Abstract
Factor VIII is activated by limited proteolytic cleavage at Arg372, Arg740, and Arg1689 by thrombin or factor Xa. Recently, it has been reported that the C2 domain and the acidic region comprising residues 389–394 in the A2 domain interacted with thrombin to facilitate cleavage at Arg1689 and Arg740, respectively. However, a thrombin-interactive site(s) responsible for cleavage at Arg372 remain to be determined. In the current study, several approaches were employed to examine the thrombin-interactive site(s). Thrombin-catalyzed factor VIII activation was inhibited by the addition of one anti-A2 monoclonal antibody 413 with an epitope of residues 484–509 in a dose-dependent manner using one-stage clotting assay. SDS-PAGE analysis revealed that cleavage rate at Arg372 in the heavy chain by thrombin selectively decreased by ~25% in the presence of the antibody. However, cleavage at Arg740 was not affected. Direct binding experiments by both surface plasmon resonance-based assay and ELISA using anhydro-thrombin (Ah-thrombin), catalytically inactive derivative of thrombin in which the active-site serine was converted to dehydroalanine, showed that anti-A2 antibody 413 inhibited the A2 subunit binding to Ah-thrombin. A synthetic peptide corresponding to the 484–509 residues also inhibited the A2/Ah-thrombin binding by ~40%. Mutant A2 molecules where the basic residues in the A2 domain were converted to alanine were evaluated for the binding of Ah-thrombin. Among the tested mutants, K466A mutant possessed ~330-fold lower affinity than the wild type A2 (Kd; 18400 nM, 55 nM, respectively), and the affinities of R471A, R484A, R489A, R490A, H497A, K499A, and K523A were decreased by 2–5-fold (Kd; 259, 212, 183, 163, 123, 255, and 165 nM, respectively). These findings demonstrate that clustered basic residues, in particular Lys466, encompassed the 484–509 region play a key role of thrombin-binding site within the A2 domain, responsible for thrombin-catalyzed factor VIII activation by cleavage at Arg372.
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