FG 寡糖对血小板蛋白酶激活受体的阻断作用及机制研究
文献类型：学位论文
| 作者 | 李素娟 |
| 答辩日期 | 2022-05 |
| 授予单位 | 中国科学院大学 |
| 导师 | 赵金华 |
| 关键词 | 血小板活化，蛋白酶激活受体，凝血酶外位点 II，血栓，岩藻糖化糖胺 聚糖 Platelet activation, Protease-activated receptor, Thrombin exosite II, Thrombosis, Fucosylated glycosaminoglycan |
| 英文摘要 | 血小板在动脉粥样斑块部位的不当活化和聚集会导致动脉血栓。凝血酶是一种重要的凝血因子，也是最有效的血小板激动剂。凝血酶诱导的血小板活化由蛋白酶激活受体(PARs)介导，该受体属于G蛋白偶联受体家族。人血小板表达PAR1和PAR4(二者形成稳定的异二聚体复合物)，它们可以单独传导跨膜信号。阻断PAR1-PAR4复合物为预防动脉血栓提供了一个新的治疗机会。 岩藻糖基化糖胺聚糖(FG)，又称岩藻糖基化硫酸软骨素(FCS)，是一种结构特殊的糖胺聚糖，由类似硫酸软骨素的主链和独特的硫酸化岩藻糖侧链组成。我们发现FG寡糖能抑制凝血酶诱导的血小板活化，FG的抗血小板作用未见报道。 (1) P-选择素(P-selectin, CD62P)是血小板活化的敏感标记物，在血小板活化过程中，CD62P从细胞质α-颗粒转移到膜表面。本研究通过微孔板结合实验和HL-60细胞结合实验筛选了本课题组制备的FG寡糖，发现具有二糖侧链结构的寡糖具有更强的抑制活性，这为后续进一步发现具有更强抑制活性的FG寡糖或在糖合成及修饰方面具有指导意义。 (2) 在研究FG寡糖对血小板P-选择素的抑制活性中发现，FG寡糖对凝血酶诱导的血小板CD62P的表达具有很强的抑制作用，此抑制作用与FG寡糖的P-选择素抑制活性无关(FG寡糖对P-选择素的抑制作用主要是通过抑制P-选择素与其配体的结合)。流式细胞术分析FG寡糖对不同激动剂诱导的血小板激活的影响，发现FG寡糖特异性抑制凝血酶诱导的血小板激活，对其它血小板激动剂如胶原、ADP、U46619(血栓素A2激动剂)、TRAP(PAR1激动剂)和AY-HN2(PAR4激动剂)诱导的血小板激活无抑制作用。通过筛选发现HS-11是具有强效抑制活性的最小的FG结构片段。HS-11不影响凝血酶的催化活性(包括对小分子生色底物的裂解和对生理性大分子底物的裂解)，即不影响凝血酶在正常生理止血中的凝血功能。 (3) 大分子相互作用分析方法测定HS-11与凝血酶的结合亲和力，结果显示，HS-11与凝血酶结合具有高的亲和力(KD为47.04 ± 0.84 nM)，与AT不结合；采用阿加曲班(结合于凝血酶活性位点)、适配体HD1(结合于凝血酶exosite I)和适配体HD22(结合于凝血酶exosite II)通过竞争性结合实验确定HS-11与凝血酶的结合位点，结果显示，HS-11不与活性位点结合，可结合外位点I和外位点II，Ki分别是14.95 ± 2.18 μM and 2.55 ± 0.39 μM。TT实验进一步验证HS-11与凝血酶的外位点II结合的重要性。 (4) 使用PAR1抑制剂沃拉帕沙(Vorapaxar, VOR)和PAR4抑制剂BMS- 986120(BMS)分别阻断PAR1通路和PAR4通路后分析HS-11对凝血酶诱导的血小板活化的影响，发现HS-11主要通过PAR4途径抑制凝血酶诱导的血小板活化；但与PAR4拮抗剂BMS相比，HS-11可以完全抑制凝血酶诱导的血小板活化而BMS只能部分抑制；HS-11对凝血酶诱导的钙动员和蛋白磷酸化也表现出与PAR4相似的抑制效应。这些结果提示，外位点II在PAR1和PAR4的同时激活中起重要作用。 (5)血小板聚集和体外血栓形成模型评估HS-11的药效学作用，结果显示，与PAR1抑制剂VOR和PAR4抑制剂BMS相比，HS-11能够完全抑制凝血酶诱导的血小板聚集。在体外血栓形成模型中，HS-11显示出较VOR和BMS更强的抑制活性。由此可见，通过凝血酶外位点II抑制凝血酶-PARs介导的血小板激活可能可以为动脉血栓的治疗提供了新的机会。 本论文报道了FG寡糖HS-11，通过直接与凝血酶外位点II结合，阻断凝血酶与PAR1-PAR4复合物的相互作用，完全抑制血小板信号转导，包括细胞内Ca2+动员和蛋白质磷酸化。HS-11能有效抑制凝血酶-PARs介导的血小板聚集并减少离体血栓形成模型中的血栓形成。这些发现首次揭示了FG寡糖通过与凝血酶外位点II结合同时阻断PAR1和PAR4受体介导的血小板活化，并揭示了凝血酶外位点II在PAR1和PAR4的同时激活中起重要作用，这种抗血栓方式为动脉血栓的治疗提供了新的机会。; Platelet inappropriate activation and aggregation can lead to arterial thrombosis, which often occurs in atherosclerotic plaques, causing myocardial infarction and stroke. Thrombin is an essential coagulation factor, as well as the most potent platelet agonist. Thrombin-induced platelet activation is mediated by protease-activated receptors (PARs), which belong to the family of G protein-coupled receptor. Human platelets express PAR1 and PAR4 that form stable heterodimeric complexes, and they can alone mediate transmembrane signaling by thrombin, respectively. Blocking