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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃德富 | |
| dc.contributor.author | Tzu-Yu Pan | en |
| dc.contributor.author | 潘姿瑜 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:46:04Z | - |
| dc.date.copyright | 2015-09-24 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17914 | - |
| dc.description.abstract | Disintegrins是從蛇毒蛋白中發現的強效小分子血小板凝集抑制劑，其作用機轉為抑制血小板上的integrin αIIbβ3，並被認為具有開發成為抗血栓作用劑的潛力。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。在本篇研究中，從日本龜殼花 (Trimeresurus flavoviridis) 蛇毒蛋白的原毒中純化得到兩個特性不同的disintegrin，分別命名為TFV-1及TFV-3。本篇的主要研究目的是探討並比較TFV-1及TFV-3這兩個dsintegrin的抗血栓作用及機轉不同之處。 經由MALDI-TOF測得TFV-1及TFV-3的分子量分別為7310及7646 Da；活性方面，TFV-1及TFV-3都呈現藥物濃度相關性地有效抑制在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。TFV-1及TFV-3對於ADP (20 μM)，thrombin (0.1 U/mL) 和collagen (10 μg/mL) 所引起血小板凝集反應的IC50分別為99 nM及17 nM，64 nM及39 nM和73 nM及28 nM。 藉由流式細胞儀的分析，觀察到TFV-3會明顯地抑制integrin αIIbβ3單株抗體7E3結合到integrin αIIbβ3上，但TFV-1卻不會影響7E3結合到integrin αIIbβ3上。另一方面，在integrin αIIbβ3單株抗體AP2存在下，TFV-3會活化integrin αIIbβ3並將FcγRII吸引過來，引起血小板的凝集反應且引發下游一連串訊息分子的磷酸化，但TFV-1觀察不到此現象。從clot retraction試驗和thromboelastography結果顯示，TFV-3會明顯地影響血液凝固的生成和凝血的過程，而TFV-1則較少影響。此外，於動物實驗的體外試驗中，TFV-1及TFV-3也都呈現藥物濃度相關性地有效抑制collagen (10 μg/mL) 在老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，TFV-3相對於TFV-1會明顯地延長尾部出血時間，然而，TFV-1及TFV-3兩者皆不會影響血小板的數目。總結上述，TFV-1及TFV-3對於integrin αIIbβ3有不同的結合位置並且對血小板活化的過程有不同的影響，由本篇研究得知，TFV-1有較小的出血傾向且較不會影響到正常的生理止血功能，因此，藉由研究TFV-1和TFV-3之間的結構差異，可以提供未來在研發較不會造成αIIbβ3構型改變及較少出血風險的新一代integrin αIIbβ3拮抗劑一些有價值的資訊。 | zh_TW |
| dc.description.abstract | Disintegrins are potent small-mass platelet inhibitors found in snake venoms, which are integrin αIIbβ3 antagonists and potential antithrombotic agents. However, current integrin αIIbβ3 antagonists have the life-threatening adverse effect of causing thrombocytopenia and bleeding. In this study, two disintegrins, TFV-1 and TFV-3, were purified from Trimeresurus flavoviridis snake venom, with different properties. The molecular weight of TFV-1 and TFV-3 were determined as 7310 Da and 7646 Da by MALDI-TOF, respectively. Both TFV-1 and TFV-3 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma and washed platelet suspension. The IC50 values of TFV-1 and TFV-3 for platelet aggregation induced by ADP (20 μM), thrombin (0.1 U/ml) and collagen (10 μg/ml) were estimated to be 99 and 17 nM，64 and 39 nM, and 73 and 28 nM, respectively. In the indirect binding assay, TFV-3 significantly inhibited 7E3, a mAb raised against integrin αIIbβ3, binding to αIIbβ3, but TFV-1 did not. On the other hand, TFV-3 induced platelet aggregation and triggered time-dependent phosphorylation of signal molecules in the presence of AP2, an integrin αIIbβ3 monoclonal antibody, due to FcγRII recruitment, but TFV-1 did not. As shown in clot retraction assay and thromboelastography, TFV-3 significantly affected the clot formation and the coagulation process, however, TFV-1 showed little effects on clot formation. Moreover, TFV-1 and TFV-3 concentration-dependently inhibited platelet aggregation in mice PRP and dose-dependently inhibited platelet aggregation of PRP in response to collagen in ex vivo model. Furthermore, TFV-3 prolonged the tail bleeding time more significantly than TFV-1 as they were intravenously administered at antithrombotic doses. However, both TFV-1 and TFV-3 did not alter the platelet counts. In summary, TFV-1 and TFV-3, two disintegrins exhibit distinct binding epitopes on integrin αIIbβ3 and different effects on platelet activation process. TFV-1 has less tendency in causing bleeding with minimal effect on normal physiological hemostasis. Therefore, the structural difference between TFV-1 and TFV-3 may provide valuable information for the future development of new integrin αIIbβ3 inhibitors with minimal alteration of integrin αIIbβ3 conformation and bleeding. