Abstract
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, Arg(336) in the A1 subunit and Arg(562) 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. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.
Highlights
FVIII is a 2332-amino-acid protein with the domain structure A1-A2-B-A3-C1-C2 (3), which is cleaved within the B domain during secretion and circulates in the blood as a heterodimer bound to von Willebrand factor
The cleavage at Arg740 leads to dissociation of the B domain from the rest of the molecule, but this cleavage is not required for FVIIIa activation (5)
FVIIIa inactivation by activated protein C (APC) and protein S was monitored with an APTT assay essentially as described previously, except that FVIII was activated with 0.8 units/ml IIa for 1 min followed by 3.2 units/ml hirudin (28)
Summary
FVIIIa can be inactivated through proteolysis by activated protein C (APC),2 which cleaves FVIIIa at Arg336 in the A1 domain and at Arg562 in the A2 domain. Using the FVIIIa concentrations for each FVIIIa variant determined in this manner, FIXa titrations were performed as described with the exception that FVIII was activated with 0.8 units/ml thrombin for 1 min (four times as much as used previously) followed by 4.8 units/ml hirudin to ensure complete inactivation of the thrombin.
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