibody. Twenty-four hours after the injection of the drug the VWF-collagen interaction recovers (Table 1 and 2). Relation between the ex vivo 82D6A3 plasma levels, VWF occupancy, and collagen binding The relationship between 82D6A3 plasma levels and VWF occupancy is illustrated in Figure 5. At plasma concentrations between 1 and 2 µg/mL 82D6A3 nearly 100% of circulating VWF was occupied (Figure 5). There was a good relation between the ex vivo VWF occupancy and the VWF binding to collagen. To obtain inhibition of VWF binding to collagen (20%-40%), a VWF occupancy of at least 80% was required, with complete inhibition at 90% to 100% occupancy (Figure 6). These data were confirmed by in vitro experiments, where different concentrations of 82D6A3 were added to baboon plasma (Figure 6). Occupancy levels of up to 60% resulted in no inhibition of the VWF binding to collagen, whereas up to complete inhibition was observed with 80% to 100% VWF-occupancy levels.
Platelet adhesion to a damaged vessel wall is the first step in arterial thrombus formation. The tethering of platelets by VWF to the collagen exposed in the damaged vessel wall is especially important under high shear conditions. Antithrombotics that interfere with the GPIb-VWF axis have been studied in animal models and were shown to be effective.13,14 The present study evaluated for the first time the antithrombotic effects of the inhibition of the VWF-collagen interaction in vivo. For this purpose, we used a monoclonal antihuman VWF antibody 82D6A3, which by binding to the VWF A3-domain, inhibits VWF binding to fibrillar collagens types I and III.17 We here showed that 82D6A3 cross-reacts with baboon VWF and inhibits baboon VWF binding to collagen type I under static conditions. Moreover, in vitro 82D6A3 is able to remove collagen-bound VWF from its surface, which in view of the presence of collagen-bound VWF in the damaged vessel wall, might be a prerequisite for a proper function in vivo. Because the subendothelium, however, not only includes different types of collagen that may interact differently with VWF, and other matrix proteins that may affect the binding of VWF, and because, in addition, it may contain different VWF forms, such as ultralarge forms unprocessed by the VWF-cleaving plasma protease, no direct extrapolation is possible. A modified Folts model was used to evaluate the antithrombotic efficacy of 82D6A3 under high shear conditions18 in baboons. This model allows study of CFRs due to platelet-dependent thrombi forming at the injured, stenotic site of the artery.21 We demonstrated that inhibition of the VWF-collagen interaction is accompanied by an antithrombotic effect in vivo. Indeed, in all 5 animals receiving doses of 300 µg/kg or higher, a 100% inhibition of the CFRs was obtained, whereas administration of 100 µg/kg resulted in 58% inhibition. Administration of the lowest 82D6A3 dose (100 µg/kg) to the baboons revealed that when 80% of the VWF-A3 domain sites were occupied (30%-36% inhibition of VWF binding to collagen), 58% reduction in CFRs was observed with no prolongation of the bleeding time. When a 100% decrease of CFRs was observed after administration of 300 µg/kg 82D6A3 (and also of 600 µg/kg) the bleeding time increased 2.7 times (60 minutes, 300 µg/kg), which was not statistically significant. At these doses, 100% VWF was occupied with 82D6A3 and 100% ex vivo inhibition of the VWF binding to collagen was observed. It is interesting to note that when we studied the effect of 300 µg/kg of an anti-GPIIb/IIIa mAb 16N7C2 in exactly the same animal model, a more than 15-fold increase in bleeding time was observed, which was statistically significant.19 Thus the therapeutic window of 82D6A3 seems to be broader when compared to the one of an anti-GPIIb/IIIa blocker in our animal model.19 This suggests that bleeding problems might be less expected when 82D6A3 is used as an antithrombotic agent. The observation that no significant decreases in platelet count and VWF-Ag levels were observed furthermore pointed out that the observed antithrombotic effect is indeed due to the specific inhibition of the VWF-collagen interaction. 82D6A3 has a high affinity for VWF (Kd 0.4 nmol17), which is reflected here by the observation that VWF is still occupied by 63% with the antibody 48 hours after its administration. On the other hand, to be functionally active, at least up to 80% VWF occupancy is needed to have an ex vivo and in vitro inhibition of VWF binding to collagen. This level was maintained for up to 5 hours after the administration of 300 and 600 µg/kg 82D6A3. The observation that up to 80% VWF occupancy is needed to be functionally relevant might be explained by the fact that when VWF is occupied for 74%, the ratio VWF monomer/mAb is 8. So too many VWF subunits are devoid of mAb to have some effect in VWF-collagen binding. It has to be noted, however, that at 100% VWF occupancy, the VWF monomer/mAb ratio (both in vitro and ex vivo) is 2.8, which demonstrates that both in vitro and ex vivo not all VWF-A3 domains are accessible for 82D6A3 binding. Although many in vitro studies demonstrated that the VWF-collagen interaction is vital for sustaining platelet adhesion under high shear conditions, this study is the first experimental proof of the in vivo importance of this interaction. It is of interest to note that for a very long time no bleeding disorders due to an isolated defect in VWF binding to collagen were known. Only recently 2 patients, mother and daughter, were identified with a moderate bleeding disorder due to Ser968Thr mutation in the A3 domain of VWF resulting in a defective binding of VWF to collagen.22 These recent results together with our data finally unequivocally demonstrate the in vivo relevance of the VWF-collagen interaction. In conclusion, blockade of VWF-collagen interaction by 82D6A3 reduced platelet thrombus formation in the injured and stenosed baboon femoral arteries in a dose-dependent manner. In view of the lack of effect on the bleeding time, it is worthwhile to develop this approach further to determine its potential to treat acute arterial thrombotic syndromes.
