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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64713完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃德富 | |
| dc.contributor.author | Ying-Ru Chen | en |
| dc.contributor.author | 陳盈如 | zh_TW |
| dc.date.accessioned | 2021-06-16T22:57:54Z | - |
| dc.date.available | 2017-09-18 | |
| dc.date.copyright | 2012-09-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-09 | |
| dc.identifier.citation | Angiolillo, D. J., Ueno, M., and Goto, S. (2010). Basic principles of platelet biology and clinical implications. Circ J 74, 597-607.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64713 | - |
| dc.description.abstract | Disintegrins是小分子量的抗血栓蛋白，最早由蛇毒蛋白中所發現，並有著強效的血小板凝集抑制活性。Disintegrins的作用機轉是抑制血小板上的GPIIb/IIIa，被認為具有開發為抗血栓抑制劑的潛力。龜殼花 (Trimeresurus mucrosquamatus) 蛇毒蛋白原毒粉末依序經由CM-Sephadex C-50離子交換層析法、Sephadex G-75 膠質過濾法、FPLC Superdex-75膠質過濾法，最後再以逆向高效液相色譜分析法進行最後純化分離，純化得到的兩種disintegrin分別為TMV-2和TMV-7。在本篇研究中，主要的目的是探討並比較TMV-2和TMV-7抗血栓作用和機轉不同之處。TMV-2和TMV-7有著不一樣的等電點，TMV-2的等電點約為4.5而TMV-7的等電點約為5-7。TMV-2和TMV-7都是單鏈的小分子，分子量經由MALDI-TOF測定分別為7663 和7672 Da。由LC-MS/MS所鑑定出的部分序列顯示，TMV-2有24%的序列相似於batroxostatin (由Bothrops atrox蛇毒蛋白中分離出的disintegrin)；而TMV-7也有24%的序列相似於batroxostatin，但有高達79%的序列相似於cotiarin (由Bothrops cotiara蛇毒蛋白中分離出的disintegrin)。TMV-2和TMV-7都能有效抑制在人類富含血小板的血漿、人類血小板懸浮液以及經elastase處理的人類血小板懸浮液中所引發的凝集反應，其抑制的程度會隨著濃度上升而增大。TMV-2和TMV-7都不會影響thromboxane A2的生成，但都會增強由collagen和thrombin所引起的P-selectin的表現。利用流式細胞儀的分析，可觀察到TMV-2對於GPIIb/IIIa的單株抗體7E3結合到GPIIb/IIIa上有明顯抑制作用，然而TMV-7卻不會抑制單株抗體7E3結合到GPIIb/IIIa上。而TMV-2和TMV-7對於另一種GPIIb/IIIa的單株抗體10E5的影響卻是會加強此單株抗體結合到GPIIb/IIIa上。在動物實驗方面，TMV-2和TMV-7也能抑制老鼠富含血小板的血漿所中所引起的血小板凝集反應，其抑制的程度也能隨著濃度上升而增大。在給予老鼠0.25μg/g的劑量之下，TMV-2會顯著的延長出血時間，然而TMV-7則不會延長出血時間。另外，TMV-2和TMV-7兩者都不會減少血小板的數量。綜合上述結果可知，TMV-2和TMV-7這兩個等電點不相同的disintegrin有著不一樣的特性。TMV-2比起TMV-7有著更強效的血小板凝集抑制效果，而且TMV-7結合到GPIIb/IIIa上的位置顯然和TMV-2及單株抗體7E3不同。另外，我們發現當TMV-2或TMV-7和單株抗體10E5一起作用時反而會引發血小板的活化。而給予老鼠0.25μg/g的劑量之下，TMV-7的副作用和TMV-2比起來顯得較少。由此我們認為TMV-2和TMV-7這兩種disintegrin之間的差異研究可利於新一代GPIIb/IIIa拮抗劑的研發，而且由TMV-2和TMV-7作用於GPIIb/IIIa的不同之處或許可以提供訊息來探討GPIIb/IIIa拮抗劑所引發血小板減少症的機轉。 | zh_TW |
| dc.description.abstract | Disintegrins are small moleculars and potent platelet inhibitors found in the snake venom. Disintegrins are GPIIb/IIIa antagonists and potential antithrombotic agents. By means of CM-Sephadex C-50, Sephadex G-75 gel filtration, FPLC Superdex 75 gel filtration and reverse phase HPLC, two disintegrins, TMV-2 and TMV-7, were purified from Trimeresurus mucrosquamatus snake venom. In this study, we investigated and compared the difference between TMV-2 and TMV-7. TMV-2 and TMV-7 had different isoelectric point (pI). The pI of TMV-2 was estimated to be around 4.5, whereas that of TMV-7 was estimated to be around 5-7. TMV-2 and TMV-7 were shown to be a single peptide chain. By MALDI-TOF, the molecular weigh of TMV-2 and TMV-7 were determined as 7663 and 7672 Da, respectively. The sequence of TMV-2 was 24% identical to batroxostatin, a disintegrin purified from the snake venom of Bothrops atrox, whereas the sequence of TMV-7 was 24% identical to batroxostatin and 79% identical to cotiarin, a disintegrin purified from the snake venom of Bothrops cotiara. Both TMV-2 and TMV-7 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma, washed human platelet suspension and elastase-treated human platelets. Also, TMV-2 and TMV-7 did not interfere with the formation of thromboxane A2. However, both TMV-2 and TMV-7 enhanced the P-selectin expression induced by collagen and thrombin. In the indirect binding assay, TMV-2 significantly inhibited 7E3, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa, but TMV-7 did not. Both TMV-2 and TMV-7 enhanced 10E5, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa. In the animal