Thrombin cleavage at the Arg372-Ser373 scissile bond.

(A) Cleavage reactions containing 50 nM human plasma FVIII and 1 nM thrombin were performed at 37°C over several time points, ranging from 2 to 120 minutes. Cleavage products were resolved by SDS-PAGE and were Western blotted using the monoclonal antibody OBT0037A, which is specific for the heavy-chain A₂ domain. Cleavage at Arg372-Ser373 gives rise to the 44-kDa A₂ fragment, detected by the antibody. (B) To directly compare the effects of the alanine-substituted thrombins on cleavage at Arg372, we performed cleavage reactions for FVIII by WT and mutant thrombins for one time point (10 minutes) in duplicate and loaded onto the same gel. The reactions were resolved by SDS-PAGE and were Western blotted as outlined in panel A. The intensity of the A₂ fragment generated by cleavage at Arg372 was determined by direct scanning of the Western blot (data not shown). A₂ band intensity generated by each mutant (the mutant Lys65Ala was included in these studies) is presented in a bar graph as a percentage of A₂band intensity with respect to WT thrombin (100%).