Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

SummaryRecent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (>80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0. 2 mg/kg with a slight prolongation of bleeding time (1. 3 times baseline value). Furthermore, at 18. 8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1. 9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5. 0 times at 0. 35 mg/kg, the lowest effective dose on platelet aggregation. Ina pharmacodynamic study,a bolus injection of OM2 at 0. 4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exerta potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

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Antiplatelet and Antithrombotic Effects of YM337, the Fab Fragment of a Humanized Anti-GPIIb/IIIa Monoclonal Antibody in Monkeys

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650342 ◽

1996 ◽

Vol 75 (04) ◽

pp. 679-684 ◽

Cited By ~ 16

Author(s):

Seiji Kaku ◽

Tomihisa Kawasaki ◽

Nami Hisamichi ◽

Yumiko Sakai ◽

Yuta Taniuchi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Arachidonic Acid ◽

Platelet Aggregation ◽

Bleeding Time ◽

Bolus Injection ◽

Ex Vivo ◽

Thrombus Formation ◽

Fab Fragment ◽

Inhibition Of Platelet Aggregation ◽

Antithrombotic Effects

SummaryThe antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.

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THE BLEEDING TENDENCY OF PROGRESSIVE RENAL FAILURE IS NOT ASSOCIATED WITH DEFECTIVE PLATELET AGGREGATION

10.1055/s-0038-1644566 ◽

1987 ◽

Author(s):

M P Gordge ◽

R W Faint ◽

P B Rylance ◽

G H Neild

Keyword(s):

Renal Failure ◽

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Plasma Creatinine ◽

Bleeding Tendency ◽

Platelet Response ◽

Normal Platelet ◽

Von Willebrand ◽

Induced Aggregation

The bleeding tendency of uraemia may be related to reduction by anaemia of erythrocyte/platelet interaction, toxic inhibition of platelet aggregation and abnormal von Willebrand Factor (vWF) mediated platelet adhesion. Our aim in this study was to determine at what stage of renal failure bleeding time becomes prolonged and to investigate the mechanisms involved.We have measured bleeding time (Simplate II), plasma levels of fibrinogen and vWF, and ex-vivo platelet responsiveness in 31 patients with chronic renal failure (CRF) of various degrees of severity and compared them with values obtained in 22 healthy controls. No patient was dialysed, nephrotic or suffering from immunological renal disease. Patients were divided into mild (plasma creatinine <300 umol/1), n=10, moderate (300-600 umol/1), n=14, or severe (>600 umol/1), n=7, CRF.Bleeding time became significantly prolonged only in severe CRF (p<0.005). Haematocrit fell as renal failure advanced, and correlated with bleeding time (r=0.40, p<0.05). Platelet counts were normal. Platelet aggregation in response to ristocetin (mediated by vWF) and ADP increased progressively (p<0.005 in severe CRF), as did spontaneous aggregation (p<0.005 in severe CRF). This was associated with an increase in plasma vWF and fibrinogen (p<0.005 in severe CRF). Collagen induced aggregation was slightly, but not significantly increased. Thromboxane (TxB2) generation in clotting blood was the only measurement that showed a reduced platelet response (p<0.025 in severe CRF).In summary, a bleeding tendency develops late in the course of progressive CRF when plasma creatinine has risen to at least 600 umol/1. Platelet aggregation is enhanced rather than reduced and platelet interaction with vWF is not defective. Anaemia appears more important than abnormal platelet aggregation in mediating uraemic bleeding, although reduced serum TxB2 generation suggests a defect in platelet response to endogenous thrombin which may also contribute. Increased platelet aggregation and fibrinogen concentrations might promote glomerular thrombosis and contribute to the progression of CRF.

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Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642390 ◽

1994 ◽

Vol 71 (01) ◽

pp. 095-102 ◽

Cited By ~ 43

Author(s):

Désiré Collen ◽

Hua Rong Lu ◽

Jean-Marie Stassen ◽

Ingrid Vreys ◽

Tsunehiro Yasuda ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Bolus Injection ◽

Cell Injury ◽

Ex Vivo ◽

Thrombus Formation ◽

Femoral Vein ◽

Thrombotic Occlusion ◽

Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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Comparative Arterial Antithrombotic Activity of Clopidogrel and Acetyl Salicylic Acid in the Pig

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646307 ◽

1992 ◽

Vol 68 (05) ◽

pp. 500-505 ◽

Cited By ~ 32

Author(s):

Ch M Samama ◽

Ph Bonnin ◽

M Bonneau ◽

G Pignaud ◽

E Mazoyer ◽

...

