The Substrate -Membrane Interaction Constrains Bond Presentation and Cleavage in the Action of Prothrombinase on Prothrombin.

Abstract Thrombin formation is catalyzed by human prothrombinase almost exclusively through sequential cleavage at Arg320 followed by cleavage at Arg271 in prothrombin. Activation site mutants in which the individual sites have been rendered uncleavable indicate that Arg271 in intact prothrombin is hydrolyzed ~26-fold more slowly than Arg320. This finding provides an adequate kinetic explanation for the observed pathway of prothrombin activation by prothrombinase via the formation of meizothrombin. However, deviations from this behavior are implied by findings in the absence of membranes or with prothrombin species that are defective in membrane binding. We have investigated the contribution of the substrate-membrane interaction to the individual cleavage reactions by comparing the action of prothrombinase on a series of recombinant prothrombin derivatives expressed in the presence or absence of reduced vitamin K to yield either fully-carboxylated (Gla) or un-carboxylated (desGla) protein. The variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with a single cleavable site at Arg320 and IIQ320 with a single cleavable site at Arg271. Quantitative analysis of 4-carboxyglutamic acid content established the intended extent of carboxylation for all Gla and desGla isoforms. Analysis of prothrombin activation by prothrombinase indicated a ~10 fold reduction in the rate of active product formation with desGla-IIWT compared to Gla-IIWT. Surprisingly, analysis by SDS-PAGE and quantitative densitometry revealed only a modest decrease (~ 2.5-fold) in the rate of consumption of desGla-IIWT. However, marked differences were evident in the intermediates produced. Bond cleavage of desGla-IIWT proceeded with a major contribution from cleavage at Arg271 followed by Arg320, in an order opposite to that observed with Gla-IIWT. Thus, the reduced rate of active product formation with desGla-IIWT reflects, in large part, the formation of a zymogen intermediate as a result of an altered order in which bonds are cleaved by prothrombinase. Comparison of the rates of cleavage of individual bonds in Gla and desGla versions of IIQ271 and IIQ320 revealed the kinetic basis for this change. Elimination of membrane binding led to a ~5 fold reduction in the rate of cleavage at Arg320 but surprisingly yielded a ~10-fold enhancement in cleavage at Arg271. Lack of Gla is not uniformly deleterious to all reactions of prothrombin activation. Compensating rate effects on the individual reactions imply a change in the restricted presentation of cleavage sites to the active site of Xa within prothrombinase upon the loss of the substrate-membrane interaction. Thus, substrate-membrane interactions appear to work in concert with exosite binding to modulate bond cleavage order by maximizing accessibility of the Arg320 bond to the active site while restricting that of Arg271. Loss of the membrane anchoring component leads to increased accessibility of the Arg271 site and a concomitant decrease in the rate of cleavage at Arg320. These observations have major implications for prothrombinase function on cell surfaces with limited binding sites for prothrombin, as well as the impact of warfarin therapy on prothrombinase function in vivo.