title: Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1.
abstract: Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Lihu
authors.initials: L
authors.lastname: Gong
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China.
authors.forename: Tu
authors.initials: T
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China.
authors.forename: Cai
authors.initials: C
authors.lastname: Yuan
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.affiliation: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
keywords: Crystal structure; Fibrinolysis; Michaëlis complex; PAI-1; Serine protease; Serpin; Structural biology; Thrombolytic agents; Tpa