Thrombin cleavage at the Arg1689-Ser1690 scissile bond.

Cleavage reactions and Western blotting conditions were similar to those in Figure 4, except the primary antibody FVIIIC, which recognizes the 80-kDa light chain, was used. (A) Western blots depicting the disappearance over time for the 80-kDa light chain caused by the specific cleavage at Arg1689-Ser1690 and the loss of the N-terminal fragment bearing the antibody epitope. (B) Percentage of FVIII light chain remaining after 2-minute incubation with WT and mutant thrombin (including Lys65Ala), as determined by measuring the band intensity, and presented on the bar graph as a percentage of total light chain (time 0).