A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization.
A novel thrombin inhibitor, Bothrops jararaca inhibitor (BjI), has been identified and purified from B. jararaca snake blood by two anionic chromatographic steps. Purified BjI showed two polypeptide chains with molecular masses of 109 and 138 kDa, by SDS-PAGE in reducing conditions. On the other hand, in nonreducing conditions the molecular masses were 150 and 219 kDa, suggesting that the polypeptide chain 109 kDa can be a dimer form linked by disulfide bond. However, the native BjI shows a molecular mass higher than 1000 kDa by gel filtration chromatography, indicating the need of a quaternary structure formation for the blood coagulation inhibition. BjI is a specific thrombin coagulant activity inhibitor that does not affect other thrombin functions, such as: amidolytic and platelet aggregation activities. BjI is not an antithrombin-like inhibitor. Fibrinogen and heparin competition ELISA assays with BjI and thrombin showed that fibrinogen does not interfere in the BjI and thrombin binding, however, heparin interferes in BjI and thrombin interaction, suggesting that BjI binds to heparin site or other sites close to it. Our findings indicate that BjI is an exosite binding thrombin inhibitor, specific upon coagulant activity thrombin inhibitor, without any anti-platelet aggregation activity.
Tanaka-Azevedo
Anita M
AM
Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brazil. amt.azevedo@uol.com.br
Tanaka
Aparecida S
AS
Sano-Martins
Ida S
IS
D000818:Animals; D000838:Anions; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D015153:Blotting, Western; D017837:Bothrops; D002241:Carbohydrates / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D051379:Mice; D010314:Partial Thromboplastin Time; D010455:Peptides / Q000737:chemistry; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011517:Prothrombin Time; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
D000838:Anions; D000925:Anticoagulants; D001798:Blood Proteins; C477871:Bothrops jararaca inhibitor; D002241:Carbohydrates; D004220:Disulfides; D010455:Peptides; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin