Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets

Abstract

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Three stages of chromatography were used to purify a 7.75 kDa peptide called Cerastokunin with pI 8.48 till homogeneity based on antitrypsin activity. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands. Cerastokunin showed a strong anticoagulant potential by completely eliminating the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. In vivo study showed a substantial reduction in tail thrombus of mice-carrageenan model; in contrast to antithrombotic medications, this antithrombosis was boosted by a greater dose of Cerastokunin. Throughout the trial course, no in vivo toxicity was observed in challenged mice at any of Cerastokunin doses up to 6 mg/kg.