The F_ab-fragment of a PAI-1 Inhibiting Antibody Reduces Thrombus Size and Restores Blood Flow in a Rat Model of Arterial Thrombosis

Summary

The effect of PRAP-1, a Fab-fragment of a PAI-1 -inhibiting polyclonal antibody, on thrombus size and arterial blood flow was studied in a rat model of arterial thrombosis. It was shown that exposure of the carotid artery to FeCl₃ led to the rapid formation of an occlusive thrombus with a morphology similar to that of arterial thrombi found in humans. Tranexamic acid (50 mg/kg), an inhibitor of fibrinolysis, increased thrombus size (p = 0.014) when given intravenously (i.v.) prior to the FeCl₃-exposure. Heparin (1000 U), when given i.v. after FeCl₃, did not affect the thrombus size per se, but caused a reduction in the interindividual variation of the size of the thrombus (p <0.05). Thus, heparin was included in all the subsequent experiments. An i.v. infusion of t-PA (1 mg/kg/h), starting before thrombus formation, induced a 3.3 fold increase in the perfusion rate (p = 0.006) and a 67% reduction in the thrombus size (p <0.001). PRAP-1, an inhibitor of rat PAI-1 activity, was given i.v. as a bolus followed by an infusion. Two doses of PRAP-1 were studied (7.5 and 15 mg/kg/h), and the administration of the PAI-1 inhibitor was started 10 min before FeCl₃. The lower PRAP-1 dose caused a 3.8 fold increase in perfusion rate (p = 0.036), a 1.44 fold increase in the time to occlusion (p = 0.034), and the thrombus size was decreased by 18% (p = 0.104). The corresponding effects of the high PRAP-1 dose were a 6.5 fold increase in perfusion rate (p <0.001), a 1.6 fold increase in time to occlusion (p = 0.038) and a 32% reduction in thrombus size (p = 0.016). It is concluded that an inhibitor of PAI-1 activity, PRAP-1, caused a moderate decrease in thrombus size and partly restores blood flow in a rat model of arterial thrombosis. This finding suggests a potential role for an inhibitor of PAI-1 in the treatment of arterial thrombosis.

