Role of Procoagulant Lipids in Human Prothrombin Activation. 1. Prothrombin Activation by Factor Xa in the Absence of Factor Va and in the Absence and Presence of Membranes

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Abstract
Activation of prothrombin by factor Xa requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg322−Ile323 produces meizothrombin (MzIIa) as an intermediate, while hydrolysis of Arg273−Thr274 produces prethrombin 2−fragment 1.2 (Pre2−F1.2). Activation by human factor Xa of human prothrombin was examined in the absence of factor Va and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzIIa, and Pre2−F1.2; a synthetic substrate (S-2238) detected thrombin or MzIIa active site formation; and SDS−PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS−PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzIIa and Pre2−F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzIIa active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M-1 s-1, for the rate of MzIIa formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane−enzyme complex. The data suggest that PS membranes (1) regulate factor Xa, (2) alter the substrate specificity of factor Xa to favor the meizothrombin intermediate, and (3) “channel” intermediate (MzIIa or Pre2−F1.2) back to the active site of factor Xa for rapid conversion to thrombin.