Download PDF
Thromb Haemost 1987; 58(03): 915-920
DOI: 10.1055/s-0038-1646015
Original Article
Schattauer GmbH Stuttgart

Human Gamma-Thrombin: Lack of Correlation Between a Platelet Functional Response and Glycoprotein V Hydrolysis

Martine Jandrot-Perrus
The Unité 150 INSERM and Unité Associée 334 CNRS, Hôpital Lariboisiére, Paris, and Laboratoire de Recherche sur I’Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France
,
Marie-Claude Guillin
The Unité 150 INSERM and Unité Associée 334 CNRS, Hôpital Lariboisiére, Paris, and Laboratoire de Recherche sur I’Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France
,
Alan T Nurden
The Unité 150 INSERM and Unité Associée 334 CNRS, Hôpital Lariboisiére, Paris, and Laboratoire de Recherche sur I’Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France
› Author Affiliations
Further Information

Publication History

Received 23 April 1987

Accepted after revision 11 June 1987

Publication Date:
28 June 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

The ability of purified human gamma-thrombin to stimulate platelet function was related to its capacity to degrade GP V. Compared to alpha-thrombin, much greater amounts of gamma- thrombin were required to induce platelet aggregation; and this also applied to secretion from dense bodies, alpha-granules and lysosomal granules. Platelet stimulation by gamma-thrombin was additionally characterized by the presence of a lag-phase. Platelets with 3H-labelled surface glycoproteins showed the same functional response to both alpha- and gamma-thrombin as unlabelled platelets. But while threshold levels of alpha-thrombin induced little GP V hydrolysis confirming McGowan et al. (1), amounts of gamma-thrombin which induced substantial degradation (e. g. 8.3 nM degraded 36% of platelet GP V in 3 min) were unable to sustain either platelet aggregation or secretion. These results suggest that protein-binding regions remote from the catalytic site of alpha-thrombin are more important for platelet activation than GP V hydrolysis. They also provide further support to the argument that GP V hydrolysis may not be the essential trigger of platelet activation by thrombin.

Keywords

Platelet aggregation - Platelet secretion - GP V hydrolysis - Human alpha-thrombin - Human gamma-thrombin
 

