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X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

ABSTRACT: The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

SUBMITTER: Law RHP

PROVIDER: S-EPMC5728053 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

Law Ruby H P RHP Wu Guojie G Leung Eleanor W W EWW Hidaka Koushi K Quek Adam J AJ Caradoc-Davies Tom T TT Jeevarajah Devadharshini D Conroy Paul J PJ Kirby Nigel M NM Norton Raymond S RS Tsuda Yuko Y Whisstock James C JC

Blood advances 20170509 12

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, i ...[more]

PMID: 29296720

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Project description:Essentials Tranexamic acid (TXA) is an antifibrinolytic drug used to reduce bleeding. Assaying plasmin generation (PG) in plasma detects clinically relevant TXA levels in vitro and ex vivo. 3.1-16.2 µg/mL TXA half-maximally inhibits PG in plasma from women undergoing cesarean delivery. PG velocity shows the strongest dose-relationship at low TXA concentrations (≤10 µg/mL). ABSTRACT: Background Tranexamic acid (TXA) is used to reduce bleeding. TXA inhibits plasmin(ogen) binding to fibrin and reduces fibrinolysis. TXA antifibrinolytic activity is typically measured by clot lysis assays; however, effects on plasmin generation (PG) are unclear due to a lack of tools to measure PG in plasma. Aims Develop an assay to measure PG kinetics in human plasma. Determine effects of TXA on PG and compare with fibrinolysis measured by rotational thromboelastometry (ROTEM). Methods We characterized effects of plasminogen, tissue plasminogen activator, fibrinogen, and α2 -antiplasmin on PG in vitro. We also studied effects of TXA on PG in plasma from 30 pregnant women administered intravenous TXA (5, 10, or 15 mg/kg) during cesarean delivery. PG was measured by calibrated fluorescence. PG parameters were compared with TXA measured by mass spectrometry and ROTEM of whole blood. Results The PG assay is specific for plasmin and sensitive to tissue plasminogen activator, fibrin(ogen), and α2 -antiplasmin. Addition of TXA to plasma in vitro dose dependently prolonged the clot lysis time and delayed and reduced PG. For all doses of TXA administered intravenously, the PG assay detected delayed time-to-peak (≤3 hours) and reduced the velocity, peak, and endogenous plasmin potential (≤24 hours) in plasma samples obtained after infusion. The PG time-to-peak, velocity, and peak correlated significantly with TXA concentration and showed less variability than the ROTEM lysis index at 30 minutes or maximum lysis. Conclusions The PG assay detects pharmacologically relevant concentrations of TXA administered in vitro and in vivo, and demonstrates TXA-mediated inhibition of PG in women undergoing cesarean delivery.

Dataset Information

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

Publications

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

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