因血栓阻塞血管的心血管疾病是全球導致死亡最大的主因，目前有許多抗血栓藥物但通常伴隨著高出血的風險性。到目前為止，使用組織型纖溶酶原激活劑 (tissue-type plasminogen activator, tPA)合併整合蛋白拮抗劑的溶栓治療，顯示可以有效減少治療心血管疾病 時所造成的不良反應 。纖溶酶原激活劑，如 Tenecteplase (TNK)、Streptokinase (SK)和 Staphylokinase (SAK)可將纖溶酶原激活成纖溶酶，從而降解富含纖維蛋白的血栓。此外在血小板表面有許多的αIIbβ3整合蛋白與止血和血栓形成息息相關 。在實驗室先前的研究中我們成功設計了一種能與整合蛋白 αIIbβ3特異性結合的去整合蛋白 突變體 RR 其具有傑出的抑制血小板凝集活性並能有效降低出血的風險。此研究的目的在於設計具整合蛋白αIIbβ3特異性結合的RR突變體與纖維蛋白特異性纖溶酶原激活劑 (TNK、 SK、 SAK)接合的融合蛋白以用於治療血栓的靶向療法 。迄今為止，我已成功在嗜甲醇酵母菌 (Pichia pastoris)中表達 SK和 SK-RR以及在哺乳動物細胞 (ExpiCHO-STM cell)中表達 TNK、 TNK-RR、 SAK和 SAK-RR。在血小板凝集試驗中 SAK-G9-RR和 TNK-G9-RR在抑制血小板凝集功能 的活性上比RR降低了 2.13倍和 14.58倍 。 在纖溶酶原激活物活性分析顯示 TNK、 TNK-G9-RR、SAK和 SAK-G9-RR的 IU/μM值分別為 21.18、 25.41、 15.84和 12.69。 在全血溶栓試驗中，TNK-G9-RR的活性比 TNK下降了 1.82倍， 而 SAK-G9-RR並沒有顯示出溶栓活性， 綜合上述結果說明 TNK-G9-RR是最具抗血栓潛力的藥物 。因此我們將TNK-G9-RR再優化，將連接序列從原本的 G9改成 (G4S)3、 (PA)3和 (EA3K)3，並透過功能性分析比較 活性。在抑制血小板凝 集的實驗中連接序列為 (G4S)3、 (PA)3和 (EA3K)3時其抑制活性 分別 比 RR降低了 8.95倍、 12.99倍和 16.91倍；在全血溶栓試驗中相較於 TNK，這些蛋白分別降低了 1.95倍、 1.67倍和 1.08倍，以上的數據表明 連接序列的種類會影響 TNK和 RR功能的執行， 此研究為未來的藥物發展奠定了基礎並為設計具有低出血風險 的 抗血栓藥物提供新的見解 。

Cardiovascular diseases (CVDs) threatens people’s life by thrombus. Many antithrombotic drugs are associated with a significant risk of bleeding. So far, a combination of thrombolytic therapy using tissue-type plasminogen activator and integrin antagonists showed a beneficial effect by reducing adverse outcomes of CVDs. Plasminogen activators, such as Tenecteplase (TNK), Streptokinase (SK), and Staphylokinase (SAK) can activate plasminogen into plasmin to degrade the fibrin-rich thrombus. Integrin αIIbβ3, on the surface of the platelet, leads to hemostasis and thrombosis. In the previous research of our laboratory, an integrin αIIbβ3-specific disintegrin mutant, RR, has been successfully designed, and it exhibited improved platelet aggregation inhibitory activity with a low risk of bleeding. In this study, I proposed to design a targeting therapeutics by fusing thrombolytic agents with integrin αIIbβ3-specific RR mutant. To date, I have successfully expressed SK and SK-RR in Pichia pastoris, as well as TNK, TNK-RR, SAK, and SAK-RR in ExpiCHO-STM cells and purified to homogeneous. Functional analysis showed that the platelet aggregation inhibitory of SAK-G9-RR and TNK-G9-RR exhibited 2.13- and 14.58-fold decreases in comparison of that of RR. The plasminogen activator activity of TNK, TNK-G9-RR, SAK, and SAK-G9-RR exhibited the activity with the IU/μM values of 21.18, 25.41, 15.84, and 12.69. The whole blood thrombolysis activity of TNK-G9-RR exhibited 1.82-fold decreases in comparison of that of TNK, however, SAK-G9-RR showed no thrombolysis activity. These results suggested that TNK-G9-RR was a potential thrombolytic agent. Furthermore, the change of linker sequence between TNK and RR from G9 to (G4S)3, (PA)3, and (EA3K)3 exhibited 8.95-, 12.99-, and 16.91-fold decreases in platelet aggregation inhibitory in comparison of that of RR, and exhibited 1.95-, 1.67-, and 1.08-fold decreases in whole blood thrombolysis activity in comparison of that of TNK. These data suggested the important role of the linker region. This study will provide new insight into the design of antithrombotic drugs with a low risk of bleeding and improved thrombolytic effect.

