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Thromb Haemost 1993; 69(05): 515-521
DOI: 10.1055/s-0038-1651643
Original Article
Thrombosis Model
Schattauer GmbH Stuttgart

Experimental Thrombosis on a Collagen Coated Arterioarterial Shunt in Rats: A Pharmacological Model to Study Antithrombotic Agents inhibiting Thrombin Formation and Platelet Deposition

Monique Freund
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Françoise Mantz
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Philippe Nicolini
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Christian Gachet
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Juliette Mulvihill
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Laurent Meyer
INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Alain Beretz
1   CNRS URA 600, Laboratoire de Pharmacologie Moléculaire et Cellulaire, Université Louis Pasteur, Faculté de Pharmacie, IIIkirch-Graffenstaden, France
,
Jean-Pierre Cazenave
1   CNRS URA 600, Laboratoire de Pharmacologie Moléculaire et Cellulaire, Université Louis Pasteur, Faculté de Pharmacie, IIIkirch-Graffenstaden, France
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Publikationsverlauf

Received 14. Juli 1992

Accepted after revision 14. Januar 1993

Publikationsdatum:
25. Juli 2018 (online)

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Summary

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 ± 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min. We conclude that this experimental arterioarterial thrombosis model is sensitive to both antiplatelet and anticoagulant agents, allowing discrimination between antiplatelet and anticoagulant mechanisms of thrombosis prevention. The technique is easily performed in a small animal, fast and reproducible, and thus comparable to primate models for use in the screening of new antithrombotic drugs.

 

