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研究生：	林娀妏 Lin, Sung-wen
論文名稱：	藉由優化龜殼花蛇毒蛋白的linker區域、KGD迴圈以及C端提升它對整合蛋白αIIbβ3的活性以及安全性 Optimization of the linker region, KGD loop and C-terminus of trimucrin to improve its integrin αIIbβ3 activity and safety
指導教授：	莊偉哲 Chuang, Woei-Jer
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	66
中文關鍵詞：	整合蛋白IIb3 、龜殼花蛇毒蛋白、KGD 迴圈、抗血小板藥物、急性冠狀動脈症候群、皮冠狀動脈介入性治療
外文關鍵詞：	Integrin αIIbβ3, Trimucrin, KGD loop, antiplatelet drug, ACS, PCI
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急性冠狀動脈症候群,又簡稱冠心病,是一個有血小板參與其中的疾病。冠心病的形成原因是因為血液中的脂質或膽固醇在冠狀動脈中堆積,造成血管管壁粥樣斑塊破裂,使得血管管壁變窄或阻塞,心積血供應減少使的心肌細胞缺氧的現象,病人會有心絞痛的症狀。那目前針對冠心病的治療以皮冠狀動脈介入性治療為主,而且病人會搭配抗血小板藥物進行服用,避免血小板在凝集而造成血管再窄化。血小板表面上有許多接受體,它們可以和不同配體去進行結合,進而引發外向訊號使得整合蛋白IIb3 從靜態模式中被活化。因此,抑制整合蛋白IIb3 的活化可以做為潛在的藥物設計標靶。目前臨床上使用的抗血小板藥物,舉例來說整合蛋白IIb3 結抗劑,會造成一些出血以及血小板低下症的副作用。因此, 設計一種能夠抑制活化狀態的整合蛋白,並且有出血風險低的藥物可以做為潛在候選藥物。在這篇研究中,來自龜殼花蛇毒的去整合蛋白被用來作為藥物設計的平台,進而去談討:(1) 去整合蛋白中的連結區域、RGD 區域以及 C 端在抑制血小版中所扮演的角色 (2) 設計具有高安性全指數的整合蛋白IIb3 結抗劑。安全性數值的評估是建立在固定濃度 AP2 作用下,去整合蛋白誘導血小板表面上整合素IIb3 活化所需的濃度。在這篇研究中,我已經利用 Pichia pastoris 系統表現了十個蛋白,並且將他們純化成同質性。我發現到 51KGDYR 以及 51KGDWR 在血小板凝集試驗中具有相似的抑制活性,他們的 IC50 分別是 104.4 nM 以及 61.3 nM,而 51KGDRR 的 IC50 則為 90.0 nM。另外,他們的安全性指數和 51KGDRR (>3366) 相比之下都很低,分別是 8 以及 278。此外,若將 C 端突變成 69NRLYG、 69NRLY、69NRFH 在血小板抑制活性方面都展現相似的抑制程度,而且都具有滿高的安全性指數。如果將連結區域突變成 41IKKGT 以及 41MKEGT 在血小板凝集試驗中表現不錯的抑制活性,他們的 IC50 分別是 56.9nM 和 84.1nM,而且 41IKKGT 以及 41IEEGT 具有滿高的安全性指數。總結來說,我們發現到位在 KGD 區域 C 端的鄰近氨基酸在安全性指數上扮演很重要的角色。本研究的結果將作為設計出血危險性低的抗血小板藥物的基礎。

Acute coronary syndrome (ACS), a platelet-associated disease, is caused by fat or cholesterol accumulates at coronary artery resulting in the rapture of plaque on the vessel wall and the formation of thrombus. Most common therapy for ACS is percutaneous coronary interventions (PCI). Patients receiving PCI will also take anti-platelet drugs because of restenosis of blood vessel. It is known that many ligands bind to receptors on platelet surface will cause outside-in signal transduction and activate resting form of integrin IIb3. Therefore, suppression of the activation of integrin IIb3 in platelets can be a potential drug-target. Current antiplatelet drugs, such as integrin IIb3 antagonist, have side effects of bleeding and thrombocytopenia. As a result, designing a drug that can inhibit active form of integrin IIb3 with low risk of bleeding may be a potential candidate. In this study, trimucrin (Tmu), a disintegrin from snake venom, is used as protein scaffold to study: (i) the roles of linker region, the KGD loop and C-terminus of disintegrin in inhibiting platelet aggregation, and to (ii) design integrin IIb3 antagonist with high safety index. The safety index value evaluates the level of antagonist-induced integrin IIb3 activation by disintegrins in the presence of AP2. In this study I have successfully expressed ten mutants in Pichia pastoris and purified them to homogeneity. I found that 51KGDYR and 51KGDWR mutants exhibited higher activity in inhibiting platelet aggregation with the IC50 values of 104.4 nM and 61.3 nM in comparison of 51KGDRR mutant with the IC50 value of 90.0 nM. In contrast, their safety index values were only 8.1 and 277.9. The C-terminal 69NRLYG, 69NRLY, and 69NRFH mutants exhibited similar activity in inhibiting platelet aggregation and they remained similar safety index values. Mutation of linker region with 41IKKGT and 41MKEGT showed better activity in suppressing platelet aggregation with 56.9 nM and 84.1 nM. Moreover, 41IKKGT and 41IEEGT exhibited high safety index values. In summary, we found that the C-terminal residues adjacent to the KGD motif play the most important role in safety index. The results of this study will serve as a molecular basis for design of the antiplatelet drugs with low risk of bleeding.

