Download PDF
Thromb Haemost 1998; 80(05): 840-844
DOI: 10.1055/s-0037-1615368
Review Article
Schattauer GmbH

Platelet Aggregation Induced In Vitro by Rabbit Plasma Clot-associated Thrombin, and Its Inhibition by Thrombin Inhibitors

Eric Gandossi
1   From the Thrombosis and Haematology Department, Cardiovascular Research, Synthélabo Recherche, Chilly Mazarin, France
,
Catherine Lunven
1   From the Thrombosis and Haematology Department, Cardiovascular Research, Synthélabo Recherche, Chilly Mazarin, France
,
Christiane Gauffeny
2   From the Pathology Group, Drug Safety Dept., Synthélabo Recherche, Gargenville, France
,
Nigel O. Roome
2   From the Pathology Group, Drug Safety Dept., Synthélabo Recherche, Gargenville, France
,
Christopher N. Berry
1   From the Thrombosis and Haematology Department, Cardiovascular Research, Synthélabo Recherche, Chilly Mazarin, France
› Author Affiliations
Further Information

Publication History

Received 18 February 1998

Accepted after resubmission 24 July 1998

Publication Date:
07 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied.

Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50 of 14 ± 3 nM compared to an IC50 of 12 ± 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 ± 0.4 nM compared to 1.6 ± 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50 = 69 ± 9 mU/ml) than against thrombin in solution (IC50 = 15 ± 5 mU/ml).

This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.

 