models, TMV-2 and TMV-7 dose-dependently inhibited platelet aggregation in mice PRP. Furthemore, TMV-2 (0.25μg/g) prolonged the bleeding time more significantly than TMV-7 (0.25μg/g) as they were intravenously administered. However, both TMV-2 and TMV-7 did not alter the platelet counts. In conclusion, TMV-2 and TMV-7, two disintegrins with different isoelectric point, have the different characters. Although both TMV-2 and TMV-7 are GPIIb/IIIa inhibitor, the inhibitory effect of TMV-2 was more potent than TMV-7. Also, our data revealed that the binding site of TMV-7 on GPIIb/IIIa was different from TMV-2 and mAb 7E3. Moreover, combination of TMV-2 or TMV-7 with mAb 10E5 could lead to platelet activation. Finally, we found that TMV-7 had fewer side effects than TMV-2 at the same dosage. Therefore, the difference between TMV-2 and TMV-7 may provide valuable information for the development of GPIIb/IIIa inhibitors and the mechanisms involved in GPIIb/IIIa inhibitor-induced thrombocytopenia. | en |
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| dc.description.tableofcontents | Contents
誌謝 ....................................................i 中文摘要 ................................................ii Abstract ................................................iv Contents ................................................vi Figures .................................................x Tables ..............................................xiii Abbreviations .....................................xiv Chapter 1 Introduction ..............................1 1.1 The role of platelets in haemostasis and thrombosis .................................................1 1.2 Stages in the formation of platelet plug...............2 1.2.1 The initiation phase ..............................2 1.2.2 The extension phase ..............................3 1.2.3 The perpetuation phase ..............................3 1.3 Integrins ............................................4 1.4 Platelet integrin : GPIIb/IIIa structure and ligand binding .................................................5 1.5 Platelet integrin : activation of GPIIb/IIIa receptor...................................................6 1.6 Disintegrins .......................................6 1.7 GPIIb/IIIa inhibitors ..............................8 1.7.1 Monoclonal antibody: Abciximab ....................8 1.7.2 Drugs designed based on snake venom disintegrins : Eptifibatide and Tirofiban ..............................9 1.8 Thrombocytopenia induced by GPIIb/IIIa inhibitors ................................................10 1.9 Aims ................................................11 Chapter 2 Materials and methods ...................15 2.1 Materials ......................................15 2.2 Purification of platelet aggregation inhibitor from Trimeresurus mucrosquamatus snake venom .........16 2.2.1 Sephadex G-75 column chromatography .........16 2.2.2 FPLC Superdex 75 HR 10/300 GL ...................17 2.2.3 Reverse-phase HPLC C18 column ...................17 2.3 BCA assay for protein quantification .........18 2.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ................................................18 2.5 2D gel electrophoresis (2D SDS-PAGE) .........18 2.6 Coomassie blue staining ............................19 2.7 In gel digestion ............................19 2.8 Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) .........20 2.9 Liquid chromatography–mass spectrometry (LC-MS / MS) ...............................................20 2.10 Preparation of human platelet-rich plasma .........21 2.11 Preparation of human platelet suspension .........21 2.12 Elastase-treated platelet suspension .........22 2.13 Platelet aggregation ............................22 