  • References

  • 1 Henry RL. Methods for inducing experimental thrombosis. Angiology 1962; 13: 554-577
    CrossrefPubMedGoogle Scholar
  • 2 Didisheim P. Animal models useful in the study of thrombosis and antithrombotic agents. Prog Hemostas Thromb 1972; 1: 165-196
    PubMedGoogle Scholar
  • 3 Hladovec J. Experimental arterial thrombosis in rats with continuous registration. Thromb Diath Haemorrh 1971; 26: 407-410
    PubMedGoogle Scholar
  • 4 Philp RB, Francey YI, Warren BA. Comparison of antithrombotic activity of heparin, ASA, sulfinpyrazone and VK 744 in a rat model of arterial thrombosis. Haemostasis 1978; 7: 282-293
    PubMedGoogle Scholar
  • 5 Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990; 60: 269-280
    CrossrefPubMedGoogle Scholar
  • 6 Heras M, Chesebro JH, Penny WJ, Bailey KR, Badimon L, Fuster V. Effects of thrombin inhibition on the development of acute plateletthrombus deposition during angioplasty in pigs. Heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation 1989; 3: 657-665
    PubMedGoogle Scholar
  • 7 Lam JYT, Chesebro JH, Steele PM, Heras M, Webster MWI, Badimon L, Fuster V. Antithrombotic therapy for deep arterial injury by angioplasty – Efficacy of common platelet inhibition compared with thrombin inhibition in pigs. Circulation 1991; 84: 814-820
    CrossrefPubMedGoogle Scholar
  • 8 Lassila R, Badimon JJ, Vallabhajosula S, Badimon L. Dynamic monitoring of platelet deposition on severely damaged vessel wall in flowing blood. Effects of different stenoses on thrombus growth. Arteriosclerosis 1990; 10: 306-315
    CrossrefPubMedGoogle Scholar
  • 9 Weiss HJ, Turitto VT, Vicic WJ, Baumgartner HR. Fibrin formation, fibrinopeptide A release, and platelet thrombus dimensions on subendothelium exposed to flowing native blood: greater in factor XII and XI than in factor VIII and IX deficiency. Blood 1984; 63: 1004-1014
    PubMedGoogle Scholar
  • 10 Cadroy Y, Horbett TA, Hanson SR. Discrimination between platelet mediated and coagulation mediated mechanisms in a model of complex thrombus formation in vivo. J Lab Clin Med 1989; 113: 436-448
    PubMedGoogle Scholar
  • 11 Cazenave J-P, Blondowska D, Richardson M, Kinlough-Rathbone RL, Packham MA, Mustard JF. Quantitative radioisotopic measurement and scanning electron microscopic study of platelet adherence to a collagen coated surface and to subendothelium with a rotating probe device. J Lab Clin Med 1979; 93: 60-70
    PubMedGoogle Scholar
  • 12 Mulvihill JN, Davies JA, Toti F, Freyssinet J-M, Cazenave J-P. Thrombin stimulated platelet accumulation on protein coated glass capillaries: role of adhesive platelet α-granule proteins. Thromb Haemostas 1989; 62: 989-995
    PubMedGoogle Scholar
  • 13 Badimon L, Badimon JJ, Turitto VT, Vallabhajosula S, Fuster V. Platelet thrombus formation on collagen type I. A model of deep vessel injury. Influence of blood rheology, von Willebrand factor, and blood coagulation. Circulation 1988; 78: 1431-1442
    CrossrefPubMedGoogle Scholar
  • 14 Baker HJ, Lindsey JR, Weisbroth SH. (eds). The Laboratory Rat, Vol.1 and 2. New York: Academic Press; 1979
    Google Scholar
  • 15 Gill III TJ, Smith GJ, Wissler RW, Kunz HW. The rat as an experimental animal. Science 1989; 245: 269-276
    CrossrefPubMedGoogle Scholar
  • 16 Smith LJ, Schaible KL, Fessler RG, Rachlin JR, Brown FD. Examination of the utility of the rat as an animal model for human anticoagulation. Haemostasis 1987; 17: 206-210
    PubMedGoogle Scholar
  • 17 Maffrand JP, Bernat A, Delebassée D, Cazenave J-P, Gordon JL. ADP plays a key role in thrombogenesis in rats. Thromb Haemostas 1988; 59: 225-230
    PubMedGoogle Scholar
  • 18 Goldsmith HL, Turitto VT. Rheological aspects of thrombosis and haemostasis: basic principles and applications. Thromb Haemostas 1986; 55: 415-435
    PubMedGoogle Scholar
  • 19 Mulvihill JN, Huisman HG, Cazenave J-P, van Mourik JA, van Aken WG. The use of monoclonal antibodies to human platelet membrane glycoprotein IIb-IIIa to quantitate platelet deposition on artificial surfaces. Thromb Haemostas 1987; 58: 724-731
    PubMedGoogle Scholar
  • 20 Eldrup-Jorgensen J, Connolly RJ, Mackey WC, Ramberg K, O'Donnell TF, Valeri R, Callow AD. Antiplatelet therapy and vascular grafts. Studies in a baboon ex vivo shunt. Arch Surgery 1986; 121: 778-781
    CrossrefPubMedGoogle Scholar
