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Thromb Haemost 2006; 96(02): 167-175
DOI: 10.1160/TH06-05-0266
Platelets and Blood Cells
Schattauer GmbH

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

Yutaka Matsumoto
1   Otsuka Maryland Medicinal Laboratories, Rockville, Maryland, USA
,
Hisao Takizawa
1   Otsuka Maryland Medicinal Laboratories, Rockville, Maryland, USA
,
Kazuhiro Nakama
2   Shin Nippon Biomedical Laboratories, Kagoshima, Japan
,
Xiaoqi Gong
1   Otsuka Maryland Medicinal Laboratories, Rockville, Maryland, USA
,
Yoshihisa Yamada
3   First Institute of New Drug Discovery, Otsuka Pharmaceutical Co. , Ltd. , Tokushima, Japan
,
Narendra N. Tandon
1   Otsuka Maryland Medicinal Laboratories, Rockville, Maryland, USA
,
Junichi Kambayashi
1   Otsuka Maryland Medicinal Laboratories, Rockville, Maryland, USA
› Author Affiliations
Further Information

Publication History

Received 15 May 2006

Accepted after minor revision 03 July 2006

Publication Date:
28 November 2017 (online)

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Summary

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.
    CrossrefPubMedSearch in Google Scholar
  • 2 Conti CR, Mehta JL. Acute myocardial ischemia: role of atherosclerosis, thrombosis, platelet activation, coronary vasospasm and altered arachidonic acid metabolism. Circulation 1987; 75: V84-V95.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 4 Hawiger J. Macromolecules that link platelets following vessel injury. Ann NY Acad Sci 1987; 509: 131-41.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 7 Handa M, Watanabe K, Kawai Y. et al. Platelet unresponsiveness to collagen: involvement of glycoprotein Ia-IIa (α2β1 integrin) deficiency associated witha myeloproliferative disorder. Thromb Haemost 1995; 73: 521-8.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 18 Neiswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102: 449-61.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 25 Qian MD, Villeval J-L, Xiong X. et al. Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from combinatorial phage display library. Human Antibodies 2002; 11: 97-105.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Search in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 28 Siljander PRM, Munnix ICA, Smethurst PA. et al. Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood. Blood 2004; 103: 1333-41.
    PubMedSearch in Google Scholar
  • 29 Matsumoto Y, Takizawa H, Gong X. et al. Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization. Thromb Res. 2006 Epub ahead of print.
    PubMedSearch in Google Scholar
  • 30 Morton LF, Hargreaves PG, Farndale RW. et al. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity. Biochem J 1995; 306: 337-44.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 33 Clemetson KJ, Clemetson JM. Platelet collagen receptors. Thromb Haemost 2001; 86: 189-97.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 35 Kahn ML, Zheng YW, Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
    CrossrefPubMedSearch in Google Scholar
  • 36 Marchese P, Kanaji T, Wagner DD. et al. Elevated threshold shear rate for the dependence of glycoprotein Ibα-mediated platelet thrombus formation onto immobilized von Willebrand factor in mouse blood. Blood 2004; 104: 996a.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 38 Buijs WC, Tibben JG, Boerman OC. et al. Dosimetric analysis of chimeric monoclonal antibody cMOv18 IgG in ovarian carcinoma patients after intraperitoneal and intravenous administration. Eur J Nucl Med 1998; 25: 1552-61.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar