Thromb Haemost 2019; 119(01): 077-086
DOI: 10.1055/s-0038-1676518
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

tPA Point Mutation at Autolysis Loop Enhances Resistance to PAI-1 Inhibition and Catalytic Activity

Shuangzhou Peng
1  College of Chemistry, Fuzhou University, Fuzhou, Fujian, People's Republic of China
2  State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, People's Republic of China
3  University of Chinese Academy of Sciences, Beijing, People's Republic of China
,
Guangpu Xue
1  College of Chemistry, Fuzhou University, Fuzhou, Fujian, People's Republic of China
,
Shanli Chen
1  College of Chemistry, Fuzhou University, Fuzhou, Fujian, People's Republic of China
,
Zhuo Chen
2  State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, People's Republic of China
,
Cai Yuan
4  College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, People's Republic of China
,
Jinyu Li
1  College of Chemistry, Fuzhou University, Fuzhou, Fujian, People's Republic of China
,
Mingdong Huang
1  College of Chemistry, Fuzhou University, Fuzhou, Fujian, People's Republic of China
2  State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, People's Republic of China
› Author Affiliations
Funding This work was supported by grants from National Key R&D Program of China (2017YFE0103200) and Natural Science Foundation of China (31370737, 31400637, 31570745, 31670739, U1405229), and the Strategic Priority Research Program of the CAS (XDA09030307), and the CAS/SAFEA International Partnership Program for Creative Research Teams.
Further Information

Publication History

17 May 2018

21 October 2018

Publication Date:
31 December 2018 (online)

Abstract

Recombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.