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研究生:黃懷賢
研究生(外文):Huai-Hsien Huang
論文名稱:作用於醣蛋白VI引發血小板凝集之台灣龜殼花蛇毒蛋白的特性和作用機轉探討
論文名稱(外文):Characterization and Mechanism of Action of GPVI-targeting Platelet Aggregation Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom
指導教授:黃德富
指導教授(外文):Tur-Fu Huang
口試委員:鄧哲明楊春茂顏茂雄吳文彬
口試委員(外文):Che-Ming TengChuen-Mao YangMao-Hsiung Yenwen-bin wu
口試日期:2015-07-24
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:53
中文關鍵詞:蛇毒台灣龜殼花醣蛋白VI血小板類C型凝集素蛇毒蛋白 (snacle)
外文關鍵詞:snake venomTrimeresurus mucrosquamatusglycoprotein VI (GPVI)plateletsnake venom C-type lectin-like proteins (snacles)
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出血性蛇毒蛋白影響血液凝固及血小板之凝集活性。在本篇實驗中,將Trimeresurus mucrosquamatus原毒經由CM-Sephadex C-50 陽離子交換層析法以及Sephadex G-75 膠質過濾法進行純化分離,得到一高致效引發血小板凝集之成分,暫稱為TMV inducer。以SDS-PAGE分析,其分子量大約為45kDa。TMV inducer在人類血小板懸浮液(PS)和富含血小板之血漿(PRP)中,皆呈現濃度相關性的引起血小板凝集,其EC50分別為54.36 ± 0.92及13.8 ± 0.3 ng/ml。PGE1、EDTA及BAPTA/AM幾乎完全抑制TMV inducer引起的血小板凝集,顯示TMV inducer引起的血小板凝集需要鈣離子或其他二價陽離子的參與;而Indomethacin的抑制程度則約80%。觀察TMV inducer活化血小板下游訊息傳遞的部分,Syk抑制劑-piceatannol 對TMV inducer凝集作用的抑制明顯但並不完全;PLC抑制劑-U73122、MEK抑制劑-PD98059、PI3-K抑制劑-LY294002能完全抑制TMV inducer造成之血小板凝集,但並未影響血小板之shape change;Src family kinase抑制劑-PP2則是同時延長血小板之shape change,並明顯抑制TMV inducer凝集作用。確認TMV inducer在血小板上之受體部分,單獨使用GPIa/IIa單株抗體(6F1)或GPIb單株抗體(6D1)只能些微抑制TMV inducer引起之血小板凝集,兩者並用有加成之抑制作用。TMV inducer 不會在福馬林固定之血小板上引發凝聚(aggutination)的情形。GPIIb/IIIa抗體7E3、aggrastat及GPVI單株抗體326E12、342D7能明顯抑制TMV inducer引起之血小板凝集。利用biotinylated TMV inducer來觀察,發現他會專一性的結合到血小板上的GPVI。此外,TMV inducer引起血小板訊息分子tyrosine磷酸化的情形類似於convulxin和trowaglerix,包括FcR γ-chain、PI3K、Syk、Src、LAT 以及PLCγ2等等。在動物實驗方面,TMV inducer未明顯延長小鼠尾靜脈止血時間。另一方面,TMV inducer則會使小鼠血小板數量有藥物濃度相關性的減少。綜合以上實驗結果,TMV inducer會專一性的結合到血小板上的GPVI,活化後引起許多訊息分子的磷酸化,包括FcR γ-chain、PI3K、Syk、Src、LAT 以及PLCγ2等等,最後引發功能性αIIbβ3的表現,造成鈣離子依賴性的血小板凝集。由於TMV inducer的專一性,可以用來研究其與已知作用在GPVI上的C-type lectins (例如:convulxin、trowaglerix)之間的異同,探討他們與GPVI分子層次之交互作用,提供GPVI拮抗劑之研發。

