Abstract
Background and PurposeMultiple members of the thiol isomerase (TI) family of enzymes are present in and released by platelets. Inhibition of these enzymes results in diminished platelet responses, aggregation, adhesion and thrombus formation. Recently, the therapeutic potential of TI inhibition has been recognised and drug‐development technologies were used to identify selective small molecule inhibitors. To date, few pan‐TI inhibitors have been characterised and the most studied, bacitracin, is known to be nephrotoxic, which prohibits its systemic therapeutic usage.Experimental ApproachWe therefore sought to identify novel broad‐spectrum inhibitors of these enzymes and test their effects in vivo. A total of 3,641 compounds were screened for inhibitory effects on the redox activity of ERp5, protein disulphide isomerase (PDI), ERp57, ERp72 and thioredoxin in an insulin turbidity assay. Of the lead compounds identified, zafirlukast was selected for further investigation.Key ResultsWhen applied to platelets, zafirlukast diminished platelet responses in vitro. Zafirlukast was antithrombotic in murine models of thrombosis but did not impair responses in a model of haemostasis. Since TIs are known to modulate adhesion receptor function, we explored the effects of zafirlukast on cell migration. This was inhibited independently of cysteinyl LT receptor expression and was associated with modulation of cell‐surface free thiol levels consistent with alterations in redox activity on the cell surface.Conclusion and ImplicationsWe identify zafirlukast to be a novel, potent, broad‐spectrum TI inhibitor, with wide‐ranging effects on platelet function, thrombosis and integrin‐mediated cell migration. Zafirlukast is antithrombotic but does not cause bleeding.
Highlights
Thrombosis-related pathologies such as myocardial infarction, stroke, pulmonary embolism or deep vein thrombosis are the primary cause of mortality for much of the Western world (Furie & Furie, 2012), highlighting the importance of identifying new therapeutic targets to treat these diseases
Immune and inflammatory diseases such as asthma are often associated with overproduction of cysteinyl LT (CysLT) which bind to CysLT receptors and induce immune-type signalling
Zafirlukast is generally regarded to be well tolerated in both animal safety studies and in humans with only minimal side effects being reported for the majority of patients
Summary
Thrombosis-related pathologies such as myocardial infarction, stroke, pulmonary embolism or deep vein thrombosis are the primary cause of mortality for much of the Western world (Furie & Furie, 2012), highlighting the importance of identifying new therapeutic targets to treat these diseases. One such interesting target is protein disulphide isomerase (PDI), which, when inhibited, attenuates both arterial and venous thrombosis, unlike current clinically used therapies (Cho et al, 2012; Jasuja et al, 2012). Zafirlukast, the more potent inhibitor, was found to modulate platelet function and integrin function in vitro and inhibit thrombosis in mice while bleeding was unaffected
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