C-type Lectin-Like Proteins (CTLPs) present in Bothrops jararaca venom that induce thrombocytopenia: purification and mechanism of action

Introduction
Bothrops snakes are the main cause of snakebites in Brazil, with envenomation leading to severe effects, especially hemostasis disorders. Bothrops jararaca venom induces inflammatory, coagulant, and hemorrhagic actions through components such as metalloproteinases, serine proteases, C-type lectin-like proteins (CTLPs), and phospholipases A2. CTLPs alter platelet aggregation and coagulation. Recent studies isolated and initially characterized two CTLPs (E6 and D4) from B. jararaca venom that induce thrombocytopenia. This project aims to characterize these proteins and understand how they reduce platelet counts during envenomation.

Objectives:
The aim of our work was to further purify and characterize CTLP-E6 and CTLP-D4 and elucidate which of their mechanisms affect platelets.

Methods:
In vivo experiments were approved by CEUAIB (protocol 8227240723) using C57BL/6 mice (30-35g, n=5-8/group). Human blood samples were obtained from volunteers (aged 18-45 years) in accordance with CONEP guidelines (CAAE: 52787521.0.0000.5469). Venom was subjected to anion exchange chromatography (Q-Sepharose), followed by cation exchange (SP—Sepharose). Fractions were analyzed by SDS-PAGE (15%) and tested in vivo: mice received subcutaneous injections of CTLP fractions or saline. Blood samples were collected 3 hours later to determine the platelet count and facilitate fibrinogen measurement.

Results:
Anion exchange chromatography showed most proteins of interest eluted in the flow-through. SDS-PAGE revealed the presence of CTLP-E6 and CTLP-C4 at ~30 kDa under non-reducing conditions, splitting into 16 kDa and 13 kDa bands under reducing conditions. These fractions underwent further purification by cation exchange. SDS-PAGE and optical density confirmed purification. Injected mice exhibited thrombocytopenia without fibrinogen changes, indicating successful CTLP purification.

Conclusions:
The study effectively purified CTLPs that induce thrombocytopenia. Further research will investigate their mechanisms of action.

Acknowledgments:
Financial support: CAPES, Fundação Butantan, and CNPq (grant 309980/2021-6).