Abstract：
The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. 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 with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting 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. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial\'s duration.
摘要：
当前的研究集中在分离Cerastescerastes毒液以生产第一个Kunitz型肽。基于其抗胰蛋白酶作用,Cerastokunin,一个7.75kDa的肽，通过三个层析步骤纯化直至均匀。发现Cerastokunin包括通过使用LC-MALDI-MSMS从头测序获得的67个氨基酸残基。与Kunitz型肽对齐后，有很高的相似性。Cerastokunin的3D结构具有12％的α-螺旋和21％的β-链，pI为8.48。Cerastokunin通过抑制凝血酶和胰蛋白酶的蛋白酶活性以及阻断内在和外在凝血途径而显示出有效的抗凝血作用。在PT和aPPT中，Cerastokunin以剂量依赖性方式增加血液凝固时间。使用Lys48和Gln192直接结合，Cerastokunin抑制凝血酶，通过分子对接显示的因子Xa和胰蛋白酶。一旦被凝血酶刺激，Cerastokunin表现出PAR依赖性途径血小板的剂量反应阻断。与抗血栓形成药物的作用相比，在体内研究中，Cerastokunin的浓度增加导致小鼠-角叉菜胶模型中的尾血栓减少更大。在所有Cerastokunin剂量高达6mg/kg时，在试验期间，在攻击小鼠中未观察到体内毒性。