Adivitiya, Babbal, S. Mohanty, and Y. P. Khasa. 2018. 'Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris', Appl Microbiol Biotechnol, 102: 10561-10577.
Badimon, L., and G. Vilahur. 2014. 'Thrombosis formation on atherosclerotic lesions and plaque rupture', J Intern Med, 276: 618-632.
Banerjee, A., Y. Chisti, and U. C. Banerjee. 2004. 'Streptokinase--a clinically useful thrombolytic agent', Biotechnol Adv, 22: 287-307.
Baruah, D. B., R. N. Dash, M. R. Chaudhari, and S. S. Kadam. 2006. 'Plasminogen activators: a comparison', Vascul Pharmacol, 44: 1-9.
Benjamin, E. J., M. J. Blaha, S. E. Chiuve, M. Cushman, S. R. Das, R. Deo, S. D. de Ferranti, J. Floyd, M. Fornage, C. Gillespie, C. R. Isasi, M. C. Jiménez, L. C. Jordan, S. E. Judd, D. Lackland, J. H. Lichtman, L. Lisabeth, S. Liu, C. T. Longenecker, R. H. Mackey, K. Matsushita, D. Mozaffarian, M. E. Mussolino, K. Nasir, R. W. Neumar, L. Palaniappan, D. K. Pandey, R. R. Thiagarajan, M. J. Reeves, M. Ritchey, C. J. Rodriguez, G. A. Roth, W. D. Rosamond, C. Sasson, A. Towfighi, C. W. Tsao, M. B. Turner, S. S. Virani, J. H. Voeks, J. Z. Willey, J. T. Wilkins, J. H. Wu, H. M. Alger, S. S. Wong, and P. Muntner. 2017. 'Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association', Circulation, 135: e146-e603.
Bonnard, T., L. S. Law, Z. Tennant, and C. E. Hagemeyer. 2017. 'Development and validation of a high throughput whole blood thrombolysis plate assay', Sci Rep, 7: 2346.
Bonnard, T., Z. Tennant, B. Niego, R. Kanojia, K. Alt, S. Jagdale, L. S. Law, S. Rigby, R. L. Medcalf, K. Peter, and C. E. Hagemeyer. 2017. 'Novel Thrombolytic Drug Based on Thrombin Cleavable Microplasminogen Coupled to a Single-Chain Antibody Specific for Activated GPIIb/IIIa', J Am Heart Assoc, 6.
Chen, H., W. Mo, H. Su, Y. Zhang, and H. Song. 2007. 'Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities', BMC Mol Biol, 8: 88.
Chen, X., J. L. Zaro, and W. C. Shen. 2013. 'Fusion protein linkers: property, design and functionality', Adv Drug Deliv Rev, 65: 1357-1369.
Faraji, H., M. Ramezani, B. Mashkani, H. R. Sadeghnia, H. M. Benhangi, S. Hosseini Teshnizi, and F. Soltani. 2019. 'Comparison of expression optimization of new derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK', Biotechnol Prog, 35: e2819.
Faraji, H., F. Soltani, M. Ramezani, H. R. Sadeghnia, R. Nedaeinia, H. Moghimi Benhangi, and B. Mashkani. 2020. 'Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro', Biotechnol Lett, 42: 103-114.
