PURIFICATION AND CHARACTERIZATION OF BOTHROMBIN, A FIBRINOGEN-CLOTTING SERINE-PROTEASE FROM THE VENOM OF BOTHROPS-JARARACA

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33 000 under no nreducing and 35 000 under reducing conditions on SDS polyacrylamide g el electrophoresis and specific fibrinogen-clotting activity equivalen t to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha -thrombin, bothrombin split off fibrinopeptide A without releasing fib rinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bo thrombin did not induce aggregation or serotonin release of washed nor mal platelets by itself, but did aggregate platelets in the presence o f exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which b locks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal anti body (which specifically inhibits alpha-thrombin binding to GP Ib). Pr ostaglandin E1 (1 mu M) and EDTA (10 mM) also abolished platelet aggre gation without affecting clotting activity. Washed platelets from a pa tient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, Suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid Sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides g enerated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues an d contains three Asn-linked oligosaccharide chains. The sequence is ho mologous to those of other serine proteases; in particular, batroxobin from the Bothrops atrox moojeni venom.