Cross-linking of factor VIII with EDC. Factor VIII (250 nmol/L) was incubated in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of EDC (250 μmol/L) in a reaction (100 μL) containing 20 mmol/L Mes, pH 6, and 0.01% Tween-20 for 1 hour at room temperature. After cross-linking, the pH of the reactions was raised to 7.2 and thrombin (100 nmol/L) was added (lanes 2 and 4) for 1 hour at room temperature. After addition of SDS sample buffer, reactions were subjected to electrophoresis on 8% gels, transferred to PVDF, and immunoblotted with antibody 58.12 (A) or R8B12 (B).