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 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.
Côté
H C
HC
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada.
Bajzar
L
L
Stevens
W K
WK
Samis
J A
JA
Morser
J
J
MacGillivray
R T
RT
Nesheim
M E
ME
D000990:Antithrombin III / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001667:Binding, Competitive; D001777:Blood Coagulation; D003619:Dansyl Compounds / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000502:physiology*; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D010974:Platelet Aggregation; D011486:Protein C / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000502:physiology*; D018180:Thrombomodulin / Q000378:metabolism
D003619:Dansyl Compounds; D004792:Enzyme Precursors; D006629:Hirudins; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D000990:Antithrombin III; D001120:Arginine; D013917:Thrombin; C026812:meizothrombin