2.14 Measurement of thromboxane B2 formation .........23 2.15 Analysis of P-selectin expression ...................23 2.16 Flow cytometric analysis of TMV-2 and TMV-7 binding to platelets ...............................................24 2.17 In vitro and ex vivo mouse platelet aggregation ...............................................24 2.18 Tail bleeding time in mice ..................25 2.19 Platelet counts in mice ............................25 2.20 Statistical analysis ............................26 Chapter 3 Results .....................................27 3.1 Purification of TMV-2 and TMV-7 from Trimeresurus mucrosquamatus snake venom ............................27 3.2 Determination of molecular mass of TMV-2 and TMV-7 ...............................................28 3.3 The isoelectric point (pI) of TMV-2 and TMV-7 ...............................................28 3.4 Protein identification of TMV-2 and TMV-7 .........29 3.5 Effect of TMV-2 and TMV-7 on platelet aggregation of human platelet-rich plasma ............................29 3.6 Effect of TMV-2 and TMV-7 on platelet aggregation of human platelet suspension ............................30 3.7 Effect of TMV-2 and TMV-7 on fibrinogen-induced aggregation of elastase-treated platelets .........30 3.8 Effect of TMV-2 and TMV-7 on thromboxane B2 formation ...............................................31 3.9 Effect of TMV-2 and TMV-7 on P-selectin expression ...............................................32 3.10 Effect of TMV-2 and TMV-7 on the expression of GPIIb/IIIa on unstimulated platelet ..................33 3.11 Effect of TMV-2 and TMV-7 on the expression of GPIIb/IIIa on ADP-stimulated platelets ..................33 3.12 Effect of TMV-2 and TMV7 on in vitro and ex vivo mice platelet aggregation ............................34 3.13 Effect of TMV-2 and TMV-7 on bleeding time and platelet counts .....................................35 Chapter 4 Discussion ............................71 Chapter 5 Conclusion ............................80 References .....................................82 Figures Fig. 1-1 Mechanism of platelet adhesion activation and aggregation. .....................................12 Fig. 1-2 The conformation of GPIIb/IIIa. ........13 Fig. 1-3 Cartoon models of the conformational states of GPIIb/IIIa under physiological conditions or in presence of inhibitors. .....................................14 Fig. 3-1 CM-Sephadex C-50 column chromatography of Trimeresurus mucrosquamatus (Adopted from Shu-Ya Yang) ...............................................36 Fig. 3-2 Sephadex G-75 column chromatography of fraction II. ...............................................37 Fig. 3-3 Purification of TMV-2 using reverse-phase HPLC. ...............................................38 Fig. 3-4 Sephadex G-75 column chromatography of fraction VII. (Adopted from Shu-Ya Yang) ..................39 Fig. 3-5 FPLC Superdex 75 HR 10/300 GL column chromatography of fraction VII-2. ..................40 Fig.3-6 Purification of TMV-7 using reverse-phase HPLC. ...............................................41 Fig. 3-7 Pattern of SDS-gel electrophoresis and 2D-gel electrophoresis of purified TMV-2 and TMV-7. ........42 Fig. 3-8 The molecular mass of TMV-2 analyzed by MALDI-TOF. ..............................................43 Fig. 3-9 The molecular mass of TMV-7 analyzed by MALDI-TOF. ..............................................44 Fig. 3-10 Partial sequences of TMV-2 and TMV-7 obtained from LC-MS/MS. .....................................45 Fig. 3-11 Effect of TMV-2 and TMV-7 on ADP-induced platelet aggregation of human platelet-rich plasma. ........46 Fig. 3-12 Effect of TMV-2 and TMV-7 on collagen-induced human platelet aggregation. ...........................48 Fig. 3-13 Effect of TMV-2 and TMV-7 on thrombin-induced platelet aggregation of washed human platelet suspension. ..............................................49 Fig. 3-14 Effect of TMV-2 and TMV-7 on U46619-induced human platelet aggregation. ...........................51 Fig. 3-15 Concentration-inhibition curve of TMV-2 and TMV-7 in human platelet-rich plasma and washed human platelet suspension. ....................................54 Fig. 3-16 Effect of TMV-2 and TMV-7 on fibrinogen-induced aggregation of elastase-treated platelets. .......55 Fig. 3-17 Concentration-inhibition curve of TMV-2 and TMV-7 in elastase-treated platelet suspension .......56 Fig. 3-18 Effect of TMV-2 and TMV-7 on fibrinogen-induced aggregation of elastase-treated platelets (II). .......57 Fig. 3-19 Effect of TMV-2 and TMV-7 on thromboxane B2 formation induced by collagen in washed human platelets..............................................59 Fig. 3-20 Effect of TMV-2 and TMV-7 on P-selectin expression activated by collagen. .................60 Fig. 3-21 Effect of TMV-2 and TMV-7 on P-selectin expression activated by thrombin. .................61 Fig. 3-22 Effect of TMV-2 and TMV-7 on the expression of membrane GPIIb/IIIa probed by mAb 7E3 on unstimulated platelets. ....................................62 Fig. 3-23 Effect of TMV-2 and TMV-7 on the expression of membrane GP IIb/IIIa probed by mAb 7E3 on ADP-stimulated platelets. ....................................63 Fig. 3-24 Effect of TMV-2 and TMV-7 on the expression of membrane GP IIb/IIIa probed by mAb 10E5 on unstimulated platelets. ....................................64 Fig. 3-25 Effect of TMV-2 and TMV-7 on the expression of membrane GP IIb/IIIa probed by mAb 10E5 on ADP-stimulated platelets. ....................................65 Fig. 3-26 Effect of TMV-2 and TMV-7 on in vitro mouse platelet aggregation of PRP induced by collagen. .......66 Fig. 3-27 Effect of TMV-2 and TMV-7 on ex vitro mouse platelet aggregation of PRP induced by collagen. .......67 Fig. 3-28 Effect of TMV-2 and TMV-7 on tail bleeding time in mice. .............................................69 Fig. 3-29 Effect of TMV-2 and TMV-7 on platelet count in mice. .............................................70 Fig. 4-1 The locations of the 10E5 and 7E3 epitopes on models of the ...................................79 bent and extended forms of GP IIb/IIIa. ......79 Tables Table. 4-1 The estimated isoelectric point of different disintegrins isolated from snake venoms. ......77 Table. 4-2 Comparision the IC50 of TMV-2 with TMV-7 in human platelet-rich plasma (PRP) and washed human platelets. .............................................78 Table. 4-3 Comparision the IC50 of TMV-2 with TMV-7 in elastased-treated human platelets. .................78 Table. 4-4 Comparision the IC50 of TMV-2 with TMV-7 in mice PRP. ..............................................78 | |
| dc.language.iso | en | |
| dc.subject | 抗血栓 | zh_TW |
| dc.subject | 蛇毒蛋白 | zh_TW |
| dc.subject | 龜殼花 | zh_TW |
| dc.subject | snake venom | en |
| dc.subject | Trimeresurus mucrosquamatus | en |
| dc.subject | antithrombotic | en |
| dc.title | 龜殼花蛇毒蛋白Disintegrin抗血栓活性與作用機轉之探討 | zh_TW |
| dc.title | The Antithrombotic Effects and Mechanisms of Disintegrin Purified from Trimeresurus mucrosquamatus Snake Venom | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧哲明,顏茂雄,楊春茂,吳文彬 | |
| dc.subject.keyword | 蛇毒蛋白,龜殼花,抗血栓, | zh_TW |
| dc.subject.keyword | snake venom,Trimeresurus mucrosquamatus,antithrombotic, | en |
| dc.relation.page | 92 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-09 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| 顯示於系所單位： | 藥理學科所 | |
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