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 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)
Nishida
S
S
Department of Blood Transfusion, Nara Medical College, Japan.
Fujimura
Y
Y
Miura
S
S
Ozaki
Y
Y
Usami
Y
Y
Suzuki
M
M
Titani
K
K
Yoshida
E
E
Sugimoto
M
M
Yoshioka
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000502:physiology; D017837:Bothrops; D002417:Cattle; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D011817:Rabbits; D051381:Rats; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013379:Substrate Specificity
D003435:Crotalid Venoms; D012701:Serotonin; D005340:Fibrinogen; D012697:Serine Endopeptidases; C086027:bothrombin