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Thromb Haemost 1995; 74(02): 655-659
DOI: 10.1055/s-0038-1649793
Original Article
Coagulation
Schattauer GmbH Stuttgart

Characterisation of a Novel Series of Aprotinin-derived Anticoagulants

II. Comparative Antithrombotic Effects on Primary Thrombus Formation In Vivo
Jean Marie Stassen
1   The Department of Orthopaedics and Hand Surgery, University of Umeå, Sweden
2   The Center for Molecular and Vascular Biology, University of Leuven, Belgium
,
Anne-Marie Lambeir
3   The Corvas International, Gent, Belgium
,
Ingrid Vreys
2   The Center for Molecular and Vascular Biology, University of Leuven, Belgium
,
Hans Deckmyn
2   The Center for Molecular and Vascular Biology, University of Leuven, Belgium
,
Gaston Matthyssens
3   The Corvas International, Gent, Belgium
,
Åke Nyström
1   The Department of Orthopaedics and Hand Surgery, University of Umeå, Sweden
,
Jos Vermylen
2   The Center for Molecular and Vascular Biology, University of Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 03 February 1995

Accepted after resubmission 24 March 1995

Publication Date:
06 July 2018 (online)

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Summary

Upon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters.

Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model.

It is concluded that acceptable interference at the initial level of the blood coagulation, e. g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.

 

