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Meizothrombin and meizothrombin(desF1) are intermediates formed during the conversion of prothrombin to thrombin by factor Xa, factor Va, phospholipids, and Ca2+ (prothrombinase). These intermediates are active toward synthetic peptide substrates but have limited ability to interact with platelets or macromolecular substrates such as fibrinogen. Meizothrombin and meizothrombin(desF1) activate protein C, however, and may exert primarily an anticoagulant effect. In this study, we investigated the inhibition of meizothrombin and meizothrombin(desF1) by two glycosaminoglycan-dependent protease inhibitors, heparin cofactor II (HCII) and antithrombin (AT). Purified recombinant meizothrombin and meizothrombin(desF1) were inhibited by HCII in the presence of dermatan sulfate with maximal second-order rate constants of 8 x 10(6) M-1.min-1 and 1.8 x 10(7) M-1.min-1, respectively, but were inhibited less than one-tenth as fast by AT in the presence of heparin. Similarly, the products of the prothrombinase reaction were inhibited in situ more effectively by HCII than by AT. When HCII and dermatan sulfate were present continuously during the prothrombinase reaction, meizothrombin was trapped as a sodium dodecyl sulfate-stable complex with HCII and no amidolytic activity could be detected with a thrombin substrate. Our findings indicate that HCII is an effective inhibitor of meizothrombin and meizothrombin(desF1) and, therefore, might regulate the anticoagulant activity of these proteases.

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. 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-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition 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 rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

When a recombinant variant of prothrombin with the cleavage site mutations R155A, R271A, and R284A (rMZ) is exposed to either prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated that is stable for weeks in the presence of Ca2+ (Côté, H. C. F., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A. (1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca2+ however, rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain at Arg55 in the sequence RTPR downward arrowDKL, yielding a molecule with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics are first order regardless of the rMZa concentration, indicating an intramolecular cleavage. This cleavage does not occur at Ca2+ concentrations in excess of 1.0 mM. To assess the role of the F1 domain in rMZa activity, another variant lacking the R155A mutation (rMZdesF1) was expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to 90% that of recombinant thrombin as Ca2+, Mg2+, or Mn2+ concentrations are varied from 0 to 10 mM. Km and kcat values for rMZa in the absence and presence of 5 mM Ca2+ are 1.9 and 2.2 microM and 65 and 105 s-1. TAME esterase activity of rMZa also increases with 5 mM Ca2+. No such metal ion-dependent effects are obtained with either thrombin or rMZdesF1a. Fibrinogen clotting activities, relative to that of thrombin, increase in a manner analogous to those obtained with small substrates, for rMZa and rMZa* but not rMZdesF1a. Complexes of the active site probe dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent decreases in fluorescence intensity, suggesting that metal ion binding in the F1 domain alters the environment of the probe at the active site. These results indicate that in the absence of divalent cations, the activity of rMZa is inhibited, perhaps by obstruction of the active site by the F1 domain, and that Ca2+ binding to the F1 domain modulates the properties of not only the F1 domain but also the protease domain.

Meizothrombin is a transient intermediate produced during the activation of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, we have expressed a mutant human prothrombin cDNA (R155A, R271A, R284A) with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 micrograms/ml of carboxylated mutant prothrombin. After purification, the mutant prothrombin was activated by the prothrombinase complex or by ecarin, resulting in the formation of a meizothrombin-like molecule. Electrophoretic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4 degrees C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pentanediyl) amide confirmed the conversion of prothrombin via meizothrombin. Compared with human plasma-derived thrombin, recombinant meizothrombin demonstrated approximately 7% clotting activity, 100% p-toluene-sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.