CHINESE ABSTRACT................... I 
ABSTRACT........................ II 
ACKNOWNLEDGEMENT.................. IV 
TABLE OF CONTENTS ..................V 
TABLES 36 ....................... VI 
LIST OF TABLES....................VII 
LIST OF FIGURES...................VIII 
ABBREVIATION ...................... IX 
CHAPTER I INTRODUCTION ..............1 
1.1 Platelet and cardiovascular diseases.............1 
1.2 Acute coronary syndrome and treatment ..........1 
1.3 The relationship between platelet aggregation and integrin IIb32 1.4 Usage of integrin IIb3 antagonists in clinic ........... 4
1.5 Side effect of integrin IIb3 antagonists: bleeding and thrombocytopenia ..................... 5 
1.6 The challenges of integrin IIb3 antagonists in drug design....6 
1.7 Integrin activation .....................6 
1.8 Disintegrins serve as a scaffold for the design of IIb3 antagonists
...........................8
1.8.1 Rhodostomin (Rho).......................9
1.8.2 Trimucrin (Tmu)........................9
1.9 Evaluation of safety index value for integrinIIb3 antagonists ..10
CHAPTER II RATIONALE AND SPECIFIC AIMS.......11 CHAPTER III MATERIALS AND METHODS .........14 
3.1 Construction of Tmu mutants ...............14
3.1.1 Fast cloning based on quick change site-directed mutagenesis ....14
3.1.2 Transformation of plasmid into E.coli XL1-blue strain.......14
3.1.3 Transformation of linearized plasmid into Pichia pastoris X-33 strain 16
3.2 Protein expression of Tmu mutants ............17
3.2.1 Small scale expression of Tmu mutants ...............17
3.2.2 Large scale expression of Tmu mutants...............18
3.3 Purification of Tmu mutants .................18 
3.3.1 Purification of Tmu mutants by CaptoMMC chromatography...18
3.3.2 Purification of Tmu mutants by reverse-phase high performance liquid chromatography ..........................19 
3.3.3 Mass spectrometric measurement ...............19
3.4 Platelet aggregation assay ................20
3.4.1 List of solution recipes ......................20
3.4.2 Preparation of platelet-rich and platelet-poor plasma .........20
3.5 Platelet activation assay ...................21 
3.6 Cell adhesion assay .....................21 
CHAPTER IV RESULTS.................23 
4.1 Expression, purification and mass characterization of Tmu mutants..........................23 
4.2 The effects of linker, KGD motif and C-terminal region of Tmu mutants on platelet aggregation and safety index ..........24
4.2.1 The effects of KGD region of Tmu on platelet aggregation and safety index ...............................24
4.2.2 The effects of linker region of Tmu on platelet aggregation and safety index ...............................24 
4.2.3 The effects of C-terminal region of Tmu on platelet aggregation and safety index ............................25
CHAPTER V DISCUSSION .................26 
5.1 The C-terminal residues adjacent to the KGD motif play the most important role in safety index................26 
5.2 Combination of the mutation on linker region and C-terminus may improve its activity .................. 27 
CHAPTER VI CONCLUSIONS AND FUTURE PERSPECTIVE28 References ........................30 
TABLES...........................36 
FIGURES.................. .45 
Lists of APPENDIX TABLES .............. 55 
Lists of APPENDIX FIGURES.................. 58
                                    

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. (2002). "Integrins. " In Molecular Biology of the Cell, ed. B Alberts, A Johnson, J Lewis, M Raff, K Roberts, P Walter, pp. 1113–18. New York: Garland Sci. 4th ed.
