FG寡糖抗凝抗血栓作用的活性特点与机制

	FG寡糖抗凝抗血栓作用的活性特点与机制
	蔡英
导师	赵金华
关键词	FG寡糖，内源性因子X酶，抗凝抗血栓活性特点，止血功能 FG oligosaccharides, Intrinsic tenase, The characteristics of anticoagulant and antithrombotic activity, Haemostatic function
摘要	选择性的内源性凝血因子抑制剂是低出血倾向抗凝药物研发的主流方向，内源性因子X酶(Intrinsic tenase, FIXa-FVIIIa-PL-Ca2+ complex, iXase)是内源性凝血途径的末位限速酶，有望成为新型低出血倾向抗凝药物的潜在靶点。海参来源的糖胺聚糖(Fucosylated glycosaminoglycan, FG)的解聚产物具有显著的iXase抑制活性、抗凝活性及低出血倾向的抗血栓活性。本文采用现代药理学技术，从靶点蛋白、纯因子系统、离体血浆及活体动物水平系统分析了FG寡糖作用于靶蛋白的分子机制及其抗凝、抗血栓活性特征。研究结果表明，FG寡糖通过与因子IXa(Active factor IXa, FIXa)的外位点结合来产生选择性的iXase抑制活性，并由此抑制内源性凝血途径及微量组织因子(Tissue factor, TF)诱导的凝血放大反应，继而抑制病理性血栓形成，而对外源性凝血途径介导的止血功能影响较小。本文主要的研究方法与结果如下： (1) FG寡糖的抗凝活性及其对凝血因子活性的影响质控血浆的凝血时间测定结果显示：FG寡糖可链长依赖性地延长质控血浆的活化部分凝血活酶时间(Activated partial thromboplastin time, APTT)，对凝血酶原时间(Prothrombin time, PT)/凝血酶时间(Thrombin time, TT)无显著影响。生色底物法分析FG寡糖对凝血因子活性的影响，结果显示：FG寡糖同样具有链长依赖性的iXase抑制活性，对外源性因子X酶(Extrinsic tenase, TF-FVIIa-PL-Ca2+ complex, eXase)活性以及抗凝血酶(Antithrombin, AT)存在下的因子Xa(Active factor X, FXa)/凝血酶(Thrombin, active factor II, FIIa)的活性无显著影响。 (2) FG寡糖抑制iXase的机制研究生物膜干涉(Bio-layer interferometry, BLI)技术分析显示，FG寡糖可高亲和力结合FIXa和酶原FIX SP-EGF2，与五个FIX SP-EGF2突变体结合的亲和力均下降。竞争性BLI分析显示，FG寡糖和低分子肝素(Low-molecular-weight heparin, LMWH)结合FIXa/FIX SP-EGF2的位点存在重叠。上述结果提示：与FIXa的外位点结合是FG寡糖抑制iXase的分子机制的基础，外位点中的五个关键氨基酸对FIXa与FG寡糖结合的重要性为R233 ＞ R165 ＞ K230 ＞ K241 ＞ R170。荧光共振能量转移(Fluorescence resonance energy transfer, FRET)法分析显示，FG寡糖虽然抑制了功能性iXase复合物的形成，但其与FIXa的结合并不改变FIXa-PL及iXase复合物中FIXa活性位点到磷脂层的空间距离。 (3) FG寡糖抑制凝血酶生成的活性及机制研究自动校正的血浆FIIa生成(Calibrated automated thrombogram, CAT)实验结果显示，FG寡糖(HS17和HS8)显著抑制内源性凝血途径(诱导剂为微量TF、APTT试剂及0.1% FIXa)依赖的人血浆和大鼠血浆FIIa生成，但对主要依赖外源性凝血途径(诱导剂为大量TF)的人血浆和大鼠血浆FIIa生成的影响相对较弱。 (4) FG寡糖的抗血栓活性及其对止血功能的影响在结扎合并TF诱导的大鼠下腔静脉血栓形成模型中，FG寡糖HS17剂量依赖地抑制微量TF所致的血栓形成(ED50, 1.285 mg/kg sc)。在ED50的5倍、10倍剂量下，HS17不影响大量TF暴露所致的血栓形成或影响较小，而LMWH显著抑制该条件下的血栓形成，提示在较高的等效抗血栓剂量下，HS17对大量TF暴露所启动的生理性止血塞形成的影响应当较LMWH小。 (5) FG寡糖抑制血栓形成效应与其体内抗凝活性之间的相关性研究 HS17剂量依赖地抑制微量TF诱导的大鼠血浆FIIa生成，其抑制效应与该化合物在相应剂量下的抗血栓活性一致。在ED50的5倍、10倍剂量下，HS17对大量TF暴露所致的FIIa生成和血栓形成的影响较小，而LMWH显著抑制相同条件下的血浆FIIa生成和血栓形成。此外，HS17剂量依赖地延长大鼠血浆的APTT，且对PT及TT均无影响，而在ED50的5倍、10倍剂量下，LMWH除延长血浆的APTT外，也显著延长了血浆的TT。综上所述，FG寡糖具有基于iXase抑制活性的抗凝、抗血栓活性以及与此相关的低止血功能影响的活性特点，该特点清晰呈现了FG寡糖从抗凝靶点到抗凝活性、抗血栓作用之间的药理学逻辑关联。此研究结果可以为FG寡糖的潜在研发价值提供充分和可靠的应用基础资料，同时为新型iXase抑制剂的研发提供新的思路和技术方法。; Selective intrinsic coagulation factor inhibitors are the mainstream direction for developing anticoagulants with low bleeding tendency. The intrinsic tenase (FIXa-FVIIIa-PL-Ca2+ complex, iXase), the terminal rate-limiting enzyme of the intrinsic coagulation pathway, could be a promising target for novel anticoagulants. The depolymerized products of fucosylated glycosaminoglycan (FG) derived from sea cucumber have potent anti-iXase activity, anticoagulation