We thank Miss Griet Vandecasteele, Mr Stephan Vauterin, Mr Seb Lamprecht, and Mr Jan P. Roodt for their excellent technical assistance and Dr Jef Arnout for help with the statistical analysis.
Submitted October 11, 2001; accepted January 8, 2002.
Supported by a EU-Biomed grant (PC 96.3517) and a Bilateral Collaboration grant between the Flemish and South African Government (BIL 98/64). D.W. was a Junior Fellow of the KU Leuven. D.W. and K.V. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hans Deckmyn, Laboratory for Thrombosis Research-IRC, K U Leuven Campus Kortrijk, E. Sabbelaan 53, B-8500 Kortrijk, Belgium; e-mail: hans.deckmyn{at}kulak.ac.be.
1. Sixma JJ, Wester J. The hemostatic plug. Semin Hematol. 1977;14:265-299[Medline] [Order article via Infotrieve]. 2. Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell. 1996;84:289-297[CrossRef][Medline] [Order article via Infotrieve]. 3. Kehrel B. Platelet-collagen interactions. Semin Thromb Hemost. 1995;21:123-129[Medline] [Order article via Infotrieve].
4.
Monnet E, Fauvel-Lafeve F.
A new platelet receptor specific to type III collagen. Type III collagen-binding protein.
J Biol Chem.
2000;275:10912-10917 5. Phillips DR, Charo IF, Scarborough RM. GPIIb-IIIa: the responsive integrin. Cell. 1991;65:359-362[CrossRef][Medline] [Order article via Infotrieve]. 6. Berndt MC, Ward CM, Booth WJ, Castaldi PA, Mazurov AV, Andrews RK. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin. Biochemistry. 1992;31:11144-11151[CrossRef][Medline] [Order article via Infotrieve]. 7. Lankhof H, van Hoeij M, Schiphorst ME, et al. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost. 1996;75:950-958[Medline] [Order article via Infotrieve].
8.
Siedlecki CA, Lestini BJ, Kottke-Marchant KK, Eppell SJ, Wilson DL, Marchant RE.
Shear-dependent changes in the three-dimensional structure of human von Willebrand factor.
Blood.
1996;88:2939-2950
9.
Peng M, Lu W, Beviglia L, Niewiarowski S, Kirby EP.
Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib.
Blood.
1993;81:2321-2328
10.
Chang MC, Lin HK, Peng HC, Huang TF.
Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox.
Blood.
1998;91:1582-1589
11.
Miller JL, Thiam-Cisse M, Drouet LO.
Reduction in thrombus formation by PG-1 F(ab')2, an anti-guinea pig platelet glycoprotein Ib monoclonal antibody.
Arterioscler Thromb.
1991;11:1231-1236
12.
McGhie AI, McNatt J, Ezov N, et al.
Abolition of cyclic flow variations in stenosed, endothelium-injured coronary arteries in nonhuman primates with a peptide fragment (VCL) derived from human plasma von Willebrand factor-glycoprotein Ib binding domain.
Circulation.
1994;90:2976-2981
13.
Cauwenberghs N, Meiring M, Vauterin S, et al.
Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates.
Arterioscler Thromb Vasc Biol.
2000;20:1347-1353
14.
Cadroy Y, Hanson SR, Kelly AB, et al.
Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates.
Blood.
1994;83:3218-3224 15. Yamamoto H, Vreys I, Stassen JM, Yoshimoto R, Vermylen J, Hoylaerts MF. Antagonism of vWF inhibits both injury induced arterial and venous thrombosis in the hamster. Thromb Haemost. 1998;79:202-210[Medline] [Order article via Infotrieve]. 16. Golino P, Ragni M, Cirillo P, et al. Aurintricarboxylic acid reduces platelet deposition in stenosed and endothelially injured rabbit carotid arteries more effectively than other antiplatelet interventions. Thromb Haemost. 1995;74:974-979[Medline] [Order article via Infotrieve]. 17. Hoylaerts MF, Yamamoto H, Nuyts K, Vreys I, Deckmyn H, Vermylen J. von Willebrand factor binds to native collagen VI primarily via its A1 domain. Biochem J. 1997;324:185-191. 18. Folts J. An in vivo model of experimental arterial stenosis, intimal damage, and periodic thrombosis. Circulation. 1991;83:IV3-14.
19.
Wu D, Meiring M, Kotze HF, Deckmyn H, Cauwenberghs N.
Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons.
Arterioscler Thromb Vasc Biol.
2002;22:323-328 20. Vanhoorelbeke K, Cauwenberghs N, Vauterin S, Schlammadinger A, Mazurier C, Deckmyn H. A reliable and reproducible ELISA method to measure ristocetin cofactor activity of von Willebrand factor. Thromb Haemost. 2000;83:107-113[Medline] [Order article via Infotrieve]. 21. Ikeda H, Koga Y, Kuwano K, et al. Cyclic flow variations in a conscious dog model of coronary artery stenosis and endothelial injury correlate with acute ischemic heart disease syndromes in humans. J Am Coll Cardiol. 1993;21:1008-1017[Abstract]. 22. Ribba AS, Loisel I, Lavergne JM, et al. Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen. Thromb Haemost. 2001;86:848-854[Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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 U. J.H. Sachs and B. Nieswandt In Vivo Thrombus Formation in Murine Models Circ. Res., April 13, 2007; 100(7): 979 - 991. [Abstract] [Full Text] [PDF]
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H. Deckmyn and K. Vanhoorelbeke When collagen meets VWF Blood, December 1, 2006; 108(12): 3628 - 3628. [Full Text] [PDF]
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S. Massberg, K. Schurzinger, M. Lorenz, I. Konrad, C. Schulz, N. Plesnila, E. Kennerknecht, M. Rudelius, S. Sauer, S. Braun, et al. Platelet Adhesion Via Glycoprotein IIb Integrin Is Critical for Atheroprogression and Focal Cerebral Ischemia: An In Vivo Study in Mice Lacking Glycoprotein IIb Circulation, August 23, 2005; 112(8): 1180 - 1188. [Abstract] [Full Text] [PDF]
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M. Fattorutto, H. Deckmyn, K. Vanhoorelbeke, and H. Kotze A modified Folts model or the original Folts model to evaluate new antithrombotics? Blood, January 15, 2003; 101(2): 782 - 782. [Full Text] [PDF]
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Top
Abstract
Introduction
, such as the GPIb-binding snake venom proteins echicetin and crotalin,
60
minutes to 0 minute), test agents (saline or 82D6A3) were given via an
intravenous bolus injection (t = 0) and monitoring was continued up
to 60 minutes after drug administration (t = +60 minutes). The
antithrombotic effect was quantified by comparing the number of CFRs
per hour before and after drug administration. If the last flow
reduction in the 60-minute recording period was not cyclic, it was
considered in the calculations. Blood samples for the different
laboratory measurements were drawn at different time points. The doses
of 82D6A3 were selected on the basis of preliminary dose-finding
studies. Group 1 was a control group where the baboons received saline
(n = 2). Group 2 received a high dose of 600 µg/kg 82D6A3
(n = 2). In group 3, the baboons received an initial dose of
100 µg/kg 82D6A3 and after 60 minutes an additional 200 µg/kg
82D6A3 was given (n = 3). In a preliminary study we found that 82D6A3
has a long half-life. This therefore resulted in a final dose of 300 µg/kg. All agents were diluted with saline.
) or were added to
a collagen-coated plate where VWF (final dilution of 1:46, ie,
217 ng/mL VWF) was already bound for 30 minutes (
). Bound VWF was
detected with rabbit antihuman VWF antibodies.
= P < .01. One-factor ANOVA followed by Fisher test
was used for statistical evaluation.