  • References

  • 1 McGowan EB, Ding AH, Detwiler TC. Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation. J Biol Chem 1983; 258: 11243-11248
    PubMedGoogle Scholar
  • 2 Tollefsen DM, Feagler JR, Majerus PW. The binding of thrombin to the surface of human platelets. J Biol Chem 1974; 249: 2646-2651
    PubMedGoogle Scholar
  • 3 Martin BM, Wasiewsky WW, Fenton JW, Detwiler TC. Equilibrium binding of thrombin to platelets. Biochemistry 1976; 15: 4886-4893
    CrossrefPubMedGoogle Scholar
  • 4 Harmon TJ, Jamieson GA. Thrombin binds to a high-affinity 900,000 dalton site on human platelets. Biochemistry 1985; 24: 58-64
    CrossrefPubMedGoogle Scholar
  • 5 Phillips DR, Agin PP. Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin. Biochim Bio-phys Res Comm 1977; 75: 940-947
    CrossrefPubMedGoogle Scholar
  • 6 Mosher DF, Vaheri A, Choate JJ, Gahmberg CG. Action of thrombin on surface glycoproteins of human platelets. Blood 1979; 53: 437-445
    PubMedGoogle Scholar
  • 7 Fenton JW, Landis BH, Walz DA, Finlavson JS. Human thrombins. In: Chemistry and Biology of Thrombin. Lundblad RL, Fenton JW, Mann KG. (eds). Ann Arbor Science Publishers; Ann Arbor, Mi: 1977: 43-70
    Google Scholar
  • 8 Fenton JW, Landis BH, Walz DA, Bing DH, Feinman RD, Zabinski MP, Sonder SA, Berliner LJ, Finlayson JL. Human thrombin: preparative evaluation, structural properties enzymic specificity. In: Chemistry of the human plasma proteins. Bing DH. (ed). Pergamon Press; 1979: 151-183
    Google Scholar
  • 9 Lorand L, Credo RB. Thrombin and fibrin stabilization. In: Chemistry and Biology of Thrombin. Lundblad RL, Fenton JW, Mann KG. (eds). Ann Arbor Science Publishers; Ann Arbor, Mi: 1977: 311-323
    Google Scholar
  • 10 Chang TL, Feinman RD, Landis BH, Fenton JW. Anti-thrombin reaction with alpha- and gamma-thrombins. Biochemistry 1979; 18: 113-119
    CrossrefPubMedGoogle Scholar
  • 11 Seegcrs WH, Hassoune HI, Hewctt-Emmet D, Walz DA, Andary TJ. Prothrombin and thrombin. Selected aspects of thrombin formation, properties, inhibition and immunology. Semin Thromb Hemost 1975; 1: 221-283
    PubMedGoogle Scholar
  • 12 Fasco MJ, Fenton JW. Human alpha-, beta(T)-, gamma(T)- thrombins. Fed Proc 1975; 34: 3555
    PubMedGoogle Scholar
  • 13 Takahashi K, Aiken M, Fenton JW, Walz DA. Thrombospondin fragmentation by alpha-thrombin and resistance to gamma-thrombin. Biochcm J 1984; 224: 673-676
    PubMedGoogle Scholar
  • 14 Fazal-Mohammed S, Whitworth C, Chuang H YK, Lundblad RL, Mason RG. Multiple active forms of thrombin: binding to platelets and effects on platelet function. Proc Natl Acad Sci USA 1976; 73: 1660-1663
    CrossrefPubMedGoogle Scholar
  • 15 Tam SW, Fenton JW, Detwiler TC. Platelet thrombin receptors. Binding of alpha-thrombin is coupled to signal generation by a chymotrypsin sensitive mechanism. J Biol Chem 1980; 255: 6626-6632
    PubMedGoogle Scholar
  • 16 Bezeaud A, Denninger MH, Guillin MC. Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III. protein C and thrombomodulin. Eur J Biochem 1985; 153: 491-496
    CrossrefPubMedGoogle Scholar
  • 17 Chase T, Shaw E. P’Nitrophenvl-p-guanidinobenzoate HCI: a new active site titrant for trypsin. Biochem Biophys Res Commun 1967; 29: 508-514
    CrossrefPubMedGoogle Scholar
  • 18 Elion J, Boissel JP, Le Bonniec B, Bezeaud A, Jandrot-Perrus M, Guillin MC. Proteolytic derivatives of thrombin. Ann N Y Acad Sci 1986; 485: 16-26
    CrossrefPubMedGoogle Scholar
  • 19 Boissel JP, Le Bonniec B, Rabiet MJ, Labie D, Elion J. Covalent structure of beta and gamma autolytic derivatives of human alpha- thrombin. J Biol Chem 1984; 259: 5691-5697
    PubMedGoogle Scholar
  • 20 Legrand C, Dubernard V, Nurden AT. Characteristics of collagen-induced fibrinogen binding to human platelets. Biochem Biophys Acta 1985; 812: 802-810
    CrossrefPubMedGoogle Scholar
  • 21 Patscheke H. Shape and functional properties of human platelets washed with acid citrate. Haemostasis 1981; 10: 14-27
