SDS-PAGE analysis of prothrombin activation. Prothrombin (5 μg) was activated with trocarin or human factor Xa (5 ng each) in the presence of Ca²⁺ ions (5 mmol/L), 9:1 PC:PS phospholipid vesicles (100 μmol/L), and factor Va (10 nmol/L) at 37°C for 4 hours. The reaction mixture was loaded on a nonreducing 4% to 20% gradient gel. Untreated prothrombin (lane a), cleavage products of prothrombin by trocarin (lane b), and factor Xa (lane c) were electrophoresed according to Laemmli.15