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.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin.

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 µg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1-/-) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced ß-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

The gingival vein as a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters.

Laboratory animals are still necessary in scientific investigation and vaccine testing, but while novel methodological approaches are not available for their replacement, the search for new, humane, easy, and painless methods is necessary to diminish their stress and pain. When multiple blood samples are to be collected from hamsters and guinea pigs, the number of available venipuncture sites-which are greatly diminished in these species in comparison with other rodents due to the absence of a long tail-, harasses animal caregivers and researchers. Thus, this study aimed to evaluate if gingival vein puncture could be used as an additional route to obtain multiple blood samples from anesthetized hamsters and guinea pigs in such a way that animal behavior, well-being or hematological parameters would not be altered. Thus, twelve anesthetized Syrian golden hamsters and English guinea pigs were randomly allocated in two groups: a control group, whose blood samples were not collected, and an experimental group in which blood samples (200 microliters) were collected by gingival vein puncture at weekly intervals over six weeks. Clinical assessment, body weight gain and complete blood cell count were evaluated weekly, and control and experimental animals were euthanized at week seven, when the mentolabial region was processed to histological analyses. Multiple blood sampling from the gingival vein evoked no clinical manifestations or alteration in the behavioral repertoire, nor a statistically significant difference in weight gain in both species. Guinea pigs showed no alteration in red blood cell, leukocyte or platelet parameters over time. Hamsters developed a characteristic pattern of age-related physiological changes, which were considered normal. Histological analyses showed no difference in morphological structures in the interdental gingiva, confirming that multiple blood sampling is barely traumatic. Thus, these results evidence that blood collection from multiple gingival vein puncture is minimally invasive and traumatic to hamsters and guinea pigs, and that it can be accomplished during at least six weeks.