Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom.

Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100µg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10µg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads.

Synthesis, Anticlotting and Antiplatelet Effects of 1,2,3-Triazoles Derivatives.

Cardiovascular diseases, such as thrombosis and stroke, represent the major cause of disability and death worldwide; and dysfunctions in platelet aggregation and blood coagulation processes are involved. The regular antithrombotic drugs have unsatisfactory results and may produce side effects. Therefore, alternative therapies have been extensively investigated. OBJECTIVE: The anticoagulant and antiplatelet aggregation potential of a series of six synthetic 1,2,3-triazole derivatives were investigated through in vitro models. METHODS: Coagulation tests included the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays, and were performed on a multichannel coagulometer, using human plasma. The platelet aggregation assays were carried out using human platelet-rich-plasma (PRP). Aggregation was initiated by adding ADP or collagen and monitored turbidimetrically on a Whole Blood Aggregometer. Toxicity of derivatives was evaluated on platelets and red blood cells, by measuring the release of lactate dehydrogenase and hemoglobin, respectively. Moreover, theoretical toxicity of derivatives was calculated using the software Osiris® Property Explorer. RESULTS: All the six derivatives tested inhibited, but with different potencies, the plasma coagulation assessed by the PT and TT assays, and also inhibited platelet aggregation of PRP induced by collagen or ADP. The derivatives did not interfere in the aPTT assay and did not affect the viability of platelets or red blood cells. Theoretical studies also revealed that all derivatives will likely to have low toxicity, great pharmacological and oral bioavailability profiles, and a Druglikeness and Drug score similar to some commercial anticoagulant and antiplatelet drugs. CONCLUSION: 1,2,3-triazoles are potential candidates for molecular modeling of new antithrombotic drugs.