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].