title: Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V.
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.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. agale@scripps.edu
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Gale
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cramer
authors.forename: Diana
authors.initials: D
authors.lastname: Rozenshteyn
authors.forename: Jason R
authors.initials: JR
authors.lastname: Cruz
mesh_terms: D001120:Arginine / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D005165:Factor V / Q000737:chemistry*; D015944:Factor VIIIa / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000737:chemistry*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors
chemical_list: D004220:Disulfides; D011486:Protein C; D017293:Protein S; D015944:Factor VIIIa; D005165:Factor V; D001120:Arginine; D013917:Thrombin