Reversible Thrombocytopenia of Functional Platelets after Nose-Horned Viper Envenomation is Induced by a Snaclec

Abstract

Profound and transient thrombocytopenia of functional platelets without bleeding was observed in patients envenomed by Vipera a. ammodytes (Vaa). This condition was rapidly reversed by administration of F(ab)₂ fragments of immunoglobulin G targeting the whole venom, leaving platelets fully functional. To investigate the potential role of snake venom C-type lectin-like proteins (snaclecs) in this process, Vaa-snaclecs were isolated from the crude venom using different liquid chromatographies. The purity of the isolated proteins was confirmed by Edman sequencing and mass spectrometry. The antithrombotic effect was investigated by platelet agglutination and aggregation assays and blood coagulation tests. Using flow cytometry, the platelet activation and binding of Vaa-snaclecs to various platelet receptors was analyzed. Antithrombotic efficacy was tested in vivo using a mouse model of vascular injury. Two Vaa-snaclecs were purified from the venom. One of them, Vaa-snaclec-3/2, inhibited ristocetin-induced platelet agglutination. It is a covalent heterodimer of Vaa-snaclec-3 (α-subunit) and Vaa-snaclec-2 (β-subunit). Our results suggest that Vaa-snaclec-3/2 induces platelet agglutination and consequently thrombocytopenia by binding to the platelet receptor glycoprotein Ib. Essentially, no platelet activation was observed in this process. In vivo, Vaa-snaclec-3/2 was able to protect the mouse from ferric chloride-induced carotid artery thrombosis, revealing its applicative potential in interventional angiology and cardiology.

Keywords

snake venom - reversible thrombocytopenia - snaclecs - arterial occlusion - mice - interventional cardiology - antithrombotic

Ethical Approval Statement

Study protocol on human blood was reviewed and approved by the Slovenian National Medical Ethics Committee (No. 87/07/15 and No. 0120–546/2017/5). The study was conducted according to the guidelines of the Declaration of Helsinki. All animal experiments were performed in strict accordance with the Slovenian legislation, which was harmonized with the European Communities Council guidelines (Directive 86/609/EGS of November 24, 1986 and recently adopted Directive 2010/63/EU of September 22, 2010). The permission for in vivo experiments was obtained from the Ministry of Agriculture Forestry and Food of the Republic of Slovenia, The Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection, approval number: U34401–9/2021/4. The ARRIVE guidelines have been followed.

Authors' Contribution

Conceptualization: A.L., M.B., and I.K.; data curation: M.D.B., A.L., K.P., K.R., and M.C.Ž.; formal analysis: M.D.B., A.L., K.P., K.R., H.P., A.P., A.T.B., M.C.Ž., R.F., M.B., and I.K.; funding acquisition: K.P., R.F., M.B., and I.K.; investigation: M.D.B., A.L., K.P., K.R., A.P., S.K.B., T.T., and M.C.Ž.; methodology: A.L., H.P., A.T.B., R.F., M.B., and I.K.; project administration: M.B. and I.K.; resources: H.P., A.T.B., R.F., M.B., and I.K.; supervision: A.L., H.P., A.T.B., R.F., M.B., and I.K.; validation: A.L., H.P., A.T.B., R.F., M.B., and I.K.; visualization: M.D.B., K.P., A.L., M.C.Ž., and R.F.; writing—original draft: M.D.B. and K.P.; writing—review and editing: A.L., K.R., H.P., A.P., A.T.B., M.C.Ž., R.F., M.B., and I.K.

^* These authors contributed equally to this work.

Publikationsverlauf

Eingereicht: 10. Mai 2024

Angenommen: 20. August 2024

Accepted Manuscript online:
03. September 2024

Artikel online veröffentlicht:
23. September 2024

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