  • References

  • 1 Francis CW, Markham REJ, Barlow GH, Florack TM, Dobrzynski DM, Marder VJ. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med 1983; 102: 220-30.
    PubMedSearch in Google Scholar
  • 2 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by antithrombin III, but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 72: 381-6.
    PubMedSearch in Google Scholar
  • 3 Berry CN, Girardot C, Lecoffre C, Lunven C. Effects of the synthetic thrombin inhibitor argatroban on fibrin- or clot-incorporated thrombin: Comparaison with heparin and recombinant hirudin. Thromb Haemost 1994; 72: 381-6.
    Thieme ConnectPubMedSearch in Google Scholar
  • 4 Kumar R, Béguin S, Hemker HC. The influence of fibrinogen and fibrin thrombin generation – Evidence for feedback activation of the clotting system by clot bound thrombin. Thromb Haemost 1994; 72: 713-21.
    Thieme ConnectPubMedSearch in Google Scholar
  • 5 Kumar R, Béguin S, Hemker HC. The effect of fibrin clots and clot-bound thrombin on the development of platelet procoagulant activity. Thromb Haemost 1995; 74: 962-8.
    Thieme ConnectPubMedSearch in Google Scholar
  • 6 Mirshahi M, Soria J, Soria C, Faivre R, Lu H, Courtney M, Roitsch C, Tri-pier D, Caen JP. Evaluation of the inhibition by heparin and hirudin of coagulation activation during r-tPA-induced thrombolysis. Blood 1989; 74: 1025-30.
    PubMedSearch in Google Scholar
  • 7 Rydel TJ, Ravichandran KG, Tulinsky A, Bode W, Huber R, Roitsch C, Fenton II. JW. The structure of a complex of recombinant hirudin and human -thrombin. Science 1990; 249: 277-80.
    CrossrefPubMedSearch in Google Scholar
  • 8 Grutter MG, Priestle JP, Grossenbacher H, Bode W, Hofsteenge J, Stone SR. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition. EMBO J 1990; 9: 2361-5.
    PubMedSearch in Google Scholar
  • 9 Kettner C, Shaw E. D-Phe-Pro-ArgCH2C1-A selective affinity label for thrombin. Thromb Res 1979; 14: 969-73.
    CrossrefPubMedSearch in Google Scholar
  • 10 Gast A, Tschopp TB, Hilpert G, Ackermann J. Inhibition of clot-bound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma. Blood Coagulation Fibrinolysis 1994; 5: 879-87.
    CrossrefPubMedSearch in Google Scholar
  • 11 Lunven C, Gauffeny C, Lecoffre C, O’Brien DP, Roome NO, Berry CN. Inhibition by argatroban, a specific thrombin inhibitor, of platelet activation by fibrin clot-associated thrombin. Thromb Haemost 1996; 75: 154-60.
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Arocas V, Zingali RB, Guillin MC, Bon C, Jandrot-Perrus M. Bothrojaracin: a potent two-site-directed thrombin inhibitor. Biochemistry 1996; 35: 9083-9.
    CrossrefPubMedSearch in Google Scholar
  • 13 Vargas JR, Radomski M, Moncada S. The use of prostaglandin in the separation from plasma and washing of human platelets. Prostaglandins 1982; 23: 929-45.
    CrossrefPubMedSearch in Google Scholar
  • 14 Gear ARL. In vitro platelet responses: aggregation. In Platelet Responses and Metabolism. Holmsen H (eds). Florida: CRC Press, Boca Raton; 1987. pp 97-114.
    Search in Google Scholar
  • 15 Broze Jr. GJ. The tissue factor pathway of coagulation: factor VII, tissue factor, and tissue factor pathway inhibitor. In: Haemostasis and Thrombosis. Blomm AL, Forbes CD, Thomas DP, Tuddenham EGD (eds). London: Churchill Livingstone; 1994. pp 349-77.
    Search in Google Scholar
  • 16 Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest 1993; 91: 1877-83.
    CrossrefPubMedSearch in Google Scholar
  • 17 Prager N-A, Abendschein D-R, McKenzie C-R, Eisenberg P-R. Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots. Circulation 1995; 92: 962-7.
    CrossrefPubMedSearch in Google Scholar
  • 18 McKenzie CR, Abendschein DR, Eisenberg PR. Sustained inhibition of whole-blood clot procoagulant activity by inhibition of thrombus-associated factor Xa. Arterioscler Thromb Vasc Biol 1996; 16: 1285-91.
    CrossrefPubMedSearch in Google Scholar
  • 19 Ginsberg MH, Hoskins R, Sigrist P, Painter RG. Purification of a heparin-neutralizing protein from rabbit platelets and its homology with human platelet factor 4. J Biol Chem 1979; 254: 12365-71.
    PubMedSearch in Google Scholar
  • 20 Nath N, Lowery CT, Niewiarowski S. Antigenic and antiheparin properties of human platelet factor 4 (PF4). Blood 1975; 45: 537-50.
    PubMedSearch in Google Scholar
  • 21 O’Brien JR, Etherington M, Jamieson S, Lawford P. Letter: Heparin thrombin clotting-time and platelet factor 4. Lancet 1974; 2: 656-7.
    PubMedSearch in Google Scholar
  • 22 Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, Ninomiya H, Hijikata A, Okamoto S. Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl) sulfonyl]-arginyl]-2-piperidinecarboxylic acid. Biochemistry 1984; 23: 85-90.
    CrossrefPubMedSearch in Google Scholar
  • 23 Banner DW, Hadvary P. Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors. J Biol Chem 1991; 266: 20085-93.
    PubMedSearch in Google Scholar
  • 24 Okamoto S, Hijikata A, Kikumoto R, Tonomara S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y. Potent Inhibition of Thrombin by the Newly Synthesized Arginine Derivative No. 805. The Importance of Stereo-Structure of its Hydrophobic Carboxamide Portion. Biochem Biophys Res Commun 1981; 101: 440-6.
    CrossrefPubMedSearch in Google Scholar
  • 25 Tapparelli C, Metternich R, Ehrhardt C, Zurini M, Claeson G, Scully MF, Stone SR. In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor. J Biol Chem 1993; 268: 4724-41.
    PubMedSearch in Google Scholar
  • 26 Jang IK, Gold HK, Ziskind AA, Leinbach RC, Fallon JT, Collen D. Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition. Circulation 1990; 81: 219-25.
    CrossrefPubMedSearch in Google Scholar
  • 27 Kaiser B, Simon A, Markwardt F. Antithrombotic effects of recombinant hirudin in experimental angioplasty and intravascular thrombolysis. Thromb Haemost 1990; 63: 44-7.
    Thieme ConnectPubMedSearch in Google Scholar
  • 28 Kelly AB, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson SR, Harker LA. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood 1991; 77: 1006-12.
    PubMedSearch in Google Scholar
  • 29 Berry CN, Girard D, Girardot C, Lochot S, Lunven C, Visconte C. Anti-thrombotic activity of argatroban in experimental thrombosis in the rabbit. Semin Thromb Hemost 1996; 22: 233-41.
    Thieme ConnectPubMedSearch in Google Scholar