Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis

Abstract

To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes.

Keywords: Vitamin K; factor II; hemolysis; kidney; metalloproteinases; platelets.

Figure 1

Schematic representation of timing of pretreatment (saline/warfarin), treatments (saline, B. jararaca venom) and collection of blood samples. (A color version of this figure is available in the online journal.)

Figure 2

MCD curves of BjV on rat plasmas. The coagulating activity of BjV was assayed on plasma pools from saline- (black circles) and warfarin-treated (red squares) rats, as well as in normal plasma pools previously depleted of VKDCF by BaCl₂ precipitation (blue triangle). The curves are representative of two different experiments. (A color version of this figure is available in the online journal.)

Figure 3

Plasma levels of fibrinogen (a), prothrombin (b), and factor X (c) activities, and platelet counts (d) in rats pretreated or not with warfarin for three days, and injected s.c. with BjV (1.6 mg/kg) or 154 mmol/L NaCl. Blood samples were collected at 3, 6, and 24 h after venom or saline injection. Statistically significant difference between groups: †P < 0.02; *P < 0.001. Data are expressed as mean ± SEM (n = 5–6 animals/group). (A color version of this figure is available in the online journal.)

Figure 4

RBC counts (a) and plasma hemoglobin levels (b) in rats pretreated or not with warfarin for three days, and injected s.c. with BjV (1.6 mg/kg) or 154 mmol/L NaCl. Blood samples were collected at 3, 6, and 24 h after venom or saline injection. Statistically significant difference between groups: †P < 0.03; #P = 0.007; *P < 0.001. Data are expressed as mean ± SEM (n = 5–6 animals/group). (A color version of this figure is available in the online journal.)

Figure 5

Representative pictures of red blood cell morphology of experimental groups. Rats were pretreated with warfarin or saline for three days, and then injected s.c. with BjV (1.6 mg/kg, s.c.) or saline. Blood samples were collected at 3, 6, and 24 h after BjV or saline injection. Note the presence of schizocytes (black arrowheads) in Sal + BjV group at all time intervals analyzed, but not in Sal + Sal, War + BjV, and War + Sal groups at 3 h. Increased polychromasia was also observed in blood smears from Sal + BjV, particularly at 3 and 6 h. (A color version of this figure is available in the online journal.)