the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis. Fucosylated glycosaminoglycan (FG), also known as Fucosylated chondroitin sulfates (FCS), is a glycosaminoglycan with a distinct structure from sea cucumber, which consists of the chondroitin sulfate-like backbone and the unique branches of sulfated fucoses. Recently, we found that an oligosaccharide purified from depolymerized FG can inhibit thrombin-induced platelet activation; such antiplatelet effect of FG has not been reported. (1) P-selectin (CD62P) is a sensitive marker for activated platelet, which is translocated from the cytoplasm α-granules to the membrane surface during platelet activation. In this study, oligosaccharides with disaccharide side chain structure with stronger p-selectin inhibitory activity were screened from FG oligosaccharides prepared by our research group using microplate binding assay and HL-60 cell binding assay. It is significant for the subsequent discovery of FG oligosaccharides with stronger inhibitory activity or in terms of carbohydrate synthesis and modification. (2) Platelets were selected as a model to study the binding inhibition of P-selectin to its ligands. It was found that FG oligosaccharide had strong inhibitory effect on platelet CD62P expression induced by thrombin, which was different to the binding inhibition of P-selectin (The P-selectin inhibiton was mainly by inhibiting the binding of P-selectin to its ligand, but not by decreasing the expression of P-selectin) In this study, the effect of FG oligosaccharides on platelet activation induced by different agonists was detected by flow cytometry. FG oligosaccharides selectively inhibits thrombin-induced platelet activation but not collagen, ADP, U46619, TRAP and AY-HN2. HS-11 is the smallest oligosaccharide with the full antiplatelet activity from HS-5~17. Moreover, HS-11 does not affect thrombin catalytic ability for a small molecular substrate or fibrinogen. (3) Biolayer interferometry (BLI) to study the HS-11-thrombin interaction. Thrombin bound to HS-11 with a high affinity (KD of 47.04 ± 0.84 nM). In contrast, no obvious binding signal of HS-11-AT was detected. To investigate the binding site of HS-11 in thrombin, a series of competitive binding experiments were conducted using thrombin direct inhibitor argatroban which is a small molecule bound to the active site, and two DNA apta |
| 语种 | 中文 |
| 源URL | [http://ir.kib.ac.cn/handle/151853/75236]  |
| 专题 | 昆明植物研究所_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 | 李素娟. FG 寡糖对血小板蛋白酶激活受体的阻断作用及机制研究[D]. 中国科学院大学. 2022. |
入库方式： OAI收割
来源：昆明植物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。