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Thromb Haemost 2000; 83(02): 327-333
DOI: 10.1055/s-0037-1613807
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Schattauer GmbH

GPV Is a Marker of In Vivo Platelet Activation – Study in a Rat Thrombosis Model[*]

Catherine Ravanat
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Monique Freund
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Pierre Mangin
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
David O. Azorsa
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Catherine Schwartz
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Sylvie Moog
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Simone Schuhler
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Josiane Dambach
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
Jean-Pierre Cazenave
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
,
François Lanza
From INSERM U.311, Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France
› Author Affiliations
Further Information

Publication History

Received 16 June 1999

Accepted after revision 12 October 1999

Publication Date:
11 December 2017 (online)

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Summary

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life.

In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

Keywords

Rat GPV - thrombin generation - prethrombotic state - animal model - flow cytometry

* Part of this work was presented in preliminary form at the XVIth Congress of the International Society of Thrombosis and Haemostasis, Florence, June 6-12, 1997 and reproduced in abstract form in Thrombosis and Haemostasis, 7: 473, 1997


 

  • References

  • 1 Ware J. Molecular analyses of the platelet glycoprotein Ib-IX-V. Thromb Haemost 1998; 79: 466-78.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Jamieson GA. Pathophysiology of platelet thrombin receptors. Thromb Haemost 1997; 78: 242-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Mazzucato M, De Marco L, Masotti A, Pradella P, Bahou WF, Ruggeri ZM. Characterization of the initial α-thrombin interaction with glycoprotein Ibα in relation to platelet activation. J Biol Chem 1998; 23: 1880-7.
    PubMedGoogle Scholar
  • 4 Greco NJ, Tandon NN, Jones GD, Kornhauser R, Jackson B, Yamamoto N, Tanoue K, Jamieson GA. Contributions of glycoprotein Ib and the seven transmembrane domain receptor to increases cytoplasmic [Ca2+] induced by α-thrombin. Biochemistry 1996; 35: 906-14.
    CrossrefPubMedGoogle Scholar
  • 5 Vu TKH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
    CrossrefPubMedGoogle Scholar
  • 6 Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502-6.
    CrossrefPubMedGoogle Scholar
  • 7 Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese Jr RV, Tram C, Coughlin SR. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
    CrossrefPubMedGoogle Scholar
  • 8 Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-887.
    CrossrefPubMedGoogle Scholar
  • 9 Connolly TM, Condra C, Feng DM, Cook JJ, Stranieri MT, Reilly CF, Nutt RF, Gould RJ. Species variability in platelet and other cellular responsiveness to thrombin receptor-derived peptides. Thromb Haemost 1994; 72: 627-33.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Kinlough-Rathbone RL, Rand ML, Packham MA. Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets. Blood 1993; 82: 103-6.
    PubMedGoogle Scholar
  • 11 Cook NS, Zerwes HG, Tapparelli C, Powling M, Singh J, Metternich R, Hagenbach A. Platelet aggregation and fibrinogen binding in human, Rhesus monkey, guinea pig, hamster and rat blood: activation by ADP and a thrombin receptor peptide and inhibition by glycoprotein IIb/IIIa antagonists. Thromb Haemost 1993; 70: 531-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 Connolly AJ, Ishihara H, Kahn ML, Farese Jr RV, Coughlin SR. Role of the thrombin receptor in development and evidence for a second receptor. Nature 1996; 381: 516-9.
    CrossrefPubMedGoogle Scholar
  • 13 Berndt MC, Phillips DR. Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V. J Biol Chem 1981; 256: 59-65.
    PubMedGoogle Scholar
  • 14 Bienz D, Shnippering W, Clemetson KJ. Glycoprotein V is not the thrombin activation receptor on human platelets. Blood 1986; 68: 720-5.
    PubMedGoogle Scholar
  • 15 Dong JF, Sae-Tung G, Lopez JA. Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site. Blood 1997; 89: 4355-63.
    PubMedGoogle Scholar
  • 16 Modderman PW, Admiraal LG, Sonnenberg A, von dem AEGBorne. Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J Biol Chem 1992; 267: 364-69.
    PubMedGoogle Scholar