  • 21 Vogel GMT, Meuleman DG, Bourgondien FGM, Hobbelen PMJ. Comparison of two experimental thrombosis models in rats. Effects of four glycosaminoglycans. Thromb Res 1989; 54: 399-410
    CrossrefPubMedGoogle Scholar
  • 22 Hanson SR, Harker LA. Baboon models of acute arterial thrombosis. Thromb Haemostas 1987; 58: 801-805
    PubMedGoogle Scholar
  • 23 Hornstra G, Vendelmans-Starrenburg A. Induction of experimental arterial occlusive thrombi in rats. Atherosclerosis 1973; 17: 369-382
    CrossrefPubMedGoogle Scholar
  • 24 Mayne R. Collagenous proteins of blood vessels. Arteriosclerosis 1986; 6: 585-593
    CrossrefPubMedGoogle Scholar
  • 25 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-363
    CrossrefPubMedGoogle Scholar
  • 26 Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphateinduced platelet aggregation in suspensions of washed rabbit platelets. Br J Haematol 1970; 19: 07-12
    CrossrefPubMedGoogle Scholar
  • 27 Gachet C, Stierlé A, Cazenave J-P, Ohlman P, Lanza F, Bouloux C, Maffrand J-P. The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man. Biochem Pharmacol 1990; 40: 229-238
    CrossrefPubMedGoogle Scholar
  • 28 Eber M, Cazenave J-P, Grob JC, Abecassis J, Methlin G. 111indium labelling of human washed platelets: kinetics and in vivo sequestration sites. In: Blood Cells in Nuclear Medicine. Part I. Cell Kinetics and Bio-distribution. Hardeman MR, Najean Y. (eds). Boston MA: Martinus Nijhoff Publishers; 1984: 29-43
    Google Scholar
  • 29 Cazenave J-P, 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: 167-179
    PubMedGoogle Scholar
  • 30 Van Ruijven-Vermeer IAM, Nieuwenhuizen W. Purification of rat fibrinogen and its constituent chains. Biochem J 1978; 169: 653-658
    CrossrefPubMedGoogle Scholar
  • 31 Regoeczi E. Iodine-Labeled Plasma Proteins. CRC Press; Boca Raton, FL: 1984: 49-56
    Google Scholar
  • 32 Freund M, Cazenave J-P, Courtney M, Degryse E, Roitsch C, Bernat A, Delebassée D, Defreyn G, Maffrand J-P. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats. Thromb Haemostas 1990; 63: 187-192
    PubMedGoogle Scholar
  • 33 Wagner WR, Hubbel JA. Local thrombin synthesis and fibrin formation in an in vitro thrombosis model result in platelet recruitment and thrombus stabilisation on collagen in heparinized blood. J Lab Clin Med 1990; 116: 636-650
    PubMedGoogle Scholar
  • 34 Chesebro JH, Zoldhelyi P, Badimon L, Fuster V. Role of thrombin in arterial thrombosis: implication for therapy. Thromb Haemostas 1991; 66: 01-05
    PubMedGoogle Scholar
  • 35 Björk I, Olson ST, Shore JD. Molecular mechanisms of the accelerating effect of heparin on the relations between antithrombin and clotting proteinases. In: Heparin. Chemical and Biological Properties. Clinical Applications. Lane D, Lindahl U. (eds). Edward Arnold. 1989: 229-235
    Google Scholar
  • 36 Markwardt F. Hirudin and derivatives as anticoagulant agents. Thromb Haemostas 1991; 66: 141-152
    PubMedGoogle Scholar
  • 37 Badimon L, Badimon JJ, Lassila R, Heras M, Chesebro JH, Fuster V. Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudins, heparin, and calcium chelation. Blood 1991; 2: 423-434
    PubMedGoogle Scholar
  • 38 Kelly AB, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson S, Harker LA. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood 1991; 77: 1006-1012
    PubMedGoogle Scholar
  • 39 Butler KD, Ambler J, Dolan S, Giddings J, Talbot MD. A nonocclusive model of arterial thrombus formation in the rat and its modification by inhibitors of platelet function, or thrombin activity. Blood Coagulat Fibrinol 1992; 3: 155-165
    PubMedGoogle Scholar
  • 40 Hemker HC, Kessels H. Feedback mechanisms in coagulation. Haemostasis 1991; 21: 189-196
    PubMedGoogle Scholar
  • 41 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-10370
    CrossrefPubMedGoogle Scholar
  • 42 Chesebro JH, Fuster V. Dynamic thrombosis and thrombolysis. Role of antithrombins. Circulation 1991; 83: 1815-1817
    CrossrefPubMedGoogle Scholar
  • 43 Cadroy Y, Maraganore JM, Hanson SR, Harker LA. Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons. Proc Natl Acad Sci 1991; 88: 1177-1181
    CrossrefPubMedGoogle Scholar
  • 44 Badimon L, Badimon JJ, Fuster V. Thrombogenesis and inhibition of platelet aggregation. Experimental aspects and future approaches. Kardiologie 1990; 79: 133-145
    PubMedGoogle Scholar