The Contrasting Effects of Bothrops lanceolatus and Bothrops atrox Venom on Procoagulant Activity and Thrombus Stability under Blood Flow Conditions

Abstract

Background: Consumption coagulopathy and hemorrhagic syndrome are the typical features of Bothrops sp. snake envenoming. In contrast, B. lanceolatus envenoming can induce thrombotic complications. Our aim was to test whether crude B. lanceolatus and B. atrox venoms would display procoagulant activity and induce thrombus formation under flow conditions.

Methods and principal findings: Fibrin formation in human plasma was observed for B. lanceolatus venom at 250-1000 ng/mL concentrations, which also induced clot formation in purified human fibrinogen, indicating thrombin-like activity. The degradation of fibrinogen confirmed the fibrinogenolytic activity of B. lanceolatus venom. B. lanceolatus venom displayed consistent thrombin-like and kallikrein-like activity increases in plasma conditions. The well-known procoagulant B. atrox venom activated plasmatic coagulation factors in vitro and induced firm thrombus formation under high shear rate conditions. In contrast, B. lanceolatus venom induced the formation of fragile thrombi that could not resist shear stress.

Conclusions: Our results suggest that crude B. lanceolatus venom displays amidolytic activity and can activate the coagulation cascade, leading to prothrombin activation. B. lanceolatus venom induces the formation of an unstable thrombus under flow conditions, which can be prevented by the specific monovalent antivenom Bothrofav^®.

Keywords: B. atrox; B. lanceolatus; Bothrops snake envenoming; antivenom; kallikrein; procoagulant; shear stress; thrombin; thrombosis.

Figure A1

Fibrinogen clotting time. Evolution of fibrinogen clotting time, determined as the evolution of optical density at 405 nm over time in the presence of 1000 ng/mL of B. atrox (red curve), 1000 ng/mL of B. lanceolatus at 1000 ng/mL (blue), 10⁵ ng/mL (dark blue), and the control (black curve).

Figure A2

Traces displaying thrombus formation. The occlusion start time (OST) for the control, B. lanceolatus (with and without Bothrofav^®), at 1000 ng/mL is indicated. OST refers to the lag time for the flow pressure to reach 10 kPa, due to partial occlusion of the capillary. Occlusion time (OT) represents the lag time for the flow pressure to reach 60 kPa from the baseline pressure. Thrombus formation growth rates (T₁₀–T₆₀) refer to the lag time for the flow pressure to reach 60 kPa from 10 kPa of pressure.

Figure A3

Comparison of the respective abilities of B. lanceolatus and B. atrox venoms to activate coagulation factors at 1000 ng/mL, using the synthetic peptides, pNAPEP 0216, 1032, and 1266, which are the specific synthetic chromogenic substrates for the measurement of thrombin, FXa, and kallikrein activities. Results are expressed as increases in optical density (OD) at 405 nm. Data are the mean ± SD; n = 4–6; **** indicates p < 0.0001.

Figure A4

Comparisons of the respective amidolytic activity of crude B. lanceolatus and B. atrox venoms at 1000 ng/mL on the synthetic peptides, pNAPEP 0216, 1032, and 1266, which are the specific synthetic chromogenic substrate for the measurement of thrombin, FXa, and kallikrein activities. The results are expressed as increases in optical density (OD) at 405 nm. Data are mean ± SD; n = 4–6; ** and **** indicate p < 0.02 and p < 0.0001, respectively.

Figure 1

Effect of B. lanceolatus and B. atrox on fibrin formation, measured as optical density (OD) increased at 405 nm. (A) Kinetics of fibrin formation in the presence of B. lanceolatus venom. (B) Time of recalcification in the presence of B. lanceolatus venom. (C) Kinetics of fibrin formation in the presence of B. atrox venom. (D) Time of recalcification in the presence of B. atrox venom. Control 0 ng/mL (black curve), 100 ng/mL (yellow curve), 250 ng/mL (blue curve), 500 ng/mL (green curve), 1000 ng/mL (dark blue curve), and 1000 ng/mL without calcium (brown curve). Bothrofav^® prevented the procoagulant activity of crude B. lanceolatus venom at a venom concentration of 1000 ng/mL (grey curve). Data are mean ± SD; n = 6–8; ns: non-significant; *, **, *** and **** indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 compared with controls, respectively.

Figure 2

Effect of B. atrox and B. lanceolatus venoms upon purified human fibrinogen. (A) Fibrinogenolytic activity of B. atrox venom at 1000 ng/mL. (B) Fibrinogenolytic activity of B. lanceolatus venom at 1000 ng/mL and (C) at 10⁵ ng/mL, assessed by reducing SDS-gel electrophoresis (12%). Column 1: molecular mass standard; column 2: fibrinogen control; column 3: fibrinogen incubated with venom for 10 min; column 4: fibrinogen incubated with venom for 20 min; column 5: fibrinogen incubated with venom for 30 min; column 6: fibrinogen incubated with venom for 60 min; column 7: 1000 ng/mL of B. atrox and B. lanceolatus venom for (A) and (B), respectively, and 10⁵ ng/mL of B. lanceolatus venom alone.