Keyword(s):

Platelet Aggregation ◽

Femoral Artery ◽

Bleeding Time ◽

Ex Vivo ◽

Flow Reduction ◽

Cyclic Flow ◽

Left Femoral Artery ◽

Before And After ◽

The Right ◽

Induced Aggregation

SummaryWe investigated the comparative antithrombotic properties of clopidogrel, an analogue of ticlopidine, and aspirin, using the Folts' model on femoral arteries in 22 pigs. On each animal, clopidogrel or aspirin were used to treat the thrombotic process on the left femoral artery and to prevent this process on the right femoral artery. Sequentially: an injury and stenosis were carried out on the left femoral artery; the thrombotic process was monitored with a Doppler during a 30-min observation period for cyclic flow reductions or permanent cessation of flow; after the first cyclic flow reduction occurred, clopidogrel (5 mg kg-1) or aspirin (2.5, 5, 100 mg kg-1) were injected intravenously; if cyclic flow reductions were abolished, epinephrine (0.4 µg kg-1 min-1) was injected to try to restore cyclic flow reductions and/or permanent cessation of flow; then injury and stenosis were applied on the right femoral artery. Before and after injection of clopidogrel or aspirin, ear immersion bleeding times and ex-vivo platelet aggregation were performed. Clopidogrel (n = 7) abolished cyclic flow reductions in all animals and epinephrine did not restore any cyclic flow reduction. On the right femoral artery, cyclic flow reductions were efficiently prevented, even for two injuries. Basal bleeding time (5 min 28) was lengthened (>15 min, 30 min after clopidogrel and remained prolonged even after 24 h). ADP-induced platelet aggregation was inhibited (more than 78%). Comparatively, aspirin had a moderate and no dose-dependent effect. Aspirin 2.5 mg kg-1 (n = 6) abolished cyclic flow reductions in 2 animals, CFR reoccurred spontaneously in one animal and epinephrine restored it in a second animal. Aspirin 5 mg kg-1 (n = 6) abolished cyclic flow reductions in only 3 animals and epinephrine always restored it. Aspirin 100 mg kg-1 (n = 3) was unable to abolish cyclic flow reductions. On the right femoral artery, aspirin did not significantly prevent cyclic flow reductions which occurred in all animals after one (n = 14) or two injuries (n = 1), except for one animal. Basal bleeding time was lengthened but it shortened rapidly, reaching its basal value after 24 h. ADP-induced aggregation was not significantly inhibited, whereas arachidonic acid induced aggregation was always inhibited. Clopidogrel appears as a more potent antithrombotic drug than aspirin in this model, in treating and preventing spontaneous or epinephrine-induced cyclic flow reductions and lengthening bleeding time.

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INITIAL STUDIES IN MAN WITH A NOVEL THROMBOXANE RECEPTOR BLOCKING DRUG GR32191

10.1055/s-0038-1643467 ◽

1987 ◽

Author(s):

M Thomas ◽

P Lumley ◽

P Ballard ◽

J R O'Brien

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Plasma Concentrations ◽

Blocking Drug ◽

Double Blind ◽

Receptor Blocking ◽

Thromboxane Receptor ◽

Pre Treatment ◽

Induced Aggregation

In-vitro GR32191 is a potent and specific thromboxane receptor blocking drug on platelets, and vascular and airways smooth muscle (Lumley et al this meeting). We have undertaken studies in healthy male subjects (n) to examine the effects of oral GR32191 upon platelet aggregation ex-vivo and template bleeding time. Platelet aggregation was monitored in whole blood by counting platelets electronically. Concentration-effect curves to U-46619 and ADP were constructed prior to and following drug or placebo. The degree of rightward displacement of a curve due to treatment was expressed as a concentration-ratio (CR) which was calculated at the 50% aggregation level (ECso post-treatment ECso pre-treatment). Plasma concentrations of GR32191 were determined by h.p.l.c. After single doses of GR32191 mean peak CR's of 8 and 80 were achieved with 0.125 and 0.25mg/kg (n=4) and values of 74 and 234 with 0.5 and lmg/kg (n=4). Peak effects were seen within 2 hours of dosing while activity was still present between 8 and 24 hours. ADP-induced aggregation was unaffected by drug (CR<2) and placebo was without significant effect upon the sensitivity to either aggregating agent (CR<2). GR32191 was rapidly absorbed and the plasma elimination half-life was about 2 hours. GR32191 17.5mg 12-hourly for 10 days (n=6) produced a progressive antagonism of U-46619 induced aggregation which resulted in a large continuous blockade in all subjects (range of 12htrough CR's 85 to 287). However, plasma concentrations of GR32191 did not accumulate on repeated administration. In a double-blind, placebo-controlled, cross-over study (n=16), a statistically significant (p= 0.002) increase in bleeding time was seen following treatment with GR32191 40mg twice daily for 7 days (pre-treatment mean 3.79 min, post-placebo mean 3.47 min, post-GR32191 mean 5.42 min). Rectal bleeding (n=l) has occurred with GR32191 but otherwise tolerability has been good. No drug related changes have been seen in routine laboratory safety screens. Clinical studies are in progress.

