FVa2 and FVa3 interfere with thrombin-catalyzed factor V activation. Human FV was incubated with human α-thrombin at a 250:1 molar ratio, in the presence or absence of the highly purified recombinant fragments of the cofactor (A, thrombin alone; B, thrombin in the presence of 50μM FVa2; C, thrombin in the presence of 50μM FVa3). Aliquots of the reactions were taken at indicated times, separated on 4%-15% SDS-polyacrylamide gels, and silver-stained. The positions of FV, the heavy (HC) and the A3-C1-C2 light-chain doublet (LC) of factor Va are indicated to the right of each panel, along with those of FVa2/FVa3, where appropriate. Notice that FV is processed with slower kinetics in the presence of the recombinant linkers, indicating competition for the activating protease. The figures shown are representative of 4 independent experiments conducted with different preparations of FV and of recombinant FVa2/FVa3.