Time course of cleavage of recombinant factor VIII by thrombin. Recombinant factor VIII wild type (WT) and R372H (100 nM) were reacted with thrombin (2.5 nM) for the indicated times as described in “Materials and methods.” Samples were run on 8% gel followed by Western blotting using an anti-A2 (A), anti-A1 (C), or anti–light chain (D) monoclonal antibody. Panel B shows quantitative densitometry of the ratio of A2 subunit/A1-A2 subunit from blotting data obtained from panel A. ○ indicates wild type; •, R372H. The data were fitted to a straight line. SC and LCh represent a single chain and the intact light chain, respectively.