Thrombin proteolysis of FVIII WT compared with^ARG531^HIS. [³⁵S]-methionine labeled FVIII WT and ^ARG531^HIS proteins immunoprecipitated from the chase conditioned medium of transiently expressing COS-1 cells were divided into equal aliquots and incubated with thrombin (0.1 U/mL) for increasing periods of time at 37°C. Reactions were terminated with SDS-PAGE sample buffer and protein fragments were separated by 10% SDS-PAGE. Time is in minutes, with 0 representing the absence of thrombin. Mock indicates medium from cells that did not receive plasmid DNA. FVIII protein forms are indicated at the right as follows: SC, single chain; HC, heavy chain; LC, light chain; A1+A2, A1, and A2, thrombin-cleaved heavy chain fragments; LC+IIa, thrombin-cleaved light chain; FVIIIa, predicted thrombin-activated FVIII heterotrimer. Molecular weight markers (m) are indicated on the left.