Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin

Summary

Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.

Keywords

Thrombin - thrombin inhibitors - clot lysis - carboxypeptidase

• References

• 1 Bajzar L, Manuel R, Nesheim M. Purification and characterization of TAFI, a thrombin activatable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-84.
• 2 Hendriks D, Wang W, Scharpe S, Lommaert MP, van Sande M. Purification and characterization of a new arginine carboxypeptidase in human serum. Biochim Biophys Acta 1990; 1034: 86-92.
• 3 Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994; 269: 15937-44.
• 4 Eaton DLB, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833-8.
• 5 Sakharov D, Plow EF, Rijken DC. On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B. J Biol Chem 1997; 272: 14477-82.
• 6 Wang W, Boffa M, Bajzar L, Walker JB, Nesheim ME. A study on the mechanism of activated thrombin-activatable fibrinolysis inhibitor. J Biol Chem 1998; 273: 27176-81.
• 7 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombinthrombomodulin complex. J Biol Chem 1996; 271: 16603-8.
• 8 Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 1995; 96: 2534-8.
• 9 Minnema MC, Friederich PW, Levi M, von dem Borne PA, Mosnier LO, Meijers JCM, Biemond BJ, Hack CE, Bouma BN, ten Cate H. Enhancement of rabbit jugular vein thrombosis by neutralization of factor XI. J Clin Invest 1998; 101: 10-4.
• 10 Gresele P, Momi S, Berrettini M, Nenci GG, Schwarz HP, Semeraro N, Colucci M. Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein C reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation. J Clin Invest 1998; 101: 667-76.
• 11 Nesheim M. Myocardial infarction and the balance between fibrin deposition and removal. Ital Heart J 2001; 02: 641-5.
• 12 Broze Jr GJ, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase U and the premature lysis of clots from hemophilic plasma. Blood 1996; 88: 3815-23.
• 13 Bajzar L, Nesheim ME, Tracy PB. The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood 1996; 88: 2093-100.
• 14 Bajzar L, Kalafatis M, Simioni P, Tracy PB. An antifibrinolytic mechanism describing the prothrombotic effect associated with factor V Leiden. J Biol Chem 1996; 271: 22949-52.
• 15 Taube J, Mc William N, Luddington R, Byrne CD, Baglin T. Activated protein C resistance: effect of platelets, platelet-derived microparticles, and atherogenic lipoproteins. Blood 1999; 93: 3792-7.
• 16 Camire RM, Kalafatis M, Simioni P, Girolami A, Tracy PB. Platelet-derived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C. Blood 1998; 91: 2818-29.
• 17 Colucci M, Binetti BM, Semeraro N. Platelets abolish the profibrinolytic effect of activated protein C. Thromb Haemost 2000; 84: 923-4.
• 18 Armstrong PW, Collen D. Fibrinolysis for acute myocardial infarction. Circulation 2001; 103: 2987-92.
• 19 Jang IK, Gold HK, Leinbach RC, Fallon JT, Collen D. In vivo thrombin inhibition enhances and sustains arterial recanalization with recombinant tissue-type plasminogen activator. Circ Res 1990; 67: 1552-61.
• 20 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: 1020-4.
• 21 Redlitz A, Nicolini FA, Malycky JL, Topol EJ, Plow EF. Inducible carboxypeptidase activity. A role in clot lysis in vivo. Circulation 1996; 93: 1328-30.
• 22 Klement P, Liao P, Bajzar L. A novel approach to arterial thrombolysis. Blood 1999; 94: 2735-43.
• 23 Nagashima M, Werner M, Wang M, Zhato L, Light DR, Pagila R, Morser J, Verhallen P. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombosis model. Thromb Res 2000; 98: 333-42.
• 24 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibiton by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-91.
• 25 Colucci M, D’Aprile AM, Italia A, Gresele P, Morser J, Semeraro N. Thrombin activatable fibrinolysis inhibitor (TAFI) does not inhibit in vitro thrombolysis by pharmacological concentrations of t-PA. Thromb Haemost 2001; 85: 661-6.
• 26 Colucci M, Paramo JA, Collen D. Inhibition of one-chain and two-chain forms of human tissue-type plasminogen activator by the fast acting inhibitor of plasminogen activator in vitro and in vivo. J Lab Clin Med 1986; 108: 53-9.
• 27 Biggs R. Human blood coagulation, haemostasis and thrombosis. Blackwell; Oxford: 1972
• 28 Mosnier LO, Meijers JCM, Bouma BN. Regulation of fibrinolysis in plasma by TAFI and protein C is dependent on the concentration of thrombomodulin. Thromb Haemost 2001; 85: 5-11.
• 29 Bouma BN, Mosnier LO, Meijers JCM, Griffin JH. Factor XI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation. Thromb Haemost 1999; 82: 1703-8.
• 30 Mohler MA, Refino CJ, Chen SA, Chen AB, Hotchkiss AJ. D-Phe-Pro- Arg-chloromethylketone: its potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemost 1986; 56: 160-4.
• 31 Valnickova Z, Enghild JJ. Human procarboxypeptidase U, or thrombin activatable fibrinolysis inhibitor, is a substrate for transglutaminases. J Biol Chem 1998; 273: 27220-4.
• 32 Mao SS, Cooper CM, Wood T, Shafer JA, Gardell SJ. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem 1999; 274: 35046-52.
• 33 Eisemberg PR, Sobel BE, Jaffe AS. Activation of prothrombin accompanying thrombolysis with recombinant tissue-type plasminogen activator. J Am Coll Cardiol 1992; 19: 1065-69.
• 34 Owen J, Friedmann KD, Grossman BA, Wilkins C, Berke AD, Powers ER. Thrombolytic therapy with tissue plasminogen activator and streptokinase induces transient thrombin activity. Blood 1988; 72: 616-20.
• 35 Granger CP, Becker R, Tracy RP, Calif RM, Topol EJ, Pieper KS, Ross AM, Roth S, Lambrew C, Bovill EG. Thrombin generation, inhibition and clinical outcomes in patients with acute myocardial infarction treated with thrombolytic therapy and heparin: results from the GUSTO-I trial. J Am Coll Cardiol 1998; 31: 497-505.