Multipurpose HTS Coagulation Analysis: Assay Development and Assessment of Coagulopathic Snake Venoms (original) (raw)
Related papers
Toxicon, 2010
Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p < 0.0001), Hoplocephalus stephensii (Stephen's-banded snake; p < 0.0001) and Notechis scutatus (tiger snake; p ¼ 0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy.
Thrombosis Research, 2016
Background: Animal models are used to test toxic effects of snake venoms/toxins and the antivenom required to neutralise them. However, venoms that cause clinically relevant coagulopathy in humans may have differential effects in animals. We aimed to investigate the effect of different procoagulant snake venoms on various animal plasmas. Methods: Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were measured in seven animal plasmas (human, rabbit, cat, guinea pig, pig, cow and rat). In vitro clotting times were then used to calculate the effective concentration (EC 50) in each plasma for four snake venoms with different procoagulant toxins: Pseudonaja textilis, Daboia russelli, Echis carinatus and Calloselasma rhodostoma. Results: Compared to human, PT and aPTT were similar for rat, rabbit and pig, but double for cat and cow, while guinea pig had similar aPTT but double PT. Fibrinogen and D-dimer levels were similar for all species. Human and rabbit plasmas had the lowest EC 50 for P. textilis (0.1 and 0.4 μg/ml), D. russelli (0.4 and 0.1 μg/ml), E. carinatus (0.6 and 0.1 μg/ml) venoms respectively, while cat plasma had the lowest EC 50 for C. rhodostoma (11 μg/ml) venom. Cow, rat, pig and guinea pig plasmas were highly resistant to all four venoms with EC 50 10-fold that of human. Conclusions: Different animal plasmas have varying susceptibility to procoagulant venoms, and excepting rabbits, animal models are not appropriate to test procoagulant activity. In vitro assays on human plasma should instead be adopted for this purpose.
Characterization of snake venom components acting on blood coagulation and platelet function
Toxicon, 1992
C. OUYANG, C.-M. T>nvG and T.-F. HUANG. Characterization of snake venom components acting on blood coagulation and platelet function. Toxicon 30, 945-966, 1992 .-Snake venoms can affect blood coagulation and platelet function in various ways. The physicochemical properties and the mechanisms of actions of the snake venom components affecting blood coagulation and platelet function are discussed.
Acta Biochimica Polonica, 2012
Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic a...
Evaluation of the Coagulant Effect of Zanjani and Latifi Viper Snake Venom Endemic in Iran
2016
The venom of the viper is very important in pharmaceutical usage, such as in the process of coagulation during medical care. This study aims to evaluate the coagulant factor of Latifi and Zanjani viper venom. In the current study, after electrophoresis of proteins found in viper venom, all of the thick and strong bands of proteins were isolated and prepared for examination of coagulation characteristics, like pro-thrombin time (PTT) and active partial thromboplastic time (APTT), followed by further study by mass spectroscopy. In this way, 11 bands of proteins were recognized in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After PT and APTT tests, one common band in 26 KDa could lead to coagulation of blood plasma in less than one second. Mass spectroscopy identified this band as serine protease isoform4. The results confirmed the coagulation effect of a 26 KDa protein fraction of venom from Latifi and Zanjani vipers. Highlights The viper snake venom is most...
Coagulopathy Caused by the Main Anticoagulant Fractions of Echis carinatus Snake Venom on Blood
Background: The venom of Viperidae snakes is a compound liquid rich in medicinally active proteins and peptides. It is an invasive weapon for preys immobilization, killing and digestion. Materials and Methods: With a combination of gel and ion exchange chromatography ten sub-fractions were isolated from the E.carinatus venom. Three sub-fractions as anticoagulant sub-fractions were then intravenously injected to mice. Blood sampling was taken before and after injecting these three sub-fractions. The PT, PTT and FT were recorded. Results: Comparison of the PT before and after injecting three sub-fractions, showed that the blood coagulation time after injection is more than the blood normal coagulation time and also more than the coagulation time after the crude venom injection. This coagulation time difference shows the intense coagulation activity of these sub-fractions which thus significantly decrease the rate of coagulation cascade activity and lead to slow blood coagulation. Conclusion: Comparison of the PT and PTT after injecting three sub-fractions with this test normal time respectively showed that the rate of the mice blood coagulation extrinsic and intrinsic system activity rate considerably decreases. By comparing the FT after injecting with this test normal time, coagulation cascade intense inactivation and the nonproduction of fibrin can be inferred. Keywords: Snake Venom; Chromatography; Anticoagulants; Echis Carinatus; Blood Coagulation.
