tPA-induced clot lysis and F.IX immunoblotting.

Fibrin clot lysis was monitored by measurement of the absorbance at 405 nm every 2 minutes at 37°C (A). At the end of the 74-minute time course, the reactions were either solubilized directly in reducing SDS-PAGE buffer for F.IX immunoblotting, or an aliquot was diluted in HBS/BSA for F.IX clotting assays prior to solubilization and reducing SDS-PAGE/F.IX immunoblotting (B). NHP (90 μL) was combined with either (final concentrations) thrombin (6 nM) and CaCl₂ (2 mM) (closed squares in A; or lane 1 in B), or tPA (10 nM, closed circles in A; or lane 2 in B), or thrombin/CaCl₂ and 0.2 nM of tPA (closed downward triangles in A; or lane 3 in B), or 0.5 nM of tPA (closed upward triangles in A; or lane 4 in B), or 1 nM of tPA (open squares in A; or lane 5 in B), or 2 nM of tPA (open circles in A; or lane 6 in B), or 5 nM of tPA (open downward triangles in A; or lane 7 in B), or 10 nM of tPA (open upward triangles in A; or lane 8 in B). Purified F.IX (4 μM) was digested with plasmin (50 nM) for 2 minutes at 37°C and added to NHP at 0.18 μM prior to solubilization and F.IX immunoblotting (B, lane 9). The positions of prestained molecular weight standards (in kd) are shown to the left of B. The migration positions of F.IX and the 45- and 30-kd immunoreactive species are shown to the right of B.