抑制因子 X 酶的 FGAG 纯化结构序列及其对血栓形成与止血功能的影响

Thrombosis diseases continue to be the leading cause of human mortality. The antithrombotic drugs have significant clinical values, but the risks of hemorrhagic complications are major concerns with their use. Latest studies have suggested that selective inhibitors of intrinsic blood coagulation factors have a low bleeding risk and it may be new avenues for innovative anticoagulant research. Fucosylated glycaosaminoglycan (FGAG), a marine natural product, is a potent anticaogulant with the mechanism of inhibiting intrinsic factor tenase (f. Xase) which is the last enzyme target in the intrinsic coagulant passway. Our Studies on a series of homogeneous fractions of FGAG have suggested that degree of polymerization and substituent groups have significant effects on the anti-f.Xase activities. Decause all of samples used were mixture of homologous compounds, accurate mechanism of the action is remained to be revealed. Herein, the topic is to prepare a series of purified FGAG fragments with distinctive end structure by depolymerization of FGAG with β-elimination or deamination and fragments purification with affinity chromatography (AC),gel permeation chromatography(GPC) and strong anion exchange (SAX) chromatography, and to analyze the minimum structure sequence of FGAG and its substistuent group for potent anti-f.Xase activities by the methods of coagulant factors activities and interactions analysis to target protein. Furthermore, the potential application value of the f. Xase inhibitor as an anticoagulant with a low bleeding risk will be assessed by analyzing the effects of purified f.Xase inhibitor on coagulation, thrombosis and hemostasis. Our studies would be helpful to research inbitor of intrinsic coagulant factor as new types of antithrombotic drugs.

血栓性疾病是主要的人类致死病因，但临床抗血栓药物都有出血危险，低出血倾向新药发现意义重大。随着内源性凝血因子对血栓和止血功能的不同影响被揭示，其抑制剂的研发渐成热点。天然产物FGAG(岩藻糖化糖胺聚糖)具有强效抗凝活性，且机制之一是抑制内源性途径上的末位凝血因子X酶(f.Xase)。经过系列均一性成分的比较研究，我们发现FGAG聚合度和取代基对其活性的有规律和显著的影响，因受限于研究样品的混合物性质，一些关键科学问题尚待阐明。本课题拟采用β-消除、脱氨基解聚以及AC、GPC及SAX-LC分离技术，制备一系列具有特定末端的FGAG纯化结构片段，通过f.Xase抑制活性以及靶蛋白相互作用研究，阐明f.Xase抑制所需的最小结构序列及必须基团，发现并采用抑制f.Xase的纯化合物片段进行抗凝、抗血栓及出血倾向研究，评价f.Xase抑制剂对于研究和开发低出血倾向抗血栓药物的潜在应用价值。

血栓栓塞性疾病已成为全球人口死亡与致残的首位原因，目前临床抗凝药物的共性缺陷是存在严重出血危险，临床上迫切需要新型抗凝剂。低出血倾向新药发现意义重大。随着内源性凝血因子对血栓和止血功能的不同影响被揭示，选择性内源性凝血途径抑制剂是国际上抗凝药物研发的最前沿领域之一。.天然产物FGAG(岩藻糖化糖胺聚糖)具有强效抗凝活性，且机制之一是抑制内源性途径上的末位凝血因子X酶(f.Xase)。本项目在前期工作的基础上，其主要内容是，采用β-消除、脱酰脱氨基解聚方法制备具有特征末端结构的FGAG解聚产物，分离纯化一系列FGAG结构片段，通过酶学及靶蛋白相互作用研究，比较分析f.Xase抑制所需的最小结构序列及其必需基团，进而研究所得f.Xase抑制剂结合和抑制f.Xase的药理学作用机制、抗血栓活性及其对止血功能的影响。.本项目已建立和完善了通过糖苷键选择性的FGAG解聚技术，并通过所述解聚技术和色谱层析技术分离制备获得了纯化的FGAG寡糖；通过纯化FGAG寡糖的确切结构解析，澄清了本领域关于天然FGAG化学结构方面的学术疑问；此为FGAG的糖化学和糖生物学研究奠定了坚实的基础。对FGAG、其解聚产物、结构修饰产物以及纯化寡糖进行了系统的构效关系研究，揭示了链长、重要取代基对FGAG类化合物的抗凝活性及其靶点机制的影响；首次报道了选择性抑制内源性f.Xase的纯品化合物--FGAG九糖。通过纯品化合物FGAG九糖的抗凝抗血栓活性研究，揭示了选择性抑制内源性f.Xase可产生强效的抗血栓活性，而对生理性止血功能影响较小。选择性的内源性f.Xase抑制剂研究，拓宽了关于内源性凝血途径抑制剂的低出血倾向的抗血栓活性的基础研究范围，也为低出血倾向的新靶点抗血栓药物研发提供了新思路。本项目研究成果还具有重要的潜在应用价值，即以内源性Xase为靶标，研发新型低出血倾向的抗血栓药物。本项目的知识产权成果已应用于新药创制，其技术转化成果为LFG新药项目，此为已知的全球首个选择性抑制内源性f.Xase的新药研发项目。目前，LFG新药及相关知识产权转移至制药企业进行进一步的新药研发。