Abstract
Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1(336)) was previously shown to possess similar affinity for A2 and retain approximately 60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys(36) that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1(37-336)) showed little if any affinity for A2 (K(d)>2 microm), whereas factor VIIIa reconstituted with A2 plus A1(37-336)/A3-C1-C2 dimer demonstrated significant cofactor activity ( approximately 30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1(37-336) showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K(m) for factor X when A1 was cleaved at Arg(336). These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasma-based assays.
Highlights
Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A
A1 truncated at both cleavage sites (A137–336) showed little if any affinity for A2 (Kd>2 M), whereas factor VIIIa reconstituted with A2 plus A137–336/A3-C1-C2 dimer demonstrated significant cofactor activity (ϳ30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay
This procofactor is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits [4, 5]
Summary
Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A. Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains This procofactor is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits [4, 5]. Evidence suggested that the C-terminal acidic region of A1 subunit (residues 337–372) represented an A2-interactive site and participated in A2 retention following thrombin activation [17]. This region is implicated in the binding of factor X [18], the significance of this cofactor-substrate interaction is not well understood. Specific mechanisms for the loss of factor VIIIa function and down-regulation of factor Xase can be attributed to individual cleavages of the A1 subunit by factor Xa
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