Armstrong, P. C. and K. Peter. (2012). "GPIIb/IIIa inhibitors: from bench to bedside and back to bench again." Thromb Haemost 107(5): 808-814.
Bavishi, C. et al. (2017). "Duration of Dual Antiplatelet Therapy in Patients with an Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention. " Am J Med 130: 1312-1325.
Bennett, J. S. (2005). "Structure and function of the platelet integrin alphaIIbbeta3". J Clin Invest 115(12): 3363-3369.
Bledzka, K., et al. (2013). "Integrin alphaIIbbeta3: from discovery to efficacious therapeutic target." Circ Res 112(8): 1189-1200.
Bougie, D. W., et al. (2012). "Antibodies causing thrombocytopenia in patients treated with RGD-mimetic platelet inhibitors recognize ligand-specific conformers of alphaIIb/beta3 integrin." Blood 119(26): 6317-6325.
Calderwood, D. A. (2004). "Integrin activation." J Cell Sci 117(Pt 5): 657-666.
Calvete, J. J. (2004). "Structures of integrin domains and concerted conformational changes
in the bidirectional signaling mechanism of alphaIIbbeta3." Exp Biol Med (Maywood) 229(8): 732-744.
Chernyshenko, V., et al. (2017). "Antiplatelet and anti-proliferative action of disintegrin from Echis multisquamatis snake venom." Croat Med J 58(2): 118-127.
Cohen-Inbar, O., et al. (2016). "Glioblastoma multiforme targeted therapy: The Chlorotoxin story." J Clin Neurosci 33: 52-58.
Day, E. S., et al. (2002). "Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity." Cell Commun Adhes 9(4): 205-219.
Dhurat, R., et al (2014). "Principles and Methods of Preparation of Platelet-Rich Plasma: A Review and Author's Perspective." J Cutan Aesthet Surg 7(4): 189-197.
Fullard, J. F. (2004). "The role of the platelet glycoprotein IIb/IIIa in thrombosis and haemostasis." Curr Pharm Des 10(14): 1567-1576.
Gan, Z. R., et al. (1988). "Echistatin. A potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus." J Biol Chem 263(36): 19827-19832.
Gao, C., et al. (2009). "Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain." J Clin Invest 119(3): 504-511.
Gupta, S., et al. (1983). "High-performance cation-exchange chromatography of proteins." Anal Biochem 128(1): 196-201.
Hsu, C. C., et al. (2016). "Improved antithrombotic activity and diminished bleeding side effect of a PEGylated alphaIIbbeta3 antagonist, disintegrin." Thromb Res 143: 3-10.
Huang, T. F., et al. (2008). "FcgammaRII mediates platelet aggregation caused by disintegrins and GPIIb/IIIa monoclonal antibody, AP2." Exp Hematol 36(12): 1704-1713.
Hung, Y. C., et al. (2017). "Trimucrin, an Arg-Gly-Asp containing disintegrin, attenuates myocardial ischemia-reperfusion injury in murine by inhibiting platelet function." Eur J Pharmacol.
Jing, F. (2013). "Thrombosis Therapy: Focus on Antiplatelet Agents." Int J Genomics Med 1(1): 103.
King, S., et al. (2016). "Glycoprotein IIb/IIIa inhibitors: The resurgence of tirofiban." Vascul Pharmacol 78: 10-16.
Kishimoto, M., et al. (2002). "Molecular cloning and sequence analysis of cDNA encoding flavoridin, a disintegrin from the venom of Trimeresurus flavoviridis." Toxicon 40(7): 1033-1040.
Li, C. et al. (2011). "FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method." BMC Biotechnology 11(1): 92.
Lincoff, A. M., et al. (2000). "Platelet glycoprotein IIb/IIIa receptor blockade in coronary artery disease." Journal of the American College of Cardiology 35(5): 1103-1115.
Luo, B. H., et al (2006). "Integrin structures and conformational signaling." Curr Opin Cell Biol 18(5): 579-586.
McKenzie, S. E., et al. (1999). "The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: a transgenic mouse model." J Immunol 162(7): 4311-4318.
Miller, L. M., et al. (2017). "Emergence of Small-Molecule Non-RGD-Mimetic Inhibitors for RGD Integrins." J Med Chem 60(8): 3241-3251.
Minoux, H., et al. (2000). "Structural analysis of the KGD sequence loop of barbourin, an alphaIIbbeta3-specific disintegrin." J Comput Aided Mol Des 14(4): 317-327.