Guo, L., W. Bi, X. Wang, W. Xu, R. Yan, Y. Zhang, K. Zhao, Y. Li, M. Zhang, X. Cai, S. Jiang, Y. Xie, Q. Zhou, L. Lu, and B. Dang. 2021. 'Engineered trimeric ACE2 binds viral spike protein and locks it in "Three-up" conformation to potently inhibit SARS-CoV-2 infection', Cell Res, 31: 98-100.
Howarth, Jack W., Jarek Meller, R. John Solaro, Jill Trewhella, and Paul R. Rosevear. 2007. 'Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin', Journal of Molecular Biology, 373: 706-722.
Hung, Y. C., Y. J. Kuo, S. S. Huang, and T. F. Huang. 2017. 'Trimucrin, an Arg-Gly-Asp containing disintegrin, attenuates myocardial ischemia-reperfusion injury in murine by inhibiting platelet function', Eur J Pharmacol, 813: 24-32.
Ishida, Hiroaki, Alicia Garcia-Herrero, and Hans J. Vogel. 2014. 'The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition', Biochimica et Biophysica Acta (BBA) - Biomembranes, 1838: 3014-3024.
Jackson, S. P. 2011. 'Arterial thrombosis--insidious, unpredictable and deadly', Nat Med, 17: 1423-1436.
Kang, CongBao, Hong Ye, Joel Chia, Bo-Hwa Choi, Sirano Dhe-Paganon, Bernd Simon, Ulrike Schütz, Michael Sattler, and Ho Sup Yoon. 2013. 'Functional role of the flexible N-terminal extension of FKBP38 in catalysis', Scientific Reports, 3: 2985.
Kaplan, Zane, and Shaun Jackson. 2011. 'The Role of Platelets in Atherothrombosis', Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program, 2011: 51-61.
King, Shawn, Marintha Short, and Cassidy Harmon. 2016. 'Glycoprotein IIb/IIIa inhibitors: The resurgence of tirofiban', Vascular Pharmacology, 78: 10-16.
Kuo, Yu-Ju, Ching-Hu Chung, Tzu-Yu Pan, Woei-Jer Chuang, and Tur-Fu Huang. 2019. 'A Novel αIIbβ3 Antagonist from Snake Venom Prevents Thrombosis without Causing Bleeding', Toxins, 12: 11.
Li, C., A. Wen, B. Shen, J. Lu, Y. Huang, and Y. Chang. 2011. 'FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method', BMC Biotechnol, 11: 92.
Llevadot, Joan, Robert P. Giugliano, and Elliott M. Antman. 2001. 'Bolus Fibrinolytic Therapy in Acute Myocardial Infarction', JAMA, 286: 442-449.
Ma, Y‐Q, J Qin, and EF Plow. 2007. 'Platelet integrin αIIbβ3: activation mechanisms', Journal of Thrombosis and Haemostasis, 5: 1345-1352.
Marder, V. J. 2001. 'Thrombolytic therapy: 2001', Blood Reviews, 15: 143-157.
Mazur, Wojciech, Grzegorz Kaluza, and Neal S. Kleiman. 1999. 'ANTIPLATELET THERAPY FOR TREATMENT OF ACUTE CORONARY SYNDROMES', Cardiol Clin, 17: 345-357.
Menter, David, and Raymond DuBois. 2012. 'Prostaglandins in Cancer Cell Adhesion, Migration, and Invasion', International journal of cell biology, 2012: 723419.
Mican, J., M. Toul, D. Bednar, and J. Damborsky. 2019. 'Structural Biology and Protein Engineering of Thrombolytics', Comput Struct Biotechnol J, 17: 917-938.
Miele, R. G., M. Prorok, V. A. Costa, and F. J. Castellino. 1999. 'Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity', J Biol Chem, 274: 7769-7776.
Miszta, A., D. Huskens, D. Donkervoort, M. J. M. Roberts, A. S. Wolberg, and B. de Laat. 2021. 'Assessing Plasmin Generation in Health and Disease', Int J Mol Sci, 22.
