FUNCTIONAL-CHARACTERIZATION OF RECOMBINANT HUMAN MEIZOTHROMBIN AND MEIZOTHROMBIN(DESF1) - THROMBOMODULIN-DEPENDENT ACTIVATION OF PROTEIN-C AND THROMBIN-ACTIVATABLE FIBRINOLYSIS INHIBITOR (TAFI), PLATELET-AGGREGATION, ANTITHROMBIN-III INHIBITION

Recombinant human prothrombin (rII) and two mutant forms (R155A,R271A, R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian ce lls. Following activation and purification, recombinant thrombin (rIIa ) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1 ) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-depen dent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid ve sicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, r MZa was also shown to be an effective activator of TAFI (thrombin-acti vatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). Ah three enzymes were c apable of inducing platelet aggregation, but 60-fold higher concentrat ions of rMZa and rMZdesF1a were required to achieve the effects obtain ed with rIIa. Second order rate constants (M(-1). min(-1)) for inhibit ion by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4 ) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZ desF1a by AT-III is not affected by heparin. Ah three enzymes bound si milarly to hirudin. The results of this and previous studies imply tha t full-length meizothrombin has marginal procoagulant properties compa red to thrombin. However, meizothrombin has potent anticoagulant prope rties, expressed through TM-dependent activation of protein C, and can contribute to downregulation of fibrinolysis through the TM-dependent activation of TAFI.