PDF herunterladen
CC BY-NC-ND 4.0 · Thromb Haemost 2025; 125(05): 484-491
DOI: 10.1055/a-2408-9375
Cellular Haemostasis and Platelets

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

Mojca Dobaja Borak‡   *
1   Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana, Ljubljana, Slovenia
2   Doctoral School, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
,
Adrijana Leonardi‡   *
3   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
,
Kity Požek
2   Doctoral School, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
3   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
,
Katarina Reberšek
4   Department of Haematology, University Medical Centre Ljubljana, Ljubljana, Slovenia
,
Helena Podgornik
4   Department of Haematology, University Medical Centre Ljubljana, Ljubljana, Slovenia
5   Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
,
Aljaž Pirnat
6   Division of Paediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenia
,
Alenka Trampuš Bakija
6   Division of Paediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenia
,
Simona Kranjc Brezar
7   Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
,
Tomaž Trobec
8   Institute of Preclinical Sciences, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
,
Monika C. Žužek
8   Institute of Preclinical Sciences, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
,
Robert Frangež
8   Institute of Preclinical Sciences, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
,
Miran Brvar 
1   Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana, Ljubljana, Slovenia
9   Center for Clinical Physiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
,
Igor Križaj 
3   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
› Institutsangaben Funding This work was supported by grants from the Slovenian Research and Innovation Agency (P1–0207, P3–0019, P4–0053, J3–2534, 1000–22–0106).
› Weitere Informationen
Auch verfügbar auf
   Lizenzen und Reprints


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

© 2024. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Dobaja Borak M, Babić Ž, Caganova B. et al. Viper envenomation in Central and Southeastern Europe: a multicentre study. Clin Toxicol (Phila) 2023; 61 (09) 656-664
    CrossrefPubMedSuche in Google Scholar
  • 2 Kurtović T, Karabuva S, Grenc D. et al. Intravenous Vipera berus venom-specific Fab fragments and intramuscular Vipera ammodytes venom-specific F(ab')2 fragments in Vipera ammodytes-envenomed patients. Toxins (Basel) 2021; 13 (04) 279
    CrossrefPubMedSuche in Google Scholar
  • 3 Brvar M, Kurtović T, Grenc D, Lang Balija M, Križaj I, Halassy B. Vipera ammodytes bites treated with antivenom ViperaTAb: a case series with pharmacokinetic evaluation. Clin Toxicol (Phila) 2017; 55 (04) 241-248
    CrossrefPubMedSuche in Google Scholar
  • 4 Dobaja Borak M, Grenc D, Reberšek K. et al. Reversible and transient thrombocytopenia of functional platelets induced by nose-horned viper venom. Thromb Res 2023; 229: 152-154
    CrossrefPubMedSuche in Google Scholar
  • 5 Eble JA. Structurally robust and functionally highly versatile-C-type lectin (-related) proteins in snake venoms. Toxins (Basel) 2019; 11 (03) 136
    CrossrefPubMedSuche in Google Scholar
  • 6 Herrera C, Rucavado A, Warrell DA, Gutiérrez JM. Systemic effects induced by the venom of the snake Bothrops caribbaeus in a murine model. Toxicon 2013; 63: 19-31
    CrossrefPubMedSuche in Google Scholar
  • 7 Nashimoto Y, Matsushita F, Dijkstra JM. et al. Bitiscetin-3, a novel C-type lectin-like protein cloned from the venom gland of the viper Bitis arietans, induces platelet agglutination and inhibits binding of von Willebrand factor to collagen. Toxins (Basel) 2022; 14 (04) 236
    CrossrefPubMedSuche in Google Scholar
  • 8 Leonardi A, Sajevic T, Pungerčar J, Križaj I. Comprehensive study of the proteome and transcriptome of the venom of the most venomous European viper: discovery of a new subclass of ancestral snake venom metalloproteinase precursor-derived proteins. J Proteome Res 2019; 18 (05) 2287-2309
    CrossrefPubMedSuche in Google Scholar
  • 9 Latinović Z, Leonardi A, Šribar J. et al. Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: therapeutic implications. J Proteomics 2016; 146: 34-47
    CrossrefPubMedSuche in Google Scholar
  • 10 Morowski M, Vögtle T, Kraft P, Kleinschnitz C, Stoll G, Nieswandt B. Only severe thrombocytopenia results in bleeding and defective thrombus formation in mice. Blood 2013; 121 (24) 4938-4947
    CrossrefPubMedSuche in Google Scholar
  • 11 Karabuva S, Brizić I, Latinović Z, Leonardi A, Križaj I, Lukšić B. Cardiotoxic effects of the Vipera ammodytes ammodytes venom fractions in the isolated perfused rat heart. Toxicon 2016; 121: 98-104
    CrossrefPubMedSuche in Google Scholar
  • 12 Premrov Bajuk B, Zrimšek P, Kotnik T, Leonardi A, Križaj I, Jakovac Strajn B. Insect protein-based diet as potential risk of allergy in dogs. Animals (Basel) 2021; 11 (07) 1942
    CrossrefPubMedSuche in Google Scholar
  • 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227 (5259) 680-685
    CrossrefPubMedSuche in Google Scholar
  • 14 Požek K, Leonardi A, Pungerčar J. et al. Genomic confirmation of the P-IIIe subclass of snake venom metalloproteinases and characterisation of its first member, a disintegrin-like/cysteine-rich protein. Toxins (Basel) 2022; 14 (04) 232
    CrossrefPubMedSuche in Google Scholar
  • 15 Sajevic T, Leonardi A, Kovačič L. et al. VaH3, one of the principal hemorrhagins in Vipera ammodytes ammodytes venom, is a homodimeric P-IIIc metalloproteinase. Biochimie 2013; 95 (06) 1158-1170
    CrossrefPubMedSuche in Google Scholar
  • 16 Ogrizek M, Grgurevič N, Snoj T, Majdič G. Injections to pregnant mice produce prenatal stress that affects aggressive behavior in their adult male offspring. Horm Behav 2018; 106: 35-43
    CrossrefPubMedSuche in Google Scholar
  • 17 Wang X, Xu L. An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research. Thromb Res 2005; 115 (1–2): 95-100
    CrossrefPubMedSuche in Google Scholar
  • 18 Wang X, Cheng Q, Xu L. et al. Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice. J Thromb Haemost 2005; 3 (04) 695-702
    CrossrefPubMedSuche in Google Scholar
  • 19 Lu Q, Clemetson JM, Clemetson KJ. Snake venom C-type lectins interacting with platelet receptors. Toxin Rev 2007; 26: 77-93
    CrossrefPubMedSuche in Google Scholar
  • 20 Kini RM. Toxins in thrombosis and haemostasis: potential beyond imagination. J Thromb Haemost 2011; 9 (Suppl. 01) 195-208
    CrossrefPubMedSuche in Google Scholar
  • 21 Sajevic T, Leonardi A, Križaj I. Haemostatically active proteins in snake venoms. Toxicon 2011; 57 (05) 627-645
    CrossrefPubMedSuche in Google Scholar
  • 22 Clemetson KJ. Snaclecs (snake C-type lectins) that inhibit or activate platelets by binding to receptors. Toxicon 2010; 56 (07) 1236-1246
    CrossrefPubMedSuche in Google Scholar