Nedaeinia, R., H. Faraji, S. H. Javanmard, G. A. Ferns, M. Ghayour-Mobarhan, M. Goli, B. Mashkani, M. Nedaeinia, M. H. H. Haghighi, and M. Ranjbar. 2020. 'Bacterial staphylokinase as a promising third-generation drug in the treatment for vascular occlusion', Mol Biol Rep, 47: 819-841.
Parry, M. A., C. Fernandez-Catalan, A. Bergner, R. Huber, K. P. Hopfner, B. Schlott, K. H. Gührs, and W. Bode. 1998. 'The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action', Nat Struct Biol, 5: 917-923.
Parry, M. A., X. C. Zhang, and I. Bode. 2000. 'Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues', Trends Biochem Sci, 25: 53-59.
Peter, Karlheinz. 2005. 'Platelet Integrins and Signaling.' in, Platelet Function (Springer).
Rabijns, A., H. L. De Bondt, and C. De Ranter. 1997. 'Three-dimensional structure of staphylokinase, a plasminogen activator with therapeutic potential', Nat Struct Biol, 4: 357-360.
Renatus, M., W. Bode, R. Huber, J. Stürzebecher, D. Prasa, S. Fischer, U. Kohnert, and M. T. Stubbs. 1997. 'Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors', J Biol Chem, 272: 21713-21719.
Rijken, D. C., G. Wijngaards, M. Zaal-de Jong, and J. Welbergen. 1979. 'Purification and partial characterization of plasminogen activator from human uterine tissue', Biochim Biophys Acta, 580: 140-153.
Ross, A. M. 1999. 'New plasminogen activators: a clinical review', Clinical cardiology, 22: 165-171.
Schlott, B., M. Hartmann, K. H. Gührs, E. Birch-Hirschfeid, H. D. Pohl, S. Vanderschueren, F. Van de Werf, A. Michoel, D. Collen, and D. Behnke. 1994. 'High yield production and purification of recombinant staphylokinase for thrombolytic therapy', Biotechnology (N Y), 12: 185-189.
Shattil, S. J., C. Kim, and M. H. Ginsberg. 2010. 'The final steps of integrin activation: the end game', Nat Rev Mol Cell Biol, 11: 288-300.
Shattil, S. J., and P. J. Newman. 2004. 'Integrins: dynamic scaffolds for adhesion and signaling in platelets', Blood, 104: 1606-1615.
Taheri, M. N., A. Behzad-Behbahani, G. Rafiei Dehbidi, S. Salehi, and S. Sharifzadeh. 2016. 'Engineering, expression and purification of a chimeric fibrin-specific streptokinase', Protein Expr Purif, 128: 14-21.
Takeda, Soichi, Tomoko Igarashi, Hidezo Mori, and Satohiko Araki. 2006. 'Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold', The EMBO Journal, 25: 2388-2396
Vaidya, B., G. P. Agrawal, and S. P. Vyas. 2012. 'Functionalized carriers for the improved delivery of plasminogen activators', Int J Pharm, 424: 1-11.
Wander, Gurpreet Singh, and Shibba Takkar Chhabra. 2013. 'Critical analysis of various drugs used for thrombolytic therapy in acute myocardial infarction', Med Updat, 23: 109-116.
Wang, X., S. Terzyan, J. Tang, J. A. Loy, X. Lin, and X. C. Zhang. 2000. 'Human plasminogen catalytic domain undergoes an unusual conformational change upon activation', J Mol Biol, 295: 903-914.
Wu, S. C., F. J. Castellino, and S. L. Wong. 2003. 'A fast-acting, modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy', J Biol Chem, 278: 18199-18206.
Zhu, J., Y. Wan, H. Xu, Y. Wu, B. Hu, and H. Jin. 2019. 'The role of endogenous tissue-type plasminogen activator in neuronal survival after ischemic stroke: friend or foe?', Cell Mol Life Sci, 76: 1489-1506.
Zinkstok, S. M., and Y. B. Roos. 2012. 'Early administration of aspirin in patients treated with alteplase for acute ischaemic stroke: a randomised controlled trial', Lancet, 380: 731-737.
何艾樺 (2017)。 設計具有專一性辨識整合蛋白αIIbβ3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，台南市。