Purification and Characterization of Bothrombin, a Fibrinogen-Clotting Serine Protease from the Venom of Bothrops jararaca

Abstract

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent 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 fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient 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 generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity fromBothrops barnettiSnake Venom

“…TLBbar showed fibrinogenolytic activity and degrades fibrinogen A chain; its activity is time and dose dependent (Figures 7(a), and 7(b)), with similarity to Bothrombin of B. jararaca [43] and Flavoxobin of Trimeresurus avoviridis [49]. The degradation was observed by cleavage A chain with 15 g of the enzyme, which starts in 15 minutes, and at two hours there was complete degradation with maximum efficiency.…”

“…Serine proteases like-thrombin with platelet aggregation activity as Bothrombin from B. jararaca [43], Cerastocitin from Cerastes cerastes [44], and BJ-PA from B. jararaca [30], among others have been discribed. Probably TLBbar activates the thrombin receptor platelet membrane (fibrinogen, factor V, and receptors 1 and 4) coupled to G protein to initiate intracellular signals that activate the phospholipases, which cleavage membrane phospholipids releasing arachidonic acid, thromboxane A 2 , calcium, and ADP responsible for the activation of platelets [29,45].…”

Structure Insights and Fibrinogen Peptides Molecular Recognition by Bothrombin, A Snake Venom Serine Protease from Bothropus jararaca

“…Bothrombin form aggregates platelets in the presence of fibrinogen and interact with glycoprotein 1b, which activates blood cogulation factor VIII. Physiologically, it selective cleave Arg-|-Xaa bond in fibrinogen, to form fibrin and release fibrinopeptide A [5]. Thus, this enzyme encourages us to do docking analysis with fibrinopeptide and get information about its precise role in the blood coagulation process.…”

Expressão e caracterização de uma protease de interesse biotecnológico clonada da glândula de peçonha de Crotalus durissus collilineatus

“…(MARKLAND et al, 1982), da batroxobina de Bothrops atrox (NISHIDA et al, 1994) e da ancrod de Calloselasma rhodostoma (NOLAN et al, 1976).…”