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Studies on blood coagulation in patients bitten by Bothrops atrox
Abstracts / Toxicon 38 (2000) 487 595
575
J.G. Rap6so, A.C. Przytyk, D.C. Lima, C.M. Oliveira, D.L.S. Dutra, R.Q. Monteiro, R.B. Zingali (Departamento de Bioq. M~dica, ICB/CCS, UFRJ, Rio de Janeiro, RJ, Brazil). Objectives: Bothrops insularis (jararaca ilhoa) is a geographically isolated snake exclusively found in Queimada Grande island in Sao Paulo. We have analyzed the physico-chemical and biological properties of this crude venom and partially purified principles affecting blood coagulation. Methods and results: B. insularis venom showed proteins with MW ranging from 20 to 70 kDa and pI varying between 4.0 and 7.0, when analyzed by SDS-PAGE, IEF in a Phast System apparatus (Pharmacia). The analysis of human plasma coagulation (in the absence of Ca 2+) and recalcification times measured in a coagulometer showed that this venom possess a very high coagulant activity partially dependent on Ca 2+ since the recalcification time (31 s for 10 ~tg/ml) was twice lower than plasma coagulation time (56 s for 10 gg/ml). It was able to inhibit thrombin-induced platelet aggregation (ICs0 = 6 gg/ml), and it possess a notable plasminogen activating and fibrinogenolytic activities. Fractionation of crude venom, by gel filtration chromatography (Sephacryl S-200 Pharmacia), separate different coagulant activities. At the first fraction (,-~70 kDa) we observed a high coagulant activity not dependent on Ca 2+ without any direct activity upon fibrinogen. The second fraction (~30 kDa) contains a thrombin-like activity since it was able to promote fibrino-coagulation. The third fraction (~20 kDa) possess an anti-coagulant effect in part due to a fibrinogenolytic activity. The fourth fraction (eluted after vt) contains a coagulant activity not dependent on calcium. Conclusion: B. insularis has a prominent coagulant activity due in part to thrombin-like enzyme(s) and to Ca 2+ dependent components. It possess a strong anti-aggregating activity, a high proteolytic activity degrading fihrinogen, and activating plasminogen, thus indicating the presence of more than one component that possess anti-hemostatic properties. Acknowledgements: Financial support from PADCT, Finep/BID, CNPq, CEE, FAPERJ.
Studies on blood coagulation in patients bitten by Bothrops atrox. S.C. Tomy a, M.C.C. Sousa-e-Silva a, I.S. Sano-Martins a, P.P.O. Pardal b, A.S. Coimbra b, B.M. Silva b, E.S. Maciel b, J.L.C. Cardoso c, R.D.G. Theakston d (aLaboratorio Fisiopatologia; bHospital JoSo Barros Barreto, Bel6m, PA, Brazil; CHospital Vital Brazil, Instituto Butantan, SP, Brazil; dLiverpool School of Tropical Medicine, Liverpool, UK). Aim: Blood coagulation disturbances are common findings in patients envenomed by South American Bothrops snakes. This study evaluated hemostatic alterations observed in five human patients bitten by Bothrops atrox snakes admitted at Hospital Joao Barros Barreto, Bel6m, Par{t State (Brazil). Methods: The following parameters were assayed: fibrinogen (colorimetric method), factors II, V, VII, VIII, IX, X, XI, XII, alpha2-antiplasmin, fibrin(ogen) degradation products, cross-linked fibrin degradation products (Diagnostica Stago-France) and thrombin-antithrombin III complex (Behring). These assays were performed in blood collected on admission, at 12 and 24 h after antivenom therapy and discharge. Results: The levels of fibrinogen (0.09-1.28 ~tg/L) and
576
Abstracts'/Toxtcon 38 (2000) 487-595
alpha2-antiplasmin (22-58%) were decreased. Blood coagulation factors II (4670%), V (27-90%) and VIII (25-125%) were also decreased, but the other factors were normal. Fibrin(ogen) degradation products (160-1280 Ixg/mL), cross-linked fibrin degradation products (4-128 gg/mL) and thrombin-antithrombin complex (23.2 150.7 pg/L) were increased in all patients studied. Conclusion: These data suggest that B. atrox envenomation induce hypofibrinogenemia, secondary activation of fibrinolytic system and intravascular thrombin generation, similarly to Bothrops jararaca envenomation. Acknowledgements: Financial support from European Community.
