Recent 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.
Keywords
GPVI -
antibody -
platelet
References
1
Fuster V,
Badiman L,
Badiman JJ.
et al. The pathogenesis of coronary artery disease and the acute coronary syndrome. N Engl J Med 1992; 326: 310-8.
3
Baumgartner HR.
Platelet interaction with collagen fibrils in flowing blood: reaction with human platelets with alpha chymotrypsin-digested subendothelium. Thromb Haemost 1977; 31: 1-16.
5
Nieuwenhuis HK,
Akkerman J,
Houdljk WP.
et al. Human platelets showing no response to collagen failed to express glycoprotein Ia. Nature 1985; 318: 470-2.
6
Kehrel B,
Balleisen R,
Kokott R.
et al. Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder. Blood 1988; 71: 1074-8.
8
Sugiyama T,
Okuma M,
Ushikubi F.
et al. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation autoimmune thrombocytopenia. Blood 1987; 69: 1712-20.
9
Moroi M,
Jung SM,
Okuma M.
et al. A patient with platelets deficient in glycoprotein VI that lack both collagen-induced platelet aggregation and adhesion. J Clin Invest 1989; 84: 1140-5.
10
Ryo R,
Yoshida A,
Sugano W.
et al. Deficiency of P62,a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation. Am J Hematol 1992; 39: 25-31.
11
Arai M,
Yamamoto N,
Moroi M.
et al. Platelets with 10% of the normal amounts of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency. Br J Haemtol 1995; 89: 124-30.
12
Takahashi H,
Moroi M.
Antibody against platelet membrane glycoprotein VI in a patient with systemic lupus erythematosus. Am J Hematol 2001; 67: 262-7.
13
Nurden P,
Jandrot-Perrus M,
Combrie R.
et al. Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome. Blood 2004; 104: 107-14.
14
Boylan B,
Chen H,
Rathore V.
et al. Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/ FcRgamma-chain complex from the human platelet surface. Blood 2004; 104: 1350-5.
15
Chen J,
Diacovo TG,
Grenache DG.
et al. The α2 integrin sub-unit deficient mouse: A multifaceted phenotype including defects in branching morphogenesis and hemostasis. Am J Pathol 2002; 161: 337-44.
16
Kato K,
Kanaji T,
Russell S.
et al. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion. Blood 2003; 102: 1701-7.
17
Lockyer S,
Okuyama K,
Begum S.
et al. GPVIdeficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism. Thromb Res. 2005 Epub ahead of print.
19
Massburg S,
Gawaz M,
Gruner S.
et al. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo
. J Exp Med 2003; 197: 41-9.
20
Munnix IC,
Strehl A,
Kuijpers MJ.
et al. The glycoprotein VI-phospholipase Cgamma2 signaling pathway controls thrombus formation induced by collagen and tissue factor in vitro and in vivo
. Arterioscler Thromb Vasc Biol 2005; 25: 2673-8.
21
Konishi H,
Katoh Y,
Takaya N.
et al. Platelets activated by collagen through immunoreceptor tyrosine based activation motif play pivotal role in the initiation and generation of neointimal hyperplasia after vascular injury. Circulation 2002; 105: 912-6.
22
Jandrot-Perrus M,
Lagrue AH,
Okuma M.
et al. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from immunoglobulin superfamily. Blood 2000; 96: 1798-807.
23
Nieswandt B,
Schulte V,
Bergmeier W.
et al. Longterm antithrombotic protection by in-vivo depletion of platelet glycoprotein GPVI in mice. J Exp Med 2001; 193: 459-69.
24
Lecut C,
Feeney LA,
Kingsbury G.
et al. Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments. J Thromb Haemost 2003; 01: 2653-62.
26
Smethurst PA,
Joutsi-Korhonen L,
O’Connor MN.
et al. Identification of the primary collagen binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody. Blood 2004; 103: 903-11.
27
Lecut C,
Schoolmeester A,
Kuijpers MJ.
et al. Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation. Arterioscler Throm Vasc Biol 2004; 24: 1-7.
31
Polgar J,
Clemetson JM,
Kehrel BE.
et al. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem 1997; 272: 13576-83.
32
Parham P,
Androlewicz MJ,
Brodsky FM.
et al. Monoclonal antibodies: purification and application to structural functional studies of class I MHC antigens. J Immunol Meth 1982; 53: 133-73.
34
Watson SP,
Asazuma N,
Atkinson B.
et al. The role of ITAM and ITIM-coupled receptors in platelet activation by collagen. Thromb Haemost 2001; 86: 276-88.
37
Boylan B,
Berndt MC,
Kahn ML.
et al. Activationindependent, antibody-mediated removal of GPVI from circulating human platelets: Development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents. Blood. 2006 Epub ahead of print.
39
Sassoli PM,
Emmell EL,
Tam SH.
et al. 7E3 F(ab’)2, an effective antagonist of rat αIIbβ3 and αvβ3, blocks in vivo thrombus formation and in vitro angiogenesis. Thromb Haemost 2001; 85: 896-902.
40
Li H,
Lockyer S,
Matsumoto Y.
et al. A novel anti-GPVI monoclonal antibody OM4 reduces in vivo thrombosis with minimal bleeding risk in rats. Circulation. 2005 112. II-241.
41
Nguyen CM,
Harrington RA.
Glycoprotein IIb/IIIa receptor antagonists: a comparative review of their use in percutaneous coronary intervention. Am J Cardiovasc Drugs 2003; 03: 423-36.
42
The EPISTENT Investigators.
Randomised placebo-controlled and balloon-angioplasty-controlled trial to assess safety of coronary stenting with use of platelet glycoprotein-IIb/IIIa blockade. Lancet 1998; 352: 87-92.
43
The GUSTO IV-ACS Investigators.
Effect of glycoprotein IIb/IIIa receptor blocker abciximab on outcome in patients with acute coronary syndromes without early coronary revascularisation: the GUST IV-ACS randomised trial. Lancet 2001; 357: 1915-24.
44
The PURSUIT Trial Investigators.
Inhibition of platelet glycoprotein IIb/IIIa with eptifibatide in patients with acute coronary syndromes. New Engl J Med 1998; 339: 436-43.
