一种新的从尖吻腹蛇蛇毒中分离纯化的具有类凝血酶作用的C-型凝集素止血作用的分子机制

The snake venom thrombin-like enzymes (SVTLEs) , which resemble at least in part thrombin, cleave Aα or Bβ chain of ?brinogen to release ?brinopeptide A (FPA) or ?brinopeptide B (FPB), and result in the formation of ?brin monomers which link to form the ?brin polymer and cause blood clotting as procoagulants in vitro. But in vivo, the formation of fibrin monomers or polymer activates ?brinolytic system and cause benign defibrination, so they have being used in myocardial infarction, ischaemic stroke and thrombotic diseases or in the prevention of thrombus formation and improving the blood circulation by reducing blood viscosity such as Ancrod or and Batroxobin. However, in China, more than 5 SVTLEs including Reptilase have been widely used for the prevention and treatment of hemorrhage in surgery, even the mechanism to prevent hemorrhage has not been proven so far. Recently, thromblectin (TL), a new SVTLE isolated and purified from Agkistrodon acutus venom, was first discovered as a typical snake venom C-type lectin-like proteins(SVCLPs) in our preliminary studies. The analysis of crystal structure and amino acid sequence demonstrated that TL was a heterodimer of SVCLPs subunits structurally different from SVTLEs but similar to X-bp(factor X-binding protein), a SVCLP from Deinagkistrodon acutus, and shared a high homology with IX-bp and IX/X-bp from the venom of the habu snake Trimeresurus flavoviridis. The results of the enzyme avtivity in vitro indicated that TL has a fibrinogen coagulation function caused by fibrinogenlytic activity similar to SVTLEs. Especially, TL exhibits anticoagulant activition by binding factor IX or X and forming a 1:1 complex in the presence of Ca2+ ions confirmed by native PAGE and ELISA. On the basis of these new findings, one of the current work is to further investigate the characteristics and patterns of fibrin polymerization mediated by A:a or B:b interaction, in which TL shows the effect of SVTLEs to remove FPA or PFB from fibrinogen. Another work concentrates on which and how TL binds to coagulation factors except IX and X which consist of a set of γ-carboxyglutamic acid (Gla)-containing domain including factors Ⅱ,Ⅶ/Ⅶa, protein C and protein S in vitro. Finally, we try to explain why TL causes the formation of fibrin monomers or polymer but not cause defibrination by exploring a direct interaction or an indirect action through coagulation factors on fibrinolytic system (plasminogen, tPA ect) in a rabbit bleeding model of scalp dissection in vivo. These works will help to illuminate the hemostatic mechanisms and a reasonable clinical application of the thrombin-like snake venom C-type lectin-like proteins.

蛇毒类凝血酶（SVTLEs）药物在国内广泛用于手术止血，但其作用机制一直不清楚。本课题前期研究中首次发现，从尖吻蝮蛇蛇毒中分离到的"类凝血酶"蛇毒蛋白Thromblectin(TL)，是一种典型的蛇毒C-型凝集素（SVCLPs），在切割纤维蛋白原（Fib），促进纤维蛋白凝固的同时，与富含Gla的凝血因子（如因子Ⅸ、Ⅹ）结合，并抑制其功能。本课题在进一步研究TL作为SVTLEs切割Fib释放FPA或FPB，通过A:a或B:b作用凝固Fib的特点和方式的同时，研究TL作为SVCLPs在体外或兔头皮剥离模型体内，对Gla凝血因子（如Ⅱ、Ⅶ/Ⅶa、蛋白C、蛋白S等）及其复合物功能的影响，探讨TL直接或通过凝血因子对纤溶系统（纤溶酶原、tPA等）、出血局部凝血功能的调节作用，阐明TL降解Fib但无降纤作用的机制，为该类药物止血作用机理的阐明和合理临床应用提供理论依据。

本研究针对目前占国内止血药市场60%以上的蛇毒类凝血酶类药物止血作用机制不清楚这一迫切需要解决的问题，以“注射用尖吻蝮蛇血凝酶”（saculin）为代表，在前期研究的基础上进一步证实了其蛇毒类凝血酶特性。saculin为典型的丝氨酸蛋白酶，其止血活性可以被丝氨酸蛋白酶抑制剂苯甲基磺酰氟（PMSF）所抑制，但不受肝素的影响，虽然saculin不能激活FXIII，但是经saculin作用形成的纤维蛋白单体仍然可以利用已有的FXIIIa和Ca2+形成一系列共价交联使其在出血局部止血时形成的凝块不易被快速溶解，进而加强其止血作用。另一方面在确证saculin为“类凝血酶”作用的基础上，重点研究了该类药物 “C-型凝集素样”结合抑制凝血因子作用，结果表明其与因子X结合蛋白X-bp一样，saculin能够结合到FX/Xa和FIX/IXa 的Gla区形成复合物抑制凝血酶原和FX的激活，从而表现出一定的抗凝活性，但均不能与Ⅱ、Ⅶ/Ⅶa和蛋白C表现出一定的结合能力。在体内的纤溶系统中，FXa能够加速t-PA激活纤溶酶原产生纤溶酶，为此我们在体外构建这种复合物，研究结果证明saculin可以通过结合到FXa抑制FXa的催化活性从而间接的抑制纤溶酶原的激活，进一步研究表明saculin不能水解激活纤溶酶原，同时对t-PA也没有降解作用。体内试验表明，治疗剂量下，saculin对家兔血浆纤维蛋白原含量没有明显影响，这一结果在一定程度上也印证了saculin对纤溶酶原激活的抑制作用。. 本项目完成了预计的研究内容和达到了研究目的，对saculin止血作用的分子机制进行了更为详细的阐明，提出了其发挥止血作用的假设机制，为该类药物合理临床应用提供了理论依据。本课题培养了3名硕士研究生，撰写了2篇论文：.1.Xian Wu et al. Characterization of a novel snake venom thrombin-like enzyme with procoagulant activity from Agkistrodon acutus. Thromb Res. 2014(in review)..2.米鹏程等. 尖吻蝮蛇血凝酶止血作用的机制.中国新药杂志.2013.22（11）：1315-1319.