Modulation of plasminogen activator inhibitor-1 (PAI-1) by the naphthoquinone shikonin

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68 +/- 2.32 mu M. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1 mg k(-1) dose significantly prolonged tail bleeding time from 12.956 +/- 4.457 min to 26.576 +/- 2.443 min. It also reduced arterial thrombus weight from 0.01 +/- 0.001 g to 0.006 +/- 0.001 g (p < 0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1 mg kg(-1) over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis. (C) 2016 Published by Elsevier B.V.