Abstract
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.
Highlights
Recombinant human prothrombin and two mutant forms (R155A,R271A,R284A and R271A,R284A) were expressed in mammalian cells
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
Bovine meizothrombin [9] and meizothrombin(desF1) [12] can activate protein C (PC) in a thrombomodulin (TM)-dependent reaction, several studies have been unable to demonstrate significant binding between cell-surface recombinant TM and recombinant meizothrombin derived from the active site serine-to-alanine mutant human prothrombin [13, 14]
Summary
Gla, ␥-carboxyglutamic acid; DAPA, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; PCPS, phosphatidylcholine/phosphatidylserine (3:1) vesicles; S-2238, D-phenylalanyl-Lpipecoyl-L-arginyl-p-nitroaniline dihydrochloride; PPA1⁄7CK D-PhePro-Arg chloromethylketone dihydrochloride; TM, thrombomodulin (Solulin, Ref. 28); rMZ, recombinant human prothrombin (R155A,R271A,R284A); rMZa, activated rMZ; rMZdesF1, recombinant human prothrombin (R271A,R284A); rMZdesF1a, activated rMZdesF1; TAFI, thrombin-activatable fibrinolysis inhibitor; AT, antithrombin; PAGE, polyacrylamide gel electrophoresis; APC, activated protein C. Meizothrombin was shown to have vasoconstrictive activity 5-fold greater than that of ␣-thrombin [21] Together, these studies suggest that meizothrombin and/or meizothrombin(desF1) produced in vivo have functions different from those of thrombin. The present studies were undertaken to analyze their functional properties and compare them to those of recombinant human thrombin (rIIa)
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