Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom.

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: A rapid procedure.

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12 h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: a rapid procedure

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12?h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: a rapid procedure

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12?h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Avaliação dos níveis de frutosamina em gatos sob estresse agudo e crônico / Assessment of fructosamine concentrations in cats with acute and chronic-stress

Frutosaminas são proteínas séricas glicadas formadas continuamente resultantes da ligação entre a glicose e proteínas circulantes, e correspondem à avaliação glicêmica de aproximadamente uma a duas semanas em gatos. A concentração de frutosamina tem sido utilizada para a diferenciação entre a hiperglicemia persistente e transitória induzida pelo estresse, sendo considerado o teste padrão ouro para o controle da glicemia em gatos diabéticos. O objetivo deste trabalho consistiu em avaliar a influência dos estados de estresse agudo e crônico em gatos sobre os níveis séricos de frutosamina. Foram selecionados 62 felinos provenientes do atendimento no Hospital Veterinário da FMVZ - UNESP Botucatu, distribuídos em três grupos: felinos com histórico de qualquer doença ou condições de estresse, excluindose o diabetes mellitus (DM), por um período máximo de 48 horas (Grupo A, n = 21) ou por um período superior a 120 horas (Grupo B, n = 27). O terceiro grupo (Grupo C = controle) foi formado por 14 felinos saudáveis. Os grupos foram avaliados quanto às dosagens séricas de frutosamina, glicose, proteína e albumina. Foi constatado um aumento significativo nos valores de frutosamina tanto nos animais submetidos ao estresse agudo quanto crônico, porém os níveis mantiveram-se dentro do intervalo de referência. Da mesma forma, os animais, em média, também se encontravam em normoglicemia, apesar da correlação positiva entre as concentrações de glicose e frutosamina. Conclui-se que a concentração de frutosamina sofre influência dos estados de estresse agudo e crônico em gatos, mantendo-se, porém, dentro dos limites de referência, sendo, portanto, útil no diagnóstico do DM.

circulating proteins, and corresponding to the blood glucose control assessment over the last one to two weeks in cats. The fructosamine concentration has been used for differentiation between persistent and transient hyperglycemia. Therefore, the determination of fructosamine is considered the gold standard for monitoring glycemia into control in diabetic cats. The objective of this study was to evaluate the influence of acute and chronic stress of cats on serum fructosamine. 62 cats were selected from the Veterinary Hospital of FMVZ - UNESP, Botucatu campus. They were distributed into three groups: cats with a history of any illness or stress condition, excluding Diabetes Mellitus (DM), for a maximum of 48 hours (Group A, n = 21) or for a period exceeding 120 hours (Group B n = 27). The third group (Group C = control) was formed by 14 health cats. The groups were evaluated for serum fructosamine, glucose, protein and albumin. In this study, there was a significant increase in the values of fructosamine in animals subjected to acute and chronic stress, but these values remained within the reference range. The animals were, on average, normoglycemic, despite the positive correlation between fructosamine and glucose concentrations. We conclude that the fructosamine concentration is influenced by acute and chronic stress in cats, remaining, however, within the reference range, and therefore, still useful in the diagnosis of DM.