Figure 3

Activation of coagulation factors by B. lanceolatus venom. Kinetics of the activation of prothrombin (A); releases of pNA (0216) in plasmatic conditions (B), non-plasmatic conditions (C) and differences in OD between the two conditions (D) in the presence of various concentrations of B. lanceolatus venom; (E) kinetics of the activation of Factor X; releases of pNA (1032) in plasmatic conditions (F); non-plasmatic conditions (G) and differences in OD between the two conditions (H) in the presence of various concentrations of B. lanceolatus venom; (I) kinetics of the activation of prekallikrein; release of pNA (1266) in plasmatic conditions (J); non-plasmatic conditions (K) and differences in OD between the two conditions (L) in the presence of various concentrations of B. lanceolatus venom. The release of pNa in plasmatic conditions is due to either the activation of coagulation factor and/or the amidolytic activity of venom enzymes. Direct cleavage of pNAPEP 0216 (thrombin), pNAPEP 1032 (FXa), and pNAPEP 1266 (kallikrein) by crude venom (non-plasmatic conditions) indicates the increased amidolytic activity of the venom on the studied substrates. Positive differences in OD between plasmatic and non-plasmatic conditions indicate the activation of plasmatic clotting factors, i.e., prothrombin to thrombin and prekallikrein to kallikrein, respectively. Data are mean ± SD; n = 4–8; *, *** and **** indicate p < 0.05, p < 0.001 and p < 0.0001, compared with the controls, respectively.

Figure 4

Activation of coagulation factor by B. atrox venom. (A) Kinetics of the activation of prothrombin; releases of pNA (0216) in plasmatic conditions (B); non-plasmatic conditions (C) and differences in OD between the two conditions (D) in the presence of various concentrations of B. atrox venom. (E) Kinetics of the activation of Factor X; releases of pNA (1032) in plasmatic conditions (F); non-plasmatic conditions (G) and differences in OD between the two conditions (H) in the presence of various concentrations of B. atrox venom. (I) Kinetics of the activation of prekallikrein; releases of pNA (1266) in plasmatic conditions (J); non-plasmatic conditions (K) and differences in OD between the two conditions (L) in the presence of various concentrations of B. atrox venom. Release of pNa in plasmatic conditions is due to either the activation of a coagulation factor and/or the amidolytic activity of venom enzymes. The direct cleavage of pNAPEP 0216 (thrombin), pNAPEP 1032 (FXa), and pNAPEP 1266 (kallikrein) by crude venom (non-plasmatic conditions) indicates the increased amidolytic activity of the venom on the studied substrates. Positive differences in optical density between plasmatic and non-plasmatic conditions indicate the activation of plasmatic clotting factors, i.e., prothrombin to thrombin and prekallikrein to kallikrein, respectively. Data are mean ± SD; n = 4–8; **, *** and **** indicate p < 0.001, p < 0.001 and p < 0.0001 compared with the controls, respectively.

Figure 5

Assessment of the activation of purified human coating factors. (A) Activation of purified human prothrombin. (B) Activation of purified human FX in the presence of B. lanceolatus and B. atrox venoms at 1000 ng/mL. The thrombin-activator venom protease ecarin and coagulation FX activating enzyme from Russell’s viper venom (RVV-X) were used as positive controls at 1000 ng/mL and 1 UI/mL, respectively. Data are mean ± SD; n = 4–6; ns: non-significant; **, *** and **** indicate p < 0.01, p < 0.001 and p < 0.0001 compared with controls, respectively.

Figure 6

Whole blood coagulation activity (AR-Chip) in the presence of B. lanceolatus venom. (A) Thrombus formation displayed as the changes in occlusion starting time (OST, s); (B) thrombus formation growth rates (T₁₀–T₆₀ time, s); and (C) occlusion time (OT, s). Data are mean ± SD; n = 5–7; ns: non-significant, **, *** and **** indicate p < 0.01, p < 0.001, and p < 0.0001, compared with controls, respectively.

Figure 7

Comparison of whole blood coagulation activity (AR-Chip) of B. atrox and B. lanceolatus venoms. (A) Comparisons of occlusion starting times (OST, s), (B) T₁₀-T₆₀ time (s) and (C) occlusion time (OT, s) in whole blood coagulation activity (AR-Chip) in the presence of B. lanceolatus and B. atrox venoms at 500 ng/mL. Data are mean ± SD; n = 4–8; ns: non-significant; * and ** indicate p < 0.01 and p < 0.05, respectively.