2016
HIGHLIGHTS • The viper snake venom is mostly blood-oriented and leads to blood coagulation. • The snakes venom, including vipers venom endemic of Iran could be applied for pharmaceutical purposes. • A 26KD protein was the most effective component of viper venom showed plasma coagulation. • The results showed this protein was related to serine protease enzyme of snake venom. The venom of the viper is very important in pharmaceutical usage, such as in the process of coagulation during medical care. This study aims to evaluate the coagulant factor of Latifi and Zanjani viper venom. In the current study, after electrophoresis of proteins found in viper venom, all of the thick and strong bands of proteins were isolated and prepared for examination of coagulation characteristics, like pro-thrombin time (PTT) and active partial thromboplastic time (APTT), followed by further study by mass spectroscopy. In this way, 11 bands of proteins were recognized in sodium dodecyl sulphate polyacrylam...
Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas
Toxicon, 1993
M. L. SANTORO and I. S. SANO-MARTINS . Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas. Toxicon 31, 733-742, 1993 .Bothrops jararaca venom is approximately 3.5 times more effective at coagulating rabbit plasma than human plasma . To investigate this difference B.jararaca venom was treated with several enzymatic inhibitors and the minimum coagulant dose was determined both on plasma anticoagulated with sodium citrate or a mixture of sodium citrate and heparin, and on fibrinogen (both human and rabbit). On human plasma, the thrombin-like component of the venom accounted for c. 60% of the coagulant activity, such activity was negligible on rabbit plasma . The venom had little clotting activity on rabbit fibrinogen . The factor II-and X-activator components could be inhibited by EDTA, EGTA and 2-mercaptoethanol, whereas the thrombin-like activity was inhibited by PMSF . These differences show that (using human plasma) B. jararaca clotting activity is mainly due to the thrombin-like component, whereas the factor II-and X-activator components are more important on rabbit plasma . The delayed action of the thrombin-like enzyme on rabbit fibrinogen may be attributed to the difference between rabbit and human fibrinopeptide A. Thus, the increased coagulant activity on rabbit plasma may be due to a faster rate of activation of factor X, V or II by snake venom enzymes.
Procoagulant activities in venoms from central Asian snakes
Toxicon, 1991
activities in venoms from central Asian snakes. Toxicon 29, 491-502, 1991 .-The venoms from central Asian snakes (Echis carinatus, Echis multisquamatus, Vipera ursini, Vipera lebetina, Agkistrodon halys halys and Naja naja oxiana) contain several enzymes with amidolytic-and procoagulant activity . We have characterized the activities and the mol. wts of the venom enzymes that are able to convert a number of commercially available chromogenic substrates for activated coagulation factors. The chromogenic substrate cleavage patterns obtained for the crude venoms may be helpful tools in the further identification of venom fractions and venom enzymes with procoagulant activity . The crude venoms were also tested for their ability to clot fibrinogen, to lyse fibrin polymers and to activate the coagulation factors prothrombin, factor X and factor V. The products of venom-catalyzed coagulation factor activation were structurally characterized by SDS gel electrophoresis and were compared with activated coagulation factors that are generated under physiological conditions .
Bratislava Medical Journal, 2019
OBJECTIVE: Viperidae snakes are responsible for 95 % of the bites caused by exotic-bred snakes in our country. Their envenoming may be associated with a severe acute coagulation disorder-venom-induced consumption coagulopathy (VICC). Thus, its early prediction is vital for an adequate therapy including antivenom delivery. MATERIAL AND METHODS: Laboratory coagulation tests of 14 patients suffering from VICC were processed and statistically analyzed to evaluate the importance of individual parameters in the time after the bite. RESULTS: The pathological values of D-dimer (D-dim) and fi brinogen (FBG) were found to be the fi rst indicators of VICC development, with a median time of 4.55 hours since the bite, while median values for prothrombin time and international normalized ratio (PT/INR), activated partial thromboplastin time (APTT), and thrombin time (TT) were 5.9 h, 8.15 h, and 5.5 h, respectively. In the fi rst samples, the values of D-dim were found to be pathologically increased in all 14 patients, while pathological levels of FBG were seen only in 11 cases. PT/ INR and APTT were prolonged in 8 and 6 cases, respectively. CONCLUSION: An increase in D-dim values was found to be the fi rst parameter signaling developing VICC in all analyzed cases (Tab. 2, Ref. 12).