A specific plasminogen activator inhibitor‐1 antagonist derived from inactivated urokinase

Abstract

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re‐establish blood flow after thrombus formation. Plasminogen activator inhibitor‐1 (PAI‐1) inhibits urokinase‐type and tissue‐type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI‐1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI‐1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI‐1 and identified a potent PAI‐1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active‐site mutation (S195A) and four additional mutations (G37bR–R217L–C122A–N145Q). PAItrap inhibits human recombinant PAI‐1 with high potency (K d = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI‐1. In addition, PAItrap inhibits both human and murine PAI‐1, allowing the evaluation in murine models. In vivo, using a laser‐induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI‐1 inhibitors using inactivated urokinase.

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“…Angiotensin-converting enzyme inhibitors (ACE-I), insulin-sensitizing agents (including metformin and thiazolidinediones), and hormone-replacement therapy in women can have mild to moderate reduction of PAI-1 levels [ 133 ]. There are direct PAI-1 antagonists known, but none has been cleared for human use yet [ 134 , 135 ].…”

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“…It is secreted by various cells including ECs, megakaryocytes, smooth muscle cells, fibroblasts, monocytes, adipocytes, hepatocytes and other cell types. Its main function is to inhibit tissue/urokinase plasminogen activator (tPA/uPA) inducing fibrinolysis [133]. However, it also plays a role in adhesion, migration, signal transduction and anti-apoptosis [134,135].…”

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“…We also reported two proteinaceous PAI-1 inhibitors (PAItrap) derived from the inactive recombinant catalytic domain of uPA (Fig. 4b, c) 172,173 . UPA is known to bind to PAI-1 through the catalytic domain with very high affinity.…”