Introduction: At the Centre for Clinical Toxicology and Pharmacology of the University Medical Centre Ljubljana, we treat 5–10 patients annually due to bites by nose-horned viper (Vipera ammodytes ammodytes; Vaa). In patients bitten by Vaa, we have repeatedly observed the occurrence of pronounced but transient thrombocytopenia without clinically significant bleeding. The mechanism underlying this process has not yet been elucidated.
Aim: The aim of this doctoral dissertation was to investigate the mechanism of thrombocytopenia following envenomation with Vaa venom.
Methods: The doctoral dissertation was divided into clinical, biochemical, ex vivo, and in vivo studies. In the clinical part of the study, we analyzed the frequency of thrombocytopenia in patients bitten by the Vaa, the associated clinical presentation, and platelet characteristics during thrombocytopenia and after its resolution. We also assessed the impact of different types of antivenom on the course and severity of thrombocytopenia. In the biochemical part of the study, several snaclec proteins were isolated from Vaa venom, structurally and functionally characterized, and their effects on platelets were investigated using ex vivo experiments. Based on a review of the literature, we hypothesized that snaclecs might play an important role in the development of thrombocytopenia. In the final part of the study, the effects of a selected snaclec, Vaa-snaclec-3/2, were characterized in an in vivo model, and its antithrombotic potential was evaluated.
Results: In the clinical part of the dissertation, we demonstrated that thrombocytopenia occurs more frequently after Vaa bites than after Vipera berus berus bites, most likely due to differences in venom composition between the two snake species. Thrombocytopenia was transient and reversible following antivenom administration and was directly dependent on the concentration of free venom in the blood, confirming the key role of venom components in its development. The increase in platelet count after intravenous antivenom administration coincided with a decrease in circulating venom concentration. Despite severe thrombocytopenia, platelets in affected patients retained full functionality. Rotational thromboelastometry performed after normalization of platelet count yielded results within reference ranges. This indicates preserved overall hemostatic function, in agreement with aggregometry results. Flow cytometry results showed no increased exposure of P-selectin on the external platelet membrane during or after thrombocytopenia, which would otherwise indicate platelet activation. Our findings suggest that thrombocytopenia is not the result of platelet aggregation, but rather of platelet agglutination without activation.
In the biochemical part of the dissertation, several snaclecs were isolated from Vaa venom. Among them, only the heterodimeric snaclec Vaa-snaclec-3/2 specifically inhibited ristocetin-induced platelet agglutination without affecting coagulation tests. Ex vivo, the addition of Vaa-snaclec-3/2 to whole blood caused profound thrombocytopenia. Microscopic examination revealed the formation of small clusters (agglutinates) consisting of a smaller number of platelets. We demonstrated that Vaa-snaclec-3/2 binds to the platelet receptor GPIb in a concentration-dependent manner, but not to the GPIIb/IIIa, GPIX, or GPVI receptors, and does not trigger platelet activation.
In an in vivo mouse model of carotid artery thrombosis, Vaa-snaclec-3/2 induced profound but transient thrombocytopenia, thereby inhibiting thrombus formation following vascular wall injury. Administration of F(ab’)₂ antibody fragments directed against Vaa venom effectively reversed thrombocytopenia.
A clinical case of a patient with recurrent episodes of thrombocytopenia following Vaa bites and repeated administrations of Fab antivenom fragments further confirmed that thrombocytopenia is dependent on venom concentration. It also confirmed the reversibility of the process with immunotherapy, which preserves full platelet functionality.
Conclusion: Profound thrombocytopenia is a common finding in Vaa venom envenomation. This complication is rapidly reversible after administration of Fab or F(ab’)₂ fragments directed against whole Vaa venom, and platelets remain functional. Among the several snaclecs present in Vaa venom, only Vaa-snaclec-3/2 induces thrombocytopenia. The cause is the binding of Vaa-snaclec-3/2 to the platelet receptor GPIb, which triggers platelet agglutination without activation. In this dissertation, we comprehensively elucidated the mechanism of thrombocytopenia in Vaa venom envenomation. The venom component Vaa-snaclec-3/2 was identified as the key molecular factor underlying the observed process, highlighting its considerable therapeutic potential in medical procedures requiring a transient and rapidly reversible antithrombotic effect.
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