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Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653771 ◽

1995 ◽

Vol 73 (02) ◽

pp. 318-323 ◽

Cited By ~ 20

Author(s):

K Azzam ◽

L I Garfinkel ◽

C Bal dit Sollier ◽

M Cisse Thiam ◽

L Drouet

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Mesenteric Arteries ◽

Antithrombotic Effects ◽

Von Willebrand ◽

Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

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Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614243 ◽

1998 ◽

Vol 79 (01) ◽

pp. 222-227 ◽

Cited By ~ 8

Author(s):

F. Stockmans ◽

W. Deberdt ◽

Å. Nyström ◽

E. Nyström ◽

J. M. Stassen ◽

...

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Peak Plasma ◽

Thrombus Formation ◽

Plasma Concentrations ◽

Inhibitory Effect ◽

Human Platelets ◽

Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

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Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.33 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Reheman Adili ◽

Katherine Mast ◽

Theodore R Holman ◽

Michael Holinstat

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

High Shear ◽

Vessel Occlusion ◽

12 Lipoxygenase ◽

Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

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The Effects Of Ticlopidine Hydrochloride On Bleeding Time And Platelet Function In Man

10.1055/s-0038-1652495 ◽

1981 ◽

Author(s):

D J Ellis ◽

R L Roe ◽

J J Bruno ◽

B J Cranston ◽

M M McSpadden

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Bleeding Time ◽

Ex Vivo ◽

Atp Release ◽

Phosphodiesterase Inhibitor ◽

Optimal Dosage ◽

Post Dose ◽

Induced Aggregation ◽

Dose Dependent

Ticlopidine hydrochloride (T) was given to healthy male and female subjects in doses ranging from 125-500 mg/day for up to 8 days to study the rate of onset and offset of action and to define the optimal dosage for inhibition of platelet function tests.Template bleeding time (BT) using the Simplate® device and ex vivo platelet aggregation were done every day from the start of dosing through the post-dosing period until platelet function had returned to baseline.Achievement of maximal bleeding time prolongation and maximal inhibition of platelet aggregation induced by ADP, collagen, and epinephrine required 3-5 days of dosing. Rate of onset was higher at higher drug doses. Post-dose return to baseline took 4-8 days.BT prolongation was dose-dependent with 2.0 × prolongation at 125 mg/d, 2.5 × at 250 mg/d, 3.4 × at 375 mg/d and 4.7 × at 500 mg/d.At steady state, ADP aggregation (1st and 2nd phase) was inhibited by 50-60%, epinephrine aggregation was inhibited by 40-80%, and collagen aggregation was inhibited by 5580%. Inhibition was dose-dependent. Arachidonic acid (1 × 10-3M) induced aggregation was not inhibited. ATP release during collagen aggregation was inhibited by 20-50% and malondialdehyde production induced by thrombin was inhibited 20-55%, both in a dose-related fashion.Ticlopidine hydrochloride is a potent inhibitor of platelet function with a broad spectrum of activity and a delayed onset and offset of action. The drug is neither a prostaglandin synthetase nor a phosphodiesterase inhibitor and appears to act by a novel mechanism.

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The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons

Thrombosis and Haemostasis ◽

10.1160/th08-02-0073 ◽

2008 ◽

Vol 100 (10) ◽

pp. 670-677 ◽

Cited By ~ 40

Author(s):

Alexandre Fontayne ◽

Muriel Meiring ◽

Seb Lamprecht ◽

Jan Roodt ◽

Eddy Demarsin ◽

...

Keyword(s):

Monoclonal Antibody ◽

Side Effects ◽

Blood Loss ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Plasma Concentrations ◽

Platelet Glycoprotein ◽

Fab Fragment ◽

Platelet Receptor

SummaryThe Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibα and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

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