Detailed Mechanisms of the Inactivation of Factor VIIIa by Activated Protein C in the Presence of Its Cofactors, Protein S and Factor V*

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg336 in the A1 subunit and Arg562 in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg336 cleavage is 6-fold faster than Arg562 cleavage, and the Arg336 cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg562 cleavage more than Arg336 cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg562 cleavage more than Arg336 cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg336 cleavage is only twice as fast as Arg562 cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.

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