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標題: | 蛇毒蛋白Echistatin和其突變衍生物之抗血栓作用及
作用機轉之探討 The antithrombotic effects and action mechanisms of disintegrin, echistatin and its mutants |
作者: | Chiang-Hui Chen 陳薔卉 |
指導教授: | 黃德富 |
關鍵字: | 蛇毒蛋白,抗黏著蛋白,抗血栓, snake venom,disintegrin,antithrombotic, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 血小板在正常生理下的凝血功能扮演重要的角色,然而在病理情況下,不正常的血栓形成可能會造成嚴重的心血管疾病。Disintegrins 是一種從蛇毒蛋白中發現的一群具有RGD 或是KGD 氨基酸序列之胜肽血小板抑制劑,作用機轉為抑制血小板上的integrin αIIbβ3,它可專一地接在受器上,目前有以此結構為基礎發展成在臨床上使用的血小板抑制劑,然而現行臨床上使用的αIIbβ3 拮抗劑普遍存在著增加出血風險及血小板低下等危及生命的副作用。Echistatin 是一種短鏈的disintegrin,具有49 個氨基酸及4 對雙硫鍵。本篇從蘇氏鋸鱗蝰(Echis carinatus sochureki)中分離出ECS-f1 及ECS-f3,以及藉由酵母菌表現系統合成出Echistatin wild type 與數種C 端氨基酸進行變異的echistatin突變衍生物(由國立成功大學莊偉哲教授提供),探討不同echistatins 之間抗血栓作用及作用機轉。在活性方面,ECS-f1、ECS-f3、ECH WT 及echistatin 突變衍生物都具有濃度相關性地有效抑制人類富含血小板的血漿和人類血小板懸浮液中所引發的凝集反應。藉由流式細胞儀的分析,也可以觀察到echistatin 會增強在collagen 和thrombin 活化下血小板的p-selectin 表現;另外,這些echistatin 都會競爭單株抗體7E3 在integrin αIIbβ3 上的結合位置,但增加單株抗體AP5 的結合程度。而當echistatins 加入integrin αIIbβ3 的抑制性單株抗體AP2 的情況下,會造成FcγRII 的靠近,引發血小板凝集反應,造成下游訊息傳遞分子的磷酸化,例如PLCγ2、FAK、Syk 及Src。從clot retraction 試驗中可以觀察到,ECS-f3 可以顯著地抑制血塊的凝縮。動物體外實驗也可以看出,ECS-f3、ECH WT、ECHP47A、ECH K45A 也都呈現藥物濃度相關性地有效抑制collagen 在小鼠富含血小板的血漿中所引發的血小板凝集反應。另外,在尾靜脈給予抗血栓劑量下,四組也都會延長小鼠尾部出血時間,然而卻不會影響血小板數量。活體實驗中,以氯化鐵造成小鼠頸動脈傷害引發血栓產生,ECS-f3、ECH WT、ECHP47A、ECH K45A 皆可延長血栓生成及血管栓塞的時間。在本篇的實驗當中,雖自行分離出的disintegrin 和echistatin 突變衍生物的藥理作用和抗血栓作用,大體上都具有一定的有效性,但是由於C 端序列的不同,造成在抑制血栓活性、造成αIIbβ3 構型改變和其他其他實驗中具有特性上的差異。因此我們希望能夠找出C 端序列對於echistatin 的影響性,可以在未來設計較不會造成αIIbβ3 構型改變及較少出血風險的新一代integrin αIIbβ3 拮抗劑中提供一些有價值的資訊。 Physiological haemostasis is mediated by platelets and coagulation system. However, in pathological situation, inappropriate thrombus formation leads to vessels occlusion in various cardiovascular diseases. Disintegrins are small-mass platelet aggregation inhibitors found in the snake venom. Disintegrins are potent antithrombotic agents, acting through the blockage of integrin αIIbβ3, an essential fibrinogen receptor in mediating platelet aggregation. Echistatin is a small-sized disintegrin which is a 49-amino acid RGD-containing peptide linked by four disulfide bonds. In this article, we purified disintegrin ECS-f1 and ECS-f3 from snake venom of Echis Carinatus sochureki. Furthermore, previous studies support a functional role for the C-terminal residues of echistatin in modulating its binding affinity towards integrin αIIbβ3, thus, different mutants of echistatin were expressed for comparative study (by Prof. Woei-Jer Chuang, National Cheng Kung University College of Medicine). ECS-f1, ECS-f3, ECH WT and echistatin mutants concentration-dependently inhibited platelet aggregation both in human platelet-rich plasma and platelet suspension. By using flow cytometry, we found that ECS-f1, ECS-f3, ECH WT and echistatin mutants enhanced the expression of P-selectin in collagen or thrombin-stimulated human platelet. In direct binding assay, echistatin significantly inhibited 7E3, however, enhanced AP5 mAb binding to platelet. In the presence of AP2, an inhibitory mAb of integrin αIIbβ3, both ECS-f1, ECS-f3 and echistatin mutants induced platelet aggregation around two-fold IC50 value, due to FcγRII recruitment and further caused the phosphorylation of downstream signaling molecules, including PLCγ2, FAK, Syk and Src. Furthermore, ECS-f3 could significantly affect clot retraction of platelet-rich plasma. Moreover, ECS-f3, ECH WT, ECH P47A and ECH K45A concentration-dependently inhibited platelet aggregation in mice PRP in vitro and dose-dependently inhibited platelet aggregation of PRP in response to collagen and ex vivo. Furthermore, all members of echistatins significantly prolonged the mice tail bleeding time as they were intravenously administered at antithrombotic doses (300 ng/g). However, all of them did not alter the platelet counts. In the in vivo ferric chloride (FeCl3)-induced arterial thrombosis model, administration of ECS-f3, ECH WT, ECH P47A and ECH K45A could prolong the occlusion time. In summary, the purified disintegrin echistatins and the echistatin mutants exhibited antithrombotic effects as tested in platelets and mice models. However, they showed various potency in antithrombotic activity and induction of conformational change as recognized by mAb AP2, suggesting that C-terminal residues are essential for modulating its binding activity. Further investigation of the structure-activity relationship of echistatins is needed for providing more information for the design of novel integrin αIIbβ3 inhibitors. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50475 |
DOI: | 10.6342/NTU201601493 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
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