Time course of thrombin activation of recombinant factor VIII. Recombinant factor VIII wild type, R372H, and R372Q (50 nM) were reacted with thrombin (20 nM) for the indicated times, after which activation was terminated by hirudin and each sample was tested immediately for factor VIIIa activity in a one-stage clotting assay. The inset shows an expanded activation time course during the initial 10 minutes of the reaction. ○ indicates wild type; •, R372H; and □, R372Q. The zero point was taken prior to addition of thrombin.