activity and the antithrombotic activity with a low bleeding risk. In this paper, using modern pharmacological techniques, the molecular mechanism of FG oligosaccharides acting on target protein, the characteristics of their anticoagulant and antithrombotic activity were systematically analyzed from the target protein, purified coagulation factor system, isolated plasma system and living animal level. The results showed that FG oligosaccharides selectively inhibited iXase via binding to the exosite of FIXa, thereby inhibiting the intrinsic pathway and the amplification of coagulation triggered by limiting TF, and then inhibiting the pathological thrombosis. Whereas, they had a little effect on the physiological hemostasis. The main methods and results in this dissertation are listed as below. (1) The anticoagulant activity of FG oligosaccharides and their effects on the activity of coagulation factors The results about the clotting time of coagulation plasma showed that, FG oligosaccharides prolonged the activated partial thromboplastin time (APTT) in a chain length-dependent manner. And they had negligible effect on both prothrombin time (PT) and thrombin time (TT). The effects on the activity of coagulation factors were analyzed using chromogenic substrate method. The results exhibited that FG oligosaccharides possessed potent activity for inhibition iXase in a chain length-dependent manner. While they had no significant inhibitory activity for the extrinsic tenase (TF-FVIIa-PL-Ca2+ complex, eXase), both Factor Xa (FXa) and thrombin (Active factor II, FIIa) in the presence of antithrombin (AT). (2) The mechanistic study of iXase inhibition by FG oligosaccharides The experimental results based on Bio-layer interferometry (BLI) technology exhibited that FG oligosaccharide could bind FIXa and zymogen FIX SP-EGF2 with high affinity, while the affinity to bind five FIX SP-EGF2 mutants decreased. Competitive BLI study indicated that FG oligosaccharides bound to an exosite on FIXa/FIX SP-EGF2 that overlapped with the binding sites for LMWH. These above results suggested that the binding to the exosite of FIXa underlay the molecular mechanism of iXase inhibition by FG oligosaccharides, the importance of five amino acid sites in which was R233 ＞ R165 ＞ K230＞ K241 ＞ R170. The experimental results based on Fluorescence resonance energy transfer (FRET) technology showed that although FG oligosaccharide could inhibit the formation of functional iXase complex, the binding to FIXa had no significant effec
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75179
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	蔡英. FG寡糖抗凝抗血栓作用的活性特点与机制[D]. 中国科学院大学,2022.

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