• Evaluation of the clotting t#ne of bovine thrombin, reptilase and thrombin-like fi'action of Crotahts durissus terrificus venom using cryoprecipitate from humans and different animals. I.A. Thomazini-Santos a"d M.J.S.M. Giannini b' d E. Toscano c"d P.E.A. Machado a, C.R.G. Lima c, B. Barraviera c'd (~Divis~o Hemocentro da FM de Botucatu: bLaboratorio de Micologia, Campus de Araraquara; CDepartamento de Doengas Tropicais e Diagn6stico por Imagem; dCEVAP/UNESP, Botucatu, SP). The objective of the present work was to evaluate the effects of thrombin-like fraction of Crotalus durissus terrificus venom, of Reptilase ~ and of bovine thrombin on fibrinogen pool in cryoprecipitate from humans, as well as that of different animals such as bovine, equine, ovine and bubaline. The authors also verified which of the cryoprecipitates studied presented behavior similar to that extracted from humans for possible use in medical practice. Forty-eight samples of cryoprecipitates derided into five groups were studied: GT=humans, n - 1 2 (m = 375.50 +/-70.95); G2 = bovine, t7= 9 (m = 218.33 +/-4.47); G3 = equine, n = 10 (m=240.80+/-29.91); G4=ovine, 1l=10 (m=267.70+/-9.50): and Gs=bubaline, n = 7 (m-664.00+/-1.80). Fibrinogen level was determined by the method of Ratnoff and Menzie, and the clotting time was determined by the method of Rapaport and Ames. The results obtained were: the clotting time obtained with bovine thrombine, Reptilase ® and thrombin-like fraction was similar for humans and bovine. Upon comparing the clotting times obtained with thrombin-like fraction of C.d.t venom and Reptilase ® using cryoprecipitate from humans and different animals, it was verified that for humans and bovine these results were similar. The other cryoprecipitates presented differences from dilution 1:3. The authors hypothesized that the use of bovine cryoprecipitate can be indicated using either bovine thrombin, Reptilase '~ or thrombin-like fraction of C.d.t venom. Further toxicological studies are needed to validate this hypothesis. Effects of bothropic antivenom on the dynamic of microcirculatory events induced by Bothrops jararaca venom (BjV). G.F. Aguiar a, H.W. Fan b, D.F. Cardoso c, Y. Cury a, S.H.P. Farsky a (aLaborat6rio de Fisiopatologia; bHospital Vital Brazil, Instituto Butantan, Av. Vital Brazil, 1500, S~o Paulo, SP, 05503-900, Brazil.; CLaborat6rio de Imunopatologia). Objectives: Bothropic antivenom is able to
575
J.G. Rap6so, A.C. Przytyk, D.C. Lima, C.M. Oliveira, D.L.S. Dutra, R.Q. Monteiro, R.B. Zingali (Departamento de Bioq. M~dica, ICB/CCS, UFRJ, Rio de Janeiro, RJ, Brazil). Objectives: Bothrops insularis (jararaca ilhoa) is a geographically isolated snake exclusively found in Queimada Grande island in Sao Paulo. We have analyzed the physico-chemical and biological properties of this crude venom and partially purified principles affecting blood coagulation. Methods and results: B. insularis venom showed proteins with MW ranging from 20 to 70 kDa and pI varying between 4.0 and 7.0, when analyzed by SDS-PAGE, IEF in a Phast System apparatus (Pharmacia). The analysis of human plasma coagulation (in the absence of Ca 2+) and recalcification times measured in a coagulometer showed that this venom possess a very high coagulant activity partially dependent on Ca 2+ since the recalcification time (31 s for 10 ~tg/ml) was twice lower than plasma coagulation time (56 s for 10 gg/ml). It was able to inhibit thrombin-induced platelet aggregation (ICs0 = 6 gg/ml), and it possess a notable plasminogen activating and fibrinogenolytic activities. Fractionation of crude venom, by gel filtration chromatography (Sephacryl S-200 Pharmacia), separate different coagulant activities. At the first fraction (,-~70 kDa) we observed a high coagulant activity not dependent on Ca 2+ without any direct activity upon fibrinogen. The second fraction (~30 kDa) contains a thrombin-like activity since it was able to promote fibrino-coagulation. The third fraction (~20 kDa) possess an anti-coagulant effect in part due to a fibrinogenolytic activity. The fourth fraction (eluted after vt) contains a coagulant activity not dependent on calcium. Conclusion: B. insularis has a prominent coagulant activity due in part to thrombin-like enzyme(s) and to Ca 2+ dependent components. It possess a strong anti-aggregating activity, a high proteolytic activity degrading fihrinogen, and activating plasminogen, thus indicating the presence of more than one component that possess anti-hemostatic properties. Acknowledgements: Financial support from PADCT, Finep/BID, CNPq, CEE, FAPERJ.