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:46:04Z (GMT). No. of bitstreams: 1 ntu-104-R02443003-1.pdf: 3208726 bytes, checksum: b70f70f5ae51430e9b2d4633449ef770 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | Contents i Abbreviations iv Tables vi Figures vii 中文摘要 x Abstract xii Chapter 1 Introduction 1 1.1 The role of platelets in hemostasis and thrombosis 1 1.2 Platelet plug formation 2 1.3 Integrins 4 1.4 Platelet integrin αIIbβ3 structure and ligand binding 5 1.5 Disintegrins 7 1.6 Integrin αIIbβ3 antagonists 8 1.7 Thrombocytopenia induced by integrin αIIbβ3 antagonists 10 1.8 Motivation 12 Chapter 2 Materials and Methods 23 2.1 Materials 23 2.2 Methods 25 2.2.1 Purification of disintegrin from Trimeresurus flavoviridis snake venom 25 2.2.2 BCA assay for protein quantification 26 2.2.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 27 2.2.4 Coomassie blue staining 27 2.2.5 Preparation of human platelet-rich plasma (PRP) and platelet suspension 27 2.2.6 Platelet aggregation 28 2.2.7 Flow cytometric analysis of TFV-1 and TFV-3 binding to platelets 29 2.2.8 Western blot analysis 29 2.2.9 Priming assay 30 2.2.10 Clot retraction 31 2.2.11 Rotational thromboelastometry (ROTEM) 31 2.2.12 in vitro and ex vivo mouse platelet aggregation 32 2.2.13 Tail bleeding time in mice 33 2.2.14 Platelet counts in mice 33 2.2.15 Statistical analysis 34 Chapter 3 Results 35 3.1 Purification of TFV-1 and TFV-3 from Trimeresurus flavoviridis snake venom 35 3.2 Determination of molecular weight of TFV-1 and TFV-3 36 3.3 Protein identification of TFV-1 and TFV-3 36 3.4 Effect of TFV-1 and TFV-3 on platelet aggregation of human platelet-rich 37 3.5 Effect of TFV-1 and TFV-3 on platelet aggregation of human platelet 37 3.6 TFV-1 binds to different epitope of integrin αIIbβ3 from TFV-3 and 7E3 38 3.7 TFV-1 does not cause significant platelet aggregation after a combination of 38 3.8 TFV-1 has minimal priming activity compared with epitifibatide and tirofiban 39 3.9 TFV-1 shows little effects on clot retraction 41 3.10 TFV-1 has little effects on clot kinetics and propagation of thrombosis in 41 3.11 TFV-3 exhibits more potent inhibitory effects on mice platelet aggregation 42 3.12 Effects of TFV-1 and TFV-3 on tail bleeding time and platelets counts 43 Chapter 4 Discussion 72 Chapter 5 Conclusions and Perspectives 82 References 85 | |
| dc.language.iso | en | |
| dc.title | "日本龜殼花蛇毒蛋白Disintegrins,TFV-1和TFV-3的抗血栓活性與作用機轉探討" | zh_TW |
| dc.title | Two disintegrins, TFV-1 and TFV-3, purified from Trimeresurus flavoviridis snake venom, exhibit distinct effects on platelet activation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 顏茂雄,楊春茂,鄧哲明,吳文彬 | |
| dc.subject.keyword | 蛇毒蛋白,整合蛋白,血小板,血小板活化,血小板低下症, | zh_TW |
| dc.subject.keyword | Snake venom,Integrin,Platelet,Platelet activation,Thrombocytopenia, | en |
| dc.relation.page | 94 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2015-07-31 | |
| 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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| ntu-104-1.pdf 未授權公開取用 | 3.13 MB | Adobe PDF |
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