  • References

  • 1 Stassen JM, Lambeir AM, Mathyssens G, Ripka WC, Nyström A, Sixma JJ, Vermylen J. Characterisation of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties Thromb Haemost 1995; 74: 646-654
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Friedman M, Van den Bovenkamp GJ. The pathogenesis of a coronary thrombus. Am J Pathol 1966; 48: 19-44
    PubMedGoogle Scholar
  • 3 Davies MJ, Thomas AC. Plaque fissuring: The cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J 1985; 53: 363-373
    CrossrefPubMedGoogle Scholar
  • 4 Fuster V, Chesebro JH. Mechanism of unstable angina. N Engl J Med 1986; 315: 1023-1025
    CrossrefPubMedGoogle Scholar
  • 5 Folts JD, Crowell EB, Rowe GG. Platelet aggregation in partially obstructed vessels and its elimination with aspirin. Circulation 1976; 54: 365-370
    CrossrefPubMedGoogle Scholar
  • 6 Willerson JT, Hillis DL, Winniford M, Buja LM. Speculation regarding mechanisms responsible lor acute ischemic disease syndromes. J Am Coll Cardiol 1986; 8: 245-250
    CrossrefPubMedGoogle Scholar
  • 7 Gold HK, Coller BS, Vasuda T, ballon JT, Guerrero JL, Leinbaeh LC, Ziskind AA, Collen D. Rapid and sustained coronary artery rceanalization with combined bolus injection of recombinant tissue-type plasminogen activator and a monoclonal antiplatelet GPIIb/llla antibody in a canine preparation. Circulation 1988; 77: 670-677
    CrossrefPubMedGoogle Scholar
  • 8 Chiang TM, Kang AH. Isolation and purification of collagen alpha 1(1) receptor from human platelet membrane. J Biol chem 1982; 257: 7581-7586
    PubMedGoogle Scholar
  • 9 Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-von Willcbrand factor bound to subendothelium. Nature 1978; 279: 636-638
    PubMedGoogle Scholar
  • 10 Zwaal RF A. Membrane and lipid involvement in blood coagulation. Biochim Biophys Acta 1978; 515: 163-205
    CrossrefPubMedGoogle Scholar
  • 11 Osterud B, Rapaport SL. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-5264
    CrossrefPubMedGoogle Scholar
  • 12 Zur M, Nemerson Y. The esterase activity of coagulation factor VII. Evidence for intrinsic activity of the zymogen J Biol Chem 1978; 253: 2203-2209
    PubMedGoogle Scholar
  • 13 Bach RR. Initiation of coagulation by tissue factor. CRC Crit Rev Biochcm 1988; 23: 339-368
    PubMedGoogle Scholar
  • 14 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues Implications for disorders of hemostasis and thrombosis. Am J Pathol 1989; 134: 1087-1097
    PubMedGoogle Scholar
  • 15 Zwaal RF A, Bevers EM, Comfurius P. Platelets and coagulation. In: Blood coagulation. Zwaal RF A, Hcmker HC. (eds) New York: Elsevier Science Publishers; 1986. pp 141-169
    Google Scholar
  • 16 Mann KG, Nesheim ME, Fraey PB. Non enzymatic cofactors: Factor V. In: Blood Coagulation. Zwaal RF A, Hemker HC. (eds) New York: Elsevier Science Publishers; 1993. pp 15-32
    Google Scholar
  • 17 Scully MF. Kinetic considerations on the physiologic control of thrombin and factor Xa. Semin Thromb Haemost 1992; 18: 218-223
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Lankiewicz MW, Bell WR. A unique circulating inhibitor with specificity for coagulation factor X. Am J Med 1992; 93: 343-346
    CrossrefPubMedGoogle Scholar
  • 19 Imura Y, Stassen JM, Collen D. Comparative antithrombotic effects of heparin, recombinant hirudin and argatroban in a hamster femoral vein platelet-rich mural thrombosis model. J Pharmacol Exp Thcr 1992; 261: 895-898
    PubMedGoogle Scholar
  • 20 Zoldhelyi P, Fuster V, Chesebro JH. Antithrombins as conjunctive therapy in arterial thrombolysis. Cor Art Dis 1992; 3: 1003-1009
    CrossrefPubMedGoogle Scholar
  • 21 Theroux P, Latour JP. Anticoagulants and their use in acute ischemic syndrome. In: Textbook of interventional cardiology. Topol EJ. (ed) Philadelphia: W. B. Saunders; 1990. pp 24-48
    Google Scholar
  • 22 Rapold HJ, Lu HR, Wu ZM, Nijs H, Collen D. Requirement of heparin for arterial and venous thrombolysis with recombinant tissue-type plasminogen activator. Blood 1991; 77: 1020-1024
    PubMedGoogle Scholar
  • 23 Hirsh J. Heparin and low-molecular weight heparins. Cor Art Dis 1992; 3: 990-1002
    CrossrefPubMedGoogle Scholar
  • 24 Stassen JM, Rapold HJ, Vanlinthout I, Collen D. Comparative effect of enoxaparin and heparin on arterial and venous clot lysis with alteplase in dogs. Thromb Haemost 1993; 69: 454-459
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Schaffer LW, Davidson JT, Vlasuk GP, Siegl PK. Antithrombotic efficacy of recombinant tick anticoagulant peptide. A potent inhibitor of coagulation factor Xa in a primate model of arterial thrombosis Circulation 1991; 84: 1741-1748
    CrossrefPubMedGoogle Scholar
  • 26 Markwardt F. Development of hirudin as an antithrombotic agent. Semin Thromb Haemost 1989; 15: 269-282
    Article in Thieme ConnectPubMedGoogle Scholar
  • 27 Neeper MP, Waxman L, Smith DE, Schulman CA, Sardana M, Ellis RW, Schaffer LW, Siegl PK, Vlasuk GP. Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa J Biol Chem 1990; 265: 17746-17752
    PubMedGoogle Scholar
  • 28 Broze GJ. Tissue factor pathway inhibitor and the revised hypothesis of blood coagulation. Trends Cardiovasc Med 1992; 2: 72-77
    CrossrefPubMedGoogle Scholar
  • 29 Eisenberg PR. Development of novel anticoagulants for use as conjunctive therapy for thrombolysis. Cor Art Dis 1992; 3: 1010-1015
    CrossrefPubMedGoogle Scholar
  • 30 Eidt JF, Allison P, Noble S, Ashton J, Golino P, McNatt J, Buja LM, Willerson JT. Thrombin is an important mediator of platelet aggregation in stenosal canine coronary arteries with endothelial injury. J Clin Invest 1989; 84: 18-27
    CrossrefPubMedGoogle Scholar
  • 31 Heras M, Chesebro JH, Penny WJ, Bailey KR, Badimon E, Fuster V. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Heparin versus recombinant hirudin, a specific thrombin inhibitor Circulation 1989; 79: 647-665
    PubMedGoogle Scholar
  • 32 Sitko GR, Ramjit DR, Stabilito H, Lehman D, Lynch JJ, Vlasuk GP. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide. Comparison to hirudin and heparin in a canine model of acute coronary artery thrombosis Circulation 1992; 85: 805-815
    CrossrefPubMedGoogle Scholar
  • 33 De Macycr M, Lambeir AM, Laroche Y, Lasters I, Lauwereys M, Mathyssens G, Stanssens P, Ripka W, Vlasuk G. Recombinant Kiinitz inhibitors of coagulation proteases. I. Site specific mutagenesis. Thromb Haemost 1993; 69: 888 (abstract 1245)
    PubMedGoogle Scholar
  • 34 De Maeyer M, Lambeir AM, Laroche Y, Lasters I, Lauwereys M, Mathyssens G, Stanssens P, Ripka W, Vlasuk G. Recombinant Kiinitz inhibitors of coagulation proteases. II. Random mutagenesis Thromb Haemost 1993; 69: 889 (abstract 1250)
    PubMedGoogle Scholar
  • 35 Stanssens P, De Maeyer M, Lambeir AM, Laroche Y, Lauwereys M, Lasters I, Ripka WC. Design of inhibitors of serine proteases: Use of site selective and random mutagenesis to define the pharmacophore and conformational selectivity of Kunitz inhibitors towards factors Xa and VIIa. Submitted
    PubMedGoogle Scholar
  • 36 Stockmans F, Deckmyn H, Gruwez J, Vcrmylen J, Acland R. Continuous quantitative monitoring of mural, platelet-dependent, thrombus kinetics in the crushed rat femoral vein. Thromb Haemost 1991; 65: 425-431
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Imura Y, Stassen JM, Bunting S, Stockmans F, Collen D. Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in a hamster platelet-rich femoral vein thrombosis model. Blood 1992; 80: 1247-1253
    PubMedGoogle Scholar
  • 38 Laroche Y, Strome V, De Meutter J, Messens J, Lauwereys M. High-level secretion and very efficient isotopic labeling of thick anticoagulant peptide (TAP) expressed in the methlyotrophic yeast, Pischia pastoris. Bio/Technology 1994; 2: 1119-1124
    PubMedGoogle Scholar
  • 39 Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost 1987; 58: 1078-1084
    Article in Thieme ConnectPubMedGoogle Scholar
  • 40 Agnelli G, Pascucci C, Cosmi B, Nenci GG. Effects of hirudin and heparin on the binding of new fibrin to the thrombus in t-PA treated rabbits. Thromb Haemost 1991; 66: 592-597
    Article in Thieme ConnectPubMedGoogle Scholar
  • 41 Agnelli G, Renga C, Weitz JI, Nenci GG, Hirsh J. Sustained antithrombotic activity of hirudin after its plasma clearance: comparison with heparin. Blood 1992; 80: 960-965
    PubMedGoogle Scholar
  • 42 Zoldhelyi P, Webster MW, Fuster V, Grill DE, Gaspar D, Edwards SJ, Cabot CF, Chesebro JH. Recombinant hirudin in patients with chronic, stable coronary artery disease. Safety, half-life, and effect on coagulation parameters. Circulation 1993; 88: 2015-2022
    CrossrefPubMedGoogle Scholar
  • 43 Cannon CP, Maraganore JM, Loscalzo J, McAllister A, Eddings K, George D, Selwyn AP, Adelman B, Fox I, Braunwald E, Ganz P. Anticoagulant effects of hirulog, a novel thrombin inhibitor, in patients with coronary artery disease. Am J Cardiol 1993; 71: 778-782
    CrossrefPubMedGoogle Scholar
  • 44 Topol EJ, Bonan R, Jewitt D, Sigwart U, Kakkar VV, Rothman M, de Bono D, Ferguson J, Willerson JT, Strony J, Ganz P, Chen MD, Raymond R, Fox I, Maraganore JM, Adelman B. Use of a direct antithrombin, hirulog, in place of heparin during coronary angioplasty. Circulation 1993; 87: 1622-1629
    CrossrefPubMedGoogle Scholar
  • 45 Vlasuk GP, Ramjit D, Fujita T, Dunwiddie CT, Nutt EM, Smith DE, She-buski RJ. Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis. Thromb Haemost 1991; 65: 257-262
    Article in Thieme ConnectPubMedGoogle Scholar