• References

• 1 Krishnamurti C, Alving BM. Plasminogen activator inhibitor type 1:Biochemistry and evidence for modulation of fibrinolysis in vivo. Sem Thromb Hemost 1992; 18: 67-80
• 2 Schneiderman J, Loskutoff DJ. Plasminogen activator inhibitors. Trends in Cardiovasc Med 1991; 1: 099-102
• 3 Padro T, Quax PHA, van denHoogen CM, Roholl P, Verheijen JH, Emeis JJ. Tissue-type plasminogen activator and its inhibitor in rat aorta: effect of endotoxin. Arterioscler Thromb 1994; 14: 1459-1465
• 4 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Brit J Haematol 1988; 70: 327-333
• 5 Rocha E, Páramo JA. The relationship between impaired fibrinolysis and coronary heart disease: a role for PAI-1 (review). Fibrinolysis 1994; 8: 294-303
• 6 Schleef RR, Higgins DL, Pillemer E, Levitt LJ. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. J Clin Invest 1989; 83: 1747-1752
• 7 Dieval J, Nguyen G, Gross S, Delobel J, Kruithof DJ. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type 1. Blood 1991; 77: 528-532
• 8 Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Brief report: Complete deficiency of plasminogen-activator inhibitor type 1 due to a frame shift mutation. N Engl J Med 1992; 327: 1729-33
• 9 Potter vanLoon BJ, Rijken DC, Brommer EJP, van derMaas APC. The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysability. Thromb Haemost 1992; 67: 101-105
• 10 Stringer HAR, van SwietenP, Heijnen HFG, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance. Studies with thrombi generated in the Chandler loop. Arterioscler Thromb 1994; 14: 1452-1458
• 11 Keijer J, Linders M, van ZonneveldA-J, Ehrlich HJ, de BoerJ-P, Pannekoek H. The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localisation of interaction sites and physiologic relevance. Blood 1991; 78: 401-409
• 12 Hladovec J. Experimental arterial thrombosis in rats with continuous registration. Thromb Diath Haemorrh 1973; 29: 407-410
• 13 Kurz KD, Main BW, Sandusky E. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990; 60: 269-280
• 14 Kawasaki T, Kawamura S, Katoh S, Takenaka T. Experimental model of. carotid artery thrombosis in rats and the thrombolytic activity of YM866, a novel modified tissue-type plasminogen activator. Japan J Pharmacol 1993; 63: 135-142
• 15 Abrahamsson T, Björquist Deinum J, Ehnebom J, Hulander M, Legnehed A, Matsson C, Nerme V, Westin-Eriksson A, Akerblom B. An antibody fragment against PAI-1 inhibits PAI-1 activity and stimulates fibrinolysis in vitro and in vivo. Fibrinolysis 1994; 8 (Suppl. 02) 55-56
• 16 Abrahamsson T, Nerme V, Strömqvist M, Åkerblom B, Legnehed A, Petersson Westin ErikssonA. Anti-thrombotic effect of a PAI-1 inhibitor in rats given endotoxin. Thromb Haemost 1996; 75: 118-126
• 17 Green CJ. Animal anaesthesia. Laboratory animals Ltd; London: 1979
• 18 Longstaff C. Studies on the mechanisms of action of aprotinin and tranexamic acid as plasmin inhibitors and anti fibrinolytic agents. Blood Coag Fibrinol 1994; 5: 537-542
• 19 Bode C, Nordt TK, Runge MS. Thrombolytic therapy in acute myocardial infarction – selected recent developments. Ann Hematol 1994; 69: S35-S40
• 20 Lenfors S, Marberg L, Wikström S, Johnsson U, Westin ErikssonA, Gustafsson D. A new rat model of arterial thrombosis with a platelet-rich head and a erythrocyt-rich tail: thrombolysis experiments with specific thrombin inhibition. Blood Coag Fibrinol 1993; 4: 263-271
• 21 Clemensen I. Three new E-antigenic fibrinogen fractions found in an commercial plasmin preparation. Science Tools, LBK Intr J 1973; 20: 7-8
• 22 Bukh A, Ingerslev J, Stenbjerg S, Møller NPH. The multimeric structure of plasma FVIII:RAg studied by electroelution and immunoperoxidase detection. Thromb Res 1986; 43: 579-584
• 23 Wiman B, Mellbring G, Rånby M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin Chim Acta 1983; 127: 279-288
• 24 Gast A, Tschopp TB, Schmid G, Hilpert K, Ackermann J. Inhibition of clotbound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro46-6240, recombinant hirudin and heparin in human plasma. Blood Coag Fibrinol 1994; 5: 879-887
• 25 Páramo JA, Colluci M, Collen D, van derWerf F. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J 1985; 291: 573-574
• 26 Levi M, Biemond B, van ZonneveldA-J, ten Cate JW, Pannekoek H. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85: 305-312
• 27 Biemond BJ, Levi M, Coronel R, Janse MJ, ten Cate JW, Pannekoek H. Thrombolysis and reocclusion in experimental jugular vein and coronary artery thrombosis: effects of a plasminogen activator inhibitor type 1 neutralizing monoclonal antibody. Circulation 1995; 91: 1175-1181
• 28 Eitzman DT, Fay WP, Lawrence DA, Francis-Chmura AM, Shore JD, Olson ST, Ginsburg D. Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro. J Clin Invest 1995; 95: 2416-2420
• 29 Podor TJ, Hirsh J, Gelehrter TD, Zeheb R, Torry D, Guigoz Y, Sierra F, Gauldie J. Type 1 plasminogen activator inhibitor is not an acute phase reactant in rats. Lack of IL-6- and hepatocyte-dependent synthesis. J Immunol 1993; 150: 225-235
• 30 Robbie LA, Booth NA, Young S, Bennett B. A study of thrombi formed in the Chandler loop. Thromb Haemost 1995; 73: 1005 (abstract).
• 31 Mimuro J, Schleef RR, Loskutoff DJ. Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 1987; 70: 721-728
• 32 Fay WP, Murphy JG, Owen WG. High concentrations of active plasminogen activator inhibitor-1 in porcine coronary artery thrombi. Arterioscler Thromb Vase Biol 1996; 16: 1277-1284
• 33 Fay WP, Eitzman DT, Shapiro AD, Madison EL, Ginsberg D. Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms. Blood 1994; 83: 351-356
• 34 Gustafsson D, Elg M, Lenfors S, Börjesson I, Teger-Nilsson A-C. Effects of inogatran, a low-molecular-weight thrombin inhibitor, in rat models of venous and arterial thrombosis, thrombolysis and bleeding time. Blood Coag Fibrinol 1996; 7: 69-79