  • 17 Takafuta T, Fujimura K, Kawano H, Noda M, Fujimoto T, Oda K, Shimomura T, Kuramoto A. Expression of platelet membrane glycoprotein V in human megakaryocytes and megakaryocytic cell lines: a study using a novel monoclonal antibody against GPV. Thromb Haemost 1994; 72: 762-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Lanza F, Morales M, de La CSalle, Cazenave JP, Clemetson KJ, Shimomura T, Phillips DR. Cloning and characterization of the gene encoding the human platelet GPV. J Biol Chem 1993; 270: 20801-7.
    PubMedGoogle Scholar
  • 19 Ravanat C, Morales M, Azorsa DO, Moog S, Schuhler S, Grunert P, Loew D, Van Dorsselaer A, Cazenave JP, Lanza F. Gene cloning of rat and mouse platelet Glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage. Blood 1997; 89: 3253-62.
    PubMedGoogle Scholar
  • 20 Azorsa DO, Moog S, Ravanat C, Schuhler S, Folléa G, Cazenave JP, Lanza F. Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA – Its use to follow platelet storage in transfusion. Thromb Haemost 1999; 81: 131-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Katsutani S, Fujimoto TT, Noda M, Shimomura T, Takafuta T, Kimura A, Fujimura K. Cloning and characterization of the gene encoding the murine glycoprotein V: The conserved thrombin-cleaved protein on platelet surface. Thromb Res 1998; 92: 43-51.
    CrossrefPubMedGoogle Scholar
  • 22 McGowan EB, Ding A, Detwiler TC. Correlation of thrombin-induced Glycoprotein V hydrolysis and platelet activation. J Biol Chem 1983; 258: 11243-8.
    PubMedGoogle Scholar
  • 23 Wicki AN, Clemetson KJ. Structure and function of platelet membrane GPIb and V Effects of leukocyte elastase and other proteases on platelet response to von Willebrand factor and thrombin. Eur J Biochem 1985; 153: 1-11.
    CrossrefPubMedGoogle Scholar
  • 24 Michelson AD, Benoit SE, Furman MI, Barnard MR, Nurden P, Nurden AT. The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: Proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system. Blood 1996; 87: 1396-408.
    PubMedGoogle Scholar
  • 25 Ravanat C, Freund M, Schuhler S, Grunert P, Meyer L, Cazenave JP. Species specific immunoassays to measure blood platelet and coagulation activation in the rat. Thromb Haemost 1996; 76: 1090-5.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Meth 1987; 96: 271-8.
    CrossrefPubMedGoogle Scholar
  • 27 Ravanat C, Freund M, Dol F, Cadroy Y, Roussi J, Incardona F, Maffrand JP, Boneu B, Drouet L, Legrand C, Herbert JM, Cazenave JP. Crossreactivity of human molecular markers for detection of prethrombotic states in various animal species. Blood Coag Fibrinol 1995; 06: 446-55.
    CrossrefPubMedGoogle Scholar
  • 28 Cazenave JP, Hemmendinger S, Beretz A, Sutter-Bay A, Launay J. L’agrégation plaquettaire: outil d’investigation clinique et d’étude pharmacologique Méthodologie. Ann Biol Clin 1983; 41: 167-79.
    PubMedGoogle Scholar
  • 29 Michelson AD. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical applications. Blood Coag Fibrinol 1994; 05: 121-31.
    CrossrefPubMedGoogle Scholar
  • 30 Fredrich M, Zierz R, Morser J, Wydro R, Witt W. A new animal model of venous thrombosis in rats with low flow conditions in the venous blood stream. Blood Coag Fibrinol 1994; 05: 243-8.
    CrossrefPubMedGoogle Scholar
  • 31 Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, Tolstoshev P, Lecoq JP. Cloning and expression of a cDNA coding for the anticoagulant hirudin from bloodsucking leech, Hirudo medicinalis . Proc Natl Acad Sci USA 1986; 83: 1084-8.
    CrossrefPubMedGoogle Scholar
  • 32 Wada H, Sakuragawa N, Mori Y, Takagi M, Nakasaki T, Shimura M, Hiyoyama K, Nisikawa M, Gabazza EC, Deguchi K, Kazama M, Shiku H. Hemostatic molecular markers before the onset of disseminated intravascular coagulation. Am J Hematol 1999; 60: 273-8.
    CrossrefPubMedGoogle Scholar
  • 33 Bick RL. Disseminated intravascular coagulation: pathophysiological mechanisms and manifestations. Semin Thromb Hemost 1998; 24: 3-18.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 34 Cox D. Methods for monitoring platelet function. Am Heart J 1998; 135: S160-9.
    CrossrefPubMedGoogle Scholar
  • 35 Zucker MB, Katz IR. Platelet factor 4: production, structure and physiologic and immunologic action. Proc Soc Exp Biol Med 1991; 198: 693-702.
    CrossrefPubMedGoogle Scholar
  • 36 Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, Valeri CR. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA 1996; 93: 11877-82.
    CrossrefPubMedGoogle Scholar
  • 37 Berger G, Hartwell DW, Wagner DD. P-selectin and platelet clearance. Blood 1998; 92: 4446-52.
    PubMedGoogle Scholar
  • 38 Files JC, Malpass TW, Yee EK, Ritchie JL, Harker LA. Studies of human platelet α-granule release in vivo . Blood 1981; 58: 607-18.
    PubMedGoogle Scholar
  • 39 Ikeda H, Nakayama H, Oda T, Kuwano K, Muraishi A, Sugi K, Koga Y, Toshima H. Soluble form of P-selectin in patients with acute myocardial infarction. Coron Artery Dis 1994; 05: 515-8.
    PubMedGoogle Scholar