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• W2024295712 abstract "Activation of prothrombin, as catalyzed by the prothrombinase complex (factor Xa, enzyme; factor Va and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzIIa) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2).V max, K m, orV max/K m for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzIIa and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 μm, whereas MzIIa proteolysis was saturable. First order rate constants for formation of MzIIa and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-Va-Xa complex (i.e.“channeling”) also was adjusted because both the membranes and factor Va have been shown to cause channeling.k cat, K m, ork cat/K m values were reported for one lipid concentration, for which all Xa was likely incorporated into a Xa-Va complex on a PS membrane. Comparing previous results, which were obtained either with factor Va (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686–28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002)Biochemistry 41, 935–949), with results presented here we conclude that both factor Va and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor Va has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzIIa versus Pre2 pathway; and 3) PS membranes cause ∼50% of the substrate to be activated via channeling at 50 μm membrane concentration, but factor Vaextends the range of efficient channeling to much lower or higher membrane concentrations." @default.
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• W2024295712 date "2003-02-01" @default.
• W2024295712 modified "2023-09-27" @default.
• W2024295712 title "Cooperative Roles of Factor Va and Phosphatidylserine-containing Membranes as Cofactors in Prothrombin Activation" @default.
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• W2024295712 doi "https://doi.org/10.1074/jbc.m208423200" @default.
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