Studies on blood coagulation in patients bitten by Bothrops atrox. S.C. Tomy a, M.C.C. Sousa-e-Silva a, I.S. Sano-Martins a, P.P.O. Pardal b, A.S. Coimbra b, B.M. Silva b, E.S. Maciel b, J.L.C. Cardoso c, R.D.G. Theakston d (aLaboratorio Fisiopatologia; bHospital JoSo Barros Barreto, Bel6m, PA, Brazil; CHospital Vital Brazil, Instituto Butantan, SP, Brazil; dLiverpool School of Tropical Medicine, Liverpool, UK). Aim: Blood coagulation disturbances are common findings in patients envenomed by South American Bothrops snakes. This study evaluated hemostatic alterations observed in five human patients bitten by Bothrops atrox snakes admitted at Hospital Joao Barros Barreto, Bel6m, Par{t State (Brazil). Methods: The following parameters were assayed: fibrinogen (colorimetric method), factors II, V, VII, VIII, IX, X, XI, XII, alpha2-antiplasmin, fibrin(ogen) degradation products, cross-linked fibrin degradation products (Diagnostica Stago-France) and thrombin-antithrombin III complex (Behring). These assays were performed in blood collected on admission, at 12 and 24 h after antivenom therapy and discharge. Results: The levels of fibrinogen (0.09-1.28 ~tg/L) and
576
Abstracts'/Toxtcon 38 (2000) 487-595
alpha2-antiplasmin (22-58%) were decreased. Blood coagulation factors II (4670%), V (27-90%) and VIII (25-125%) were also decreased, but the other factors were normal. Fibrin(ogen) degradation products (160-1280 Ixg/mL), cross-linked fibrin degradation products (4-128 gg/mL) and thrombin-antithrombin complex (23.2 150.7 pg/L) were increased in all patients studied. Conclusion: These data suggest that B. atrox envenomation induce hypofibrinogenemia, secondary activation of fibrinolytic system and intravascular thrombin generation, similarly to Bothrops jararaca envenomation. Acknowledgements: Financial support from European Community.
• Evaluation of the clotting t#ne of bovine thrombin, reptilase and thrombin-like fi'action of Crotahts durissus terrificus venom using cryoprecipitate from humans and different animals. I.A. Thomazini-Santos a"d M.J.S.M. Giannini b' d E. Toscano c"d P.E.A. Machado a, C.R.G. Lima c, B. Barraviera c'd (~Divis~o Hemocentro da FM de Botucatu: bLaboratorio de Micologia, Campus de Araraquara; CDepartamento de Doengas Tropicais e Diagn6stico por Imagem; dCEVAP/UNESP, Botucatu, SP). The objective of the present work was to evaluate the effects of thrombin-like fraction of Crotalus durissus terrificus venom, of Reptilase ~ and of bovine thrombin on fibrinogen pool in cryoprecipitate from humans, as well as that of different animals such as bovine, equine, ovine and bubaline. The authors also verified which of the cryoprecipitates studied presented behavior similar to that extracted from humans for possible use in medical practice. Forty-eight samples of cryoprecipitates derided into five groups were studied: GT=humans, n - 1 2 (m = 375.50 +/-70.95); G2 = bovine, t7= 9 (m = 218.33 +/-4.47); G3 = equine, n = 10 (m=240.80+/-29.91); G4=ovine, 1l=10 (m=267.70+/-9.50): and Gs=bubaline, n = 7 (m-664.00+/-1.80). Fibrinogen level was determined by the method of Ratnoff and Menzie, and the clotting time was determined by the method of Rapaport and Ames. The results obtained were: the clotting time obtained with bovine thrombine, Reptilase ® and thrombin-like fraction was similar for humans and bovine. Upon comparing the clotting times obtained with thrombin-like fraction of C.d.t venom and Reptilase ® using cryoprecipitate from humans and different animals, it was verified that for humans and bovine these results were similar. The other cryoprecipitates presented differences from dilution 1:3. The authors hypothesized that the use of bovine cryoprecipitate can be indicated using either bovine thrombin, Reptilase '~ or thrombin-like fraction of C.d.t venom. Further toxicological studies are needed to validate this hypothesis. Effects of bothropic antivenom on the dynamic of microcirculatory events induced by Bothrops jararaca venom (BjV). G.F. Aguiar a, H.W. Fan b, D.F. Cardoso c, Y. Cury a, S.H.P. Farsky a (aLaborat6rio de Fisiopatologia; bHospital Vital Brazil, Instituto Butantan, Av. Vital Brazil, 1500, S~o Paulo, SP, 05503-900, Brazil.; CLaborat6rio de Imunopatologia). Objectives: Bothropic antivenom is able to