Mould, A. P., et al. (2002). "Integrin activation involves a conformational change in the alpha 1 helix of the beta subunit A-domain." J Biol Chem 277(22): 19800-19805.
Nieswandt, B., et al. (2003). "Targeting of platelet integrin alphaIIbbeta3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory FcgammaR." Int Immunol 15(3): 341-349.
Ojeda, P. G., et al. (2016). "Chlorotoxin: Structure, activity, and potential uses in cancer therapy." Biopolymers 106(1): 25-36.
Paladino, A., et al. (2017). "High Affinity vs. Native Fibronectin in the Modulation of alphavbeta3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design." PLoS Comput Biol 13(1): e1005334.
Roffi, M., et al. (2016). "2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC)." Eur Heart J 37(3): 267-315.
Rosen, E. D., et al. (2002). "FXI is essential for thrombus formation following FeCl3-induced injury of the carotid artery in the mouse." Thromb Haemost 87(4): 774-776.
Sandercock, P., et al. (2003). "Antiplatelet therapy for acute ischaemic stroke." Cochrane Database Syst Rev(2): CD000029.
Scott W., et al. (2002). "Platelets and cardiovascular disease." Eur J Cardiovasc Nurs1: 273.288.
Shattil, S. J., et al. (2010). "The final steps of integrin activation: the end game." Nat Rev Mol Cell Biol 11(4): 288-300.
Tsai, I. H., et al (1994). "Characterization of a cDNA encoding the precursor of platelet aggregation inhibition and metalloproteinase from Trimeresurus mucrosquamatus venom. " Biochim Biophys Acta 1200: 337-340
Tomiyama, Y., et al. (1992). "The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules." Blood 79(9): 2303-2312.
Tseng, Y. L., et al. (2004). "Rhodostomin, a disintegrin, inhibits adhesion of neutrophils to fibrinogen and attenuates superoxide production." J Biomed Sci 11(5): 683-691.
Van Agthoven, J. F., et al. (2014). "Structural basis for pure antagonism of integrin alphaVbeta3 by a high-affinity form of fibronectin." Nat Struct Mol Biol 21(4): 383-388.
van den Eijnden-van Raaij, A. J., et al. (1987). "Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents." Anal Biochem 163(1): 263-269.
Werle, M., et al. (2006). "Strategies to improve plasma half life time of peptide and protein drugs. " Amino acids 30: 351-367.
Wilson, S. J., et al. (2017). "Duration of dual antiplatelet therapy in acute coronary syndrome." Heart 103(8): 573-580.
Yang, L.-J., et al. (2015). "Substitution of the Echistatin Amino Acid Motif RGDD with KGDW Enhances Inhibition of Platelet Aggregation and Thrombogenesis." Int J Pept Res Ther 21(4): 451-458.
Yasuda, T., et al. (1991). "Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a canine preparation." Circulation 83(3): 1038-1047.
Ye, F., et al. (2012). "Reconstruction of integrin activation." Blood 119(1): 26-33. Zhang, K., et al (2014). "The regulation of integrin function by divalent cations."
Cell Adhesion & Migration 6(1): 20-29. 
張耀宗 (2014). 馬來腹蛇蛇毒蛋白其 RGD loop、linker 區域與 C 端突變蛋白的結構與鑑識整合蛋白活性的關連性研究. 國立成功大學基礎醫學研究所博士論文.
梁天豪 (2012). 利用馬來腹蛇蛇毒蛋白探討去整合蛋白 C 端序列對於整合蛋白的辨識及受質誘導結合位的表現. 國立成功大學生物化學暨分子生物研究所碩士論文.
陳柔瑞 (2016). 去整合蛋白的 KGD 迴圈、C 端區域以及二聚體對於整合蛋白交互作用所扮演的角色. 國立成功大學生物化學暨分子生物研究所碩士論文.
何艾樺 (2017). 設計具有專一性辨識整合蛋白llb3與低出血性風險之去整合突變蛋白. 國立成功大學生物化學暨分子生物研究所碩士論文.
蔡景皓 (2018). 利用整合突變蛋白設計具有專一性辨認整合蛋白 v6 或 llb3 之拮抗劑. 國立成功大學生物化學暨分子生物研究所碩士論文.
沈芳綺 (2010). 探討Rhodostomin的XRGDmotif在辨認整合蛋白上所扮演的角色. 國立成功大學生物化學暨分子生物研究所碩士論文.
黃俊豪 (2015). 針對癌症治療發展對於整合蛋白 v3 和/或 51 具有選擇性和親和力的去整合蛋白. 國立成功大學生物化學暨分子生物研究所碩士論文.

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