A murine experimental model of the pulmonary thrombotic effect induced by the venom of the snake Bothrops lanceolatus

Abstract

Background: The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved.

Methodology/principal findings: The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection.

Conclusions/significance: An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect.

Fig 1. SDS-PAGE separation of venoms of adult (Ad) and juvenile (Ju) specimens of B. lanceolatus.

Various amounts of venom (10, 20 and 40 μg) were separated under reducing conditions on 4–20% pre-cast gels. Molecular weight (Mw) markers were also run. Proteins were stained with Coomassie Blue R-250.

Fig 2. RP-HPLC separation of venoms of juvenile (A) and adult (B) specimens of B. lanceolatus.

Samples of 2 mg were dissolved in water containing 0.1% trifluoroacetic acid (solution A). After centrifugation, the supernatant was applied to a reverse-phase column equilibrated with solution A and separation was monitored by recording the absorbance at 215 nm. The following gradient of acetonitrile, containing 0.1% trifluoroacetic acid, was used for elution: 0% for 5 min, 0–15% for 10 min, 15–45% for 60 min, 45–70% for 10 min, and 70% for 9 min (red line).

Fig 3. In vitro procoagulant activity of venoms of juvenile and adult specimens of B. lanceolatus.

A: Procoagulant effect on plasma. Fifty μg venom, dissolved in 100 μL TBS, were added to wells in a microplate. Then, 4 μL of 0.4 M CaCl₂ were added to 100 μL of mouse citrated plasma previously incubated at 37°C, and the mixture added to wells in the plate containing the venom solution. Controls included plasma/CaCl₂ incubated with TBS with no venom. After shaking for 5 sec, the absorbances at 340 nm were recorded during 10 min as an index of the increase in turbidity of the samples. No change in absorbance was observed in control samples. B and C: Thrombin-like (pseudo-procoagulant) activity effect of venoms on bovine fibrinogen. In B, solutions containing various amounts of venom, dissolved in 100 μL TBS, were added to 100 μL of a fibrinogen solution (6 mg/mL) previously incubated at 37°C, and the change in absorbance at 340 nm were recorded at 10 min. In C, solutions containing 25 μg venom, dissolved in 100 μL TBS, were added to 100 μL of fibrinogen (6 mg/mL). The changes in absorbance at 340 nm were recorded at various time intervals. Controls included fibrinogen incubated with TBS with no venom. No change in absorbance was observed in control samples. Results are presented as mean ± SEM (n = 3). *p < 0.05.

Fig 4. Effect of i.v. injection of juvenile and adult B. lanceolatus venoms on classical clotting tests.

20 μg venom from juvenile or adult specimens, dissolved in 100 μL PBS, were injected i.v. in mice. Controls received 100 μl PBS. One hour after injection mice were bled under isoflurane anesthesia and blood was collected, added to citrate anticoagulant, and centrifuged for plasma collection to determine prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration. Results are presented as mean ± SEM (n = 4). *p < 0.01 when compared to controls.

Fig 5. Effect of i.v. injection of juvenile and adult B. lanceolatus venoms on rotational thromboelastometry parameters.

Twenty μg venom, dissolved in 100 μL PBS, was injected i.v. in mice. Controls received 100 μl of PBS. On hour after injection mice were bled under isoflurane anesthesia and blood was collected and added to citrate anticoagulant for determination of Extem, Intem and Fibtem parameters. CT: Clotting time; CFT: clot formation time; A20: clot amplitude (in mm) 20 min after CT (see Methods for details). Results are presented as mean ± SEM (n = 4). *p < 0.05, **p<0.01 when compared to control; # p < 0.05 when comparing juvenile and adult venoms. ∞: In the case of Fibtem CFT in envenomed mice, the clot did not reach an amplitude of 20 mm.

Fig 6. Representative Extem rotational thromboelastometry tracings from mice injected with PBS or venoms of juvenile and adult specimens of B. lanceolatus.