Snake venoms profoundly affect platelet aggregation and hemostasis. By using column chromatography of CM Sephadex C-50 cationic exchanger and Sephadex G-75 gel filtration, a novel TMV inducer was purified from Trimeresurus mucrosquamatus snake venom. Under reducing conditions, it migrates as a protein with a mass about 45 KDa on SDS-PAGE. TMV inducer-induced platelet aggregation of human washed platelets and platelet-rich plasma in a concentration-dependent manner with EC50, 54.36 ± 0.92 and 13.8 ± 0.3 ng/ml, respectively. Aggrastat, anti-integrin αIIbβ3 mAb 7E3 significantly inhibited platelet aggregation caused by TMV inducer. Anti-GPIb mAb 6D1 and anti-GPIa/IIa mAb 6F1 only slightly inhibited platelet aggregation caused by TMV-inducer. An additive inhibitory effect was observed when 6D1and 6F1 were added. However, anti-GPVI mAb 326E12 and 342D7 profoundly inhibited platelet aggregation. In pull-down assay, biotinylated TMV inducer specifically bound to GPVI, but not GPIb or integrin α2. TMV inducer did not elicit agglutination of fixed platelets. PGE1 and BAPTA/AM completely inhibited platelet aggregation induced by TMV inducer. EDTA and indomethacin also caused profound inhibition. Syk inhibitor, piceatannol had higher inhibitory effect (80%). PLC inhibitor U73122, MEK inhibitor PD98059 and PI3-K inhibitor LY294002 totally inhibited TMV-inducer induced platelet aggregation without inhibiting platelet shape change. Src inhibitor, PP2 delayed platelet shape change, and profoundly inhibited aggregation. To confirm the downstream signal transduction, TMV-inducer induced a time-dependent tyrosine phosphorylation of a number of proteins similar to those activated by convulxin, including PLCγ2, PI3K, Syk, Src, Fyn and LAT. In mice models, TMV inducer did not significantly prolonged mice tail bleeding time. However, TMV inducer caused a dose-dependent decrease of platelet counts. Taken together, TMV inducer, a monomeric platelet aggregating inducer, activates platelets mainly through GPVI, leading to phosphorylation of many signal molecules, including PLCγ2, PI3K, Syk, Src, LAT and Fcγ, finally inducing the exposure of functional αIIbβ3 and Ca2+- dependent platelet aggregation. This novel snake venom protein may provide an useful tool for studies of GPVI and signaling mechanisms involved as well as for comparative study with other C-type lectin GPVI agonist, like convulxin or trowaglerix, and its association of platelet receptors, such as GPIb and GPIa/IIa. These studies would provide new insights regarding the molecular interaction of these GPVI agonists, providing clues for the design of GPVI antagonists, a new class of antithrombotic agent.

口試委員會審定書....…………………………………………………………………... i
誌謝…………………………………………………………………………………….. ii
中文摘要....……………………………………………………………………………. iii
Abstract....………………………………………………………………………...…….. v
Abbreviation table....…………………………………………………………………... vii
Chapter 1 Introduction………………….…………………………………………..… 1
1-1 Formation and characterization of platelets...…………………………....... 1
1-2 The mechanism of platelet activation, thrombus formation and hemostasis 1
1-3 Atherosclerosis, atherothrombosis and antiplatelet agents………............... 2
1-4 Collagen/ GPVI/ integrin α2β1-mediated hemostasis and signaling
transduction………………………………………………………………... 3
1-5 vWF/GPIb-IX-V mediated signaling…...………..…………...…………… 4
1-6 Thrombin-mediated signaling pathway via G-protein coupled receptor
(GPCR)…………………………………………………………………..… 5
1-7 Effects of snake proteins on hemostatic system………………………..…. 6
1-8 C-type lectin-like protein (SNACLES) and snake venom platelet
aggregation inducer……………………………………………………….. 7
1-9 Specific aim of the study……….......…………………...………………… 7
Chapter 2 Materials and methods…………………………………………………… 13
Materials.…..…………………………………………………….............. 13
2-1 Purification and isolation of crude TMV………………………..……….. 14
2-2 Desalting, fraction concentrating and protein quantification…..............… 15
2-3 Protein electrophoresis analysis……………………………………..…… 15
2-3-1 SDS-PAGE…………..………………………………...…...….. 15
2-3-2 Coomassie blue staining…………………………………...…... 15
2-4 Preparation of human platelet-rich plasma (PRP) and platelet
suspension (PS).………...…………………………………………..….... 16
2-5 Measurement of platelet aggregation…………...………………............... 16
2-6 Formaldehyde-fixed platelet suspension…………………………………. 17
2-7 Pull-down assay of TMV inducer with target platelet receptors…………. 17
2-8 Binding analysis of TMV inducer on platelets………………………...… 17
2-9 Western blotting……………...………………………………………...… 18
2-10 Animal models………………………………………………………….... 19
2-10-1 Tail-bleeding time in mice……………………………………….. 19
2-10-2 Platelet counts in mice…………………………………………… 19
2-11 Statistics analysis…………………………………………………………. 19
Chapter 3 Result..…………………………………………………………………… 20
3-1 Purification of TMV inducer from Trimeresurus mucrosquamatus venom20
3-2 Electrophoresis of TMV inducer……………………………….……….... 20
3-3 Effects of TMV inducer on human platelet aggregation……….……….... 20
3-4 Effects of platelet receptor blocking antibodies on TMV inducer-induced
platelet aggregation…………………………………………………….… 21
3-5 Effect of TMV inducer in formaldehyde-fixed human platelets…………. 21
3-6 Effects of inhibitor on TMV inducer-induced platelet aggregation…….... 22
3-7 Effects of signal transduction inhibitor on TMV inducer-induced platelet
aggregation………………………………………………………………. 22
3-8 Investigation of TMV inducer receptor on platelet by pull-down assay…. 22
3-9 TMV inducer induces protein tyrosine phosphorylation in human
platelets…………………………………………………………………... 23
3-10 In vivo effects of TMV inducer in animal models……………………….. 24
Chapter 4 Discussion………………………………………………………………... 41
Chapter 5 Conclusion and perspectives……………………………………..………. 46
References…………………………………………………………48

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