Generation of FVa heavy and light chains.

(A) An immunoblot showing generation of FVa heavy chain (FVa-HC) and (B) FVa light chain (FVa-LC) with disulfide bonds intact. In both panels, consecutive 30-second bleeding-time blood samples were separated on a 5% to 15% linear gradient SDS-PAGE gel under nonreducing conditions. FVa-HC was probed with a monoclonal antibody reactive toward residues 307 to 506 of FVa, whereas FVa-LC was probed with a monoclonal antibody raised against the light chain of FVa. Wells A—blood samples taken before aspirin (ASA) ingestion (75 mg/d for 7 days); wells B—samples after aspirin administration. (C) Time courses for generation of both FVa chains in bleeding-time blood. Their relative levels are plotted versus time (seconds), taking a maximum concentration as 100. Concentration of FVa-HC before (open circles) and after aspirin treatment (closed circles). Concentration of FVa-LC before (open squares) and after aspirin treatment (closed squares). Values are plotted as means ± SEM. (D) An immunoblot of the product of FVa heavy-chain cleavage by APC (30 kd; residues 307-506). Consecutive 30-second bleeding-time blood samples were separated on a 5% to 15% linear gradient SDS-PAGE gel under nonreducing conditions. Immunoreactive fragments of FVa inactivation were probed with a monoclonal antibody against residues 307 to 506 of the heavy chain. Wells A—blood samples taken before aspirin ingestion (75 mg/d for 7 days); wells B—samples after aspirin treatment. (E) An immunoblot of FVa heavy chain (top band) and the 97-kd fragment of this chain (bottom band) in bleeding-time blood with disulfide bonds reduced. Both peptides are detected by a monoclonal antibody reactive toward residues 307 to 506 in FVa heavy chain. Wells A—blood samples taken before aspirin (ASA) treatment; wells B—samples after aspirin ingestion.