Blood was collected by cardiac puncture under inhaled isoflurane anesthesia, added to sodium citrate solution, and evaluated by rotational thromboelastometry (see Methods for details). (A) Extem tracing from a mouse receiving PBS by the i.v. route and bled 1 hr after injection. (B) and (C) Extem tracings from mice receiving 20 μg of juvenile (B) or adult (C) B. lanceolatus venoms by the i.v. route and bled 1 hr after injection. (D) and (E) Extem tracings from mice receiving 70 μg of juvenile (D) or adult (E) venoms by the i.p. route and bled 4 hr after injection. CT (clotting time, green line) is the time lapse (in sec) needed for the formation of a clot amplitude of 2 mm. CFT (clot formation time, pink) is the time lapse (in sec) between 2 mm clot amplitude and 20 mm clot amplitude. A20 (clot amplitude at 20 min) reflects the clot firmness (in mm amplitude) 20 min after CT.

Fig 7. Effects of i.p. injection of juvenile and adult B. lanceolatus venoms on classical clotting tests and platelet counts.

Mice received 70 μg venom, dissolved in 100 μL PBS, by the i.p. route. Controls received 100 μl of PBS. Four hours after injection mice were bled under isoflurane anesthesia and blood was collected and added to citrate anticoagulant for determination of prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and platelet counts (see Methods for details). Results are presented as mean ± SEM (n = 4 in the case of clotting tests and fibrinogen concentration and n = 4–11 in the case of platelet counts). *p < 0.05 when compared to control; # p < 0.05 when comparing juvenile and adult venom.

Fig 8. Effect of i.p. injection of juvenile and adult B. lanceolatus venoms on rotational thromboelastometry parameters.

Mice received 70 μg of venom, dissolved in PBS, by the i.p. route. Controls were injected with 100 μl of PBS. Four hours after injection mice were bled under isoflurane anesthesia and blood was added to citrate anticoagulant for determination of Extem, Intem and Fibtem parameters. CT: Clotting time; CFT: clot formation time; A20: clot amplitude (in mm) 20 min after CT (see Methods for details). Results are presented as mean ± SEM (n = 4). No significant differences (p > 0.05) were observed between the groups.

Fig 9. Light micrographs of sections of pulmonary tissue of mice which received an i.p. injection of venom from juvenile or adult B. lanceolatus.

Sections correspond to samples of mice receiving 100 μL PBS (A) or 70 μg of B. lanceolatus venom from either juvenile (B and C) or adult (D) specimens, dissolved in 100 μL PBS. Mice were sacrificed 4 hr after injections, and samples from pulmonary tissue were collected, added to formalin fixative and processed for embedding in paraffin. Sections from control mice receiving PBS show a normal histological pattern. Abundant thrombi are observed in sections of mice receiving juvenile venom (arrows in B and C). In contrast, samples from mice injected with adult venom are largely devoid of thrombi, and only a pale hyaline material is observed in some vessels (arrow in D). Hematoxylin-eosin staining. Bar represents 100 μm.

Fig 10. Thrombi in the pulmonary microvasculature of mice injected with juvenile B. lanceolatus venom.

Light micrographs of sections of pulmonary tissue of mice which received an i.p. injection of 100 μL PBS (A) or 70 μg of B. lanceolatus venom from juvenile specimens, dissolved in 100 μL PBS (B). Mice were sacrificed 4 hr after injections, and samples from pulmonary tissue were collected, added to formalin fixative and processed for embedding in paraffin. Sections were stained with Martius-Scarlet-Blue, which stains fibrin in red color. No fibrin is observed in samples of mice receiving PBS, whereas abundant red fibrin deposits are observed in the microvasculature of the section from mice treated with venom. Bar represents 100 μm.

Fig 11. Inhibitory action of Batimastat on the effects induced by juvenile B. lanceolatus venom in the lungs.

Light micrographs of sections of pulmonary tissue of mice which received an i.p. injection of 70 μg of B. lanceolatus venom from juvenile specimens dissolved in 100 μL PBS (A) or the same dose of venom which was previously incubated with Batimastat (250 μM final concentration) (B). Mice were sacrificed 4 hr after injections, and samples from pulmonary tissue were collected, added to formalin fixative and processed for embedding in paraffin. The section from a mouse receiving venom show abundant hemorrhage (*) and a thrombus in a blood vessel (arrow). In contrast, no hemorrhage is observed in tissue from a mouse receiving venom incubated with Batimastat, whereas thrombi are present (arrows). Hematoxylin-eosin staining. Bar represents 100 μm.