Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S

Karl Malm; Björn Dahlbäck; Björn Arnljots

doi:10.1160/TH03-03-0174

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2003; 90(02): 227-234
DOI: 10.1160/TH03-03-0174

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S

Karl Malm

¹Departments of Plastic and Reconstructive Surgery and Experimental Research,

,

Björn Dahlbäck

²Department of Laboratory Medicine, Division of Clinical Chemistry, University Hospital, Malmö, Sweden

,

Björn Arnljots

¹Departments of Plastic and Reconstructive Surgery and Experimental Research,

› Institutsangaben

Abstract

Summary

The antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, whereas bAPC combined with bPS prolonged the clotting time considerably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent anti-thrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.

Keywords

Thrombosis - activated protein C - protein S - bleeding - animal model

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Referenzen

References
1 Dahlback B. Blood coagulation. Lancet 2000; 355: 1627-32.
2 Dahlback B, Stenflo J. The Protein C Anticoagulant System. In: The Molecular Basis of Blood Diseases Stamatoyannopoulos, Majerus, Perlmutter, Varmus, eds. Saunders WB. 2001; 614-56.
3 Esmon CT. Regulation of blood coagulation. Biochim Biophys Acta 2000; 1477: 349-60.
4 Lane DA, Mannucci PM, Bauer KA. et al. Inherited thrombophilia: Part 1. Thromb Haemost 1996; 76: 651-62.
5 Lane DA, Mannucci PM, Bauer KA. et al. Inherited thrombophilia: Part 2. Thromb Haemost 1996; 76: 824-34.
6 Shen L, Dahlback B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-8.
7 Arnljots B, Dahlback B. Protein S as an in vivo cofactor to activated protein C in prevention of microarterial thrombosis in rabbits. J Clin Invest 1995; 95: 1987-93.
8 Arnljots B, Dahlback B. Antithrombotic effects of activated protein C and protein S in a rabbit model of microarterial thrombosis. Arterioscler Thromb Vasc Biol 1995; 15: 937-41.
9 Walker FJ. Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity. Thromb Res 1981; 22: 321-7.
10 Solymoss S, Tucker MM, Tracy PB. Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection. J Biol Chem 1988; 263: 14884-90.
11 Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RF, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-8.
12 He X, Dahlback B. Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin-K-dependent protein S. Eur J Biochem 1993; 217: 857-65.
13 Rosing J, Hoekema L, Nicolaes GA, Thomassen MC, Hemker HC, Varadi K. et al. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-8.
14 Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
15 Arnljots B, Bergqvist D, Dahlback B. Inhibition of microarterial thrombosis by activated protein C in a rabbit model. Thromb Haemost 1994; 72: 415-20.
16 Jackson CV, Bailey BD, Shetler TJ. Pharmacological profile of recombinant, human activated protein C (LY203638) in a canine model of coronary artery thrombosis. J Pharmacol Exp Ther 2000; 295: 967-71.
17 Foo RS, Gershlick AH, Hogrefe K. et al. Inhibition of platelet thrombosis using an activated protein C-loaded stent: in vitro and in vivo results. Thromb Haemost 2000; 83: 496-502.
18 Sakamoto T, Ogawa H, Yasue H. et al. Prevention of arterial reocclusion after thrombolysis with activated protein C. Comparison with heparin in a canine model of coronary artery thrombosis. Circulation 1994; 90: 427-32.
19 Gruber A, Griffin JH, Harker LA, Hanson SR. Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-42.
20 Gruber A, Hanson SR, Kelly AB. et al. Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis. Circulation 1990; 82: 578-85.
21 Araki H, Nishi K, Ishihara N, Okajima K. Inhibitory effects of activated protein C and heparin on thrombotic arterial occlusion in rat mesenteric arteries. Thromb Res 1991; 62: 209-16.
22 Yamashita T, Matsuoka A, Funatsu A, Yamamoto J. The antithrombotic effect of human activated protein C on He-Ne laser- induced thrombosis in rat mesenteric microvessels. Thromb Res 1994; 75: 33-40.
23 Smirnov MD, Pyzh MV, Borovikov DV. et al. Low doses of activated protein C delay arterial thrombosis in rats. Thromb Res 1990; 57: 645-50.
24 Hashimoto M, Watanabe S, Oiwa K. et al. Enhanced thrombolysis induced by argatroban or activated protein C in the presence or absence of staphylokinase, measured in an in vivo animal model using mesenteric arterioles. Haemostasis 2001; 31: 80-9.
25 Hashimoto M, Yamashita T, Oiwa K, Watanabe S, Giddings JC, Yamamoto J. Enhancement of endogenous plasminogen activator-induced thrombolysis by argatroban and APC and its control by TAFI, measured in an arterial thrombolysis model in vivo using rat mesenteric arterioles. Thromb Haemost 2002; 87: 110-3.
26 Stenflo J. A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251: 355-63.
27 Stenflo J, Jonsson M. Protein S, a new vitamin K-dependent protein from bovine plasma. FEBS Lett 1979; 101: 377-81.
28 Dahlback B, Hildebrand B, Linse S. Novel type of very high affinity calcium-binding sites in beta- hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S. J Biol Chem 1990; 265: 18481-9.
29 Soderstrom T, Hedner U, Arnljots B. Active site-inactivated factor VIIa prevents thrombosis without increased surgical bleeding: topical and intravenous administration in a rat model of deep arterial injury. J Vasc Surg 2001; 33: 1072-9.
30 Acland RD. Microsurgery Practice Manual. In. Mosby: St Louis; 1980: 58-9.
31 Marmur JD, Rossikhina M, Guha A. et al. Tissue factor is rapidly induced in arterial smooth muscle after balloon injury. J Clin Invest 1993; 91: 2253-9.
32 Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A 1989; 86: 2839-43.
33 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: 1087-97.
34 Malm K, Dahlback B, Arnljots B. Low-molecular-weight heparin (Dalteparin) effectively prevents thrombosis in a rat model of deep arterial injury. Plast Reconstr Surg 2003; 111 (05) 1659-66.