    PubMedGoogle Scholar
  • 22 Brown TR, Ho T TS, Walz DA. Improved radioimmunoassay of platelet factor 4 and beta thromboglobulin in plasma. Clin Chim Acta 1980; 101: 225-233
    CrossrefPubMedGoogle Scholar
  • 23 Dangelmaier CA, Holmsen H. Determination of acid hydrolases in human platelets. Analyt Biochem 1980; 104: 182-191
    CrossrefPubMedGoogle Scholar
  • 24 Hatton M WC, Regoeczi E. The proteolytic nature of commercial samples of galactose oxidase. Purification of the enzyme by a simple affinity method. Biochim Biophys Acta 1976; 438: 339-346
    CrossrefPubMedGoogle Scholar
  • 25 Nurden AT, Dupuis D, Kunicki TJ, Caen JP. Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional gel electrophoresis. J Clin Invest 1981; 67: 1431-1440
    CrossrefPubMedGoogle Scholar
  • 26 Bonner WA, Laskey RA. A film detection method for tritium- labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 1974; 46: 83-88
    CrossrefPubMedGoogle Scholar
  • 27 Nurden AT, Dupuis D, Pidard D, Kieffer N, Kunicki TJ, Cartron JP. Surface modifications in the platelets of a patient with beta-N- acetyl-D-galactosamine residues, the Tn-syndrome. J Clin Invest 1982; 70: 1281-1291
    CrossrefPubMedGoogle Scholar
  • 28 Berndt MC, Phillips DR. Purification and preliminary characterization of human platelet glycoprotein V. J Biol Chem 1981; 256: 59-65
    PubMedGoogle Scholar
  • 29 Phillips DR, Agin PP. Platelet plasma membrane glycoproteins - Evidence for the presence of nonequivalent disulfide bonds using non reduced/reduced two dimensional gel electrophoresis. J Biol Chem 1977; 252: 2121-2126
    PubMedGoogle Scholar
  • 30 Holmsen H, Dangelmaier CA, Holmsen HK. Thrombin-induced platelet responses differ in requirement tor receptor occupancy. Evidence for a tight coupling of occupancy and compartimentalized phosphatidic acid formation. J Biol Chem 1981; 256: 9393-9396
    PubMedGoogle Scholar
  • 31 Alexander RJ, Fenton JW, Detwiler TC. Thrombin platelet interactions: an assessment of the roles of saturable and non saturable binding in platelet activation. Arch Biochem Biophys 1983; 222: 266-275
    CrossrefPubMedGoogle Scholar
  • 32 Okumura T, Hasitz M, Jamieson GA. Platelet glycocalicin. Interaction with thrombin and role as thrombin receptor of the platelet surface. J Biol Chem 1978; 253: 3435-3443
    PubMedGoogle Scholar
  • 33 Yamamoto K, Yamamoto N, Kitagawa H, Tanque K, Kosaki G, Yamazaki H. Localization of a thrombin-binding site on human platelet membrane glycoprotein lb determined by a monoclonal antibody. Thromb Haemostas 1986; 55: 162-167
    PubMedGoogle Scholar
  • 34 White GC, Knupp CL. Glycoprotein V hydrolysis by thrombin: lack of correlation with secretion. Thromb Res 1985; 38: 641-648
    CrossrefPubMedGoogle Scholar
  • 35 Knupp CL, White GC. Effect of active-site modified thrombin on the hydrolysis of platelet-associated glycoprotein V by native thrombin. Blood 1985; 65: 578-583
    PubMedGoogle Scholar
  • 36 Bienz D, Schnippering W, Clemetson KJ. Glycoprotein V is not the thrombin activation receptor on human blood platelets. Blood 1986; 68: 266-275
    PubMedGoogle Scholar
  • 37 Bauer RS, Chang TL, Berliner LJ. Stability differences between high coagulant (alpha) and non coagulant (gamma) human thrombins. J Biol Chem 1980; 255: 5900-5903
    PubMedGoogle Scholar
  • 38 Berliner LJ. Structure-function relationship in human alpha- and gamma-thrombin. In: Molecular and Cellular Biochemistry. Martinus Nijhoff Publishers; Boston: 1984. 34 159-172
    Google Scholar
  • 39 Furie B, Bing DH, Feldmann RJ, Robison DJ, Burnier JP, Furie BC. Computer-generated models of hlnnd coagulation factor Xa, factor IX a and thrombin based upon structural homology with other serine proteases. J Biol Chem 1982; 257: 3875-3882
    PubMedGoogle Scholar
  • 40 Villanueva GB. Conformational differences between high clotting human alpha-thrombin and non clotting gamma-thrombin. Biochemistry 1981; 20: 6519-6525
    CrossrefPubMedGoogle Scholar