Abstract
Background and PurposeEthaninidothioic acid (R5421) has been used as a scramblase inhibitor to determine the role of phospholipid scrambling across a range of systems including platelet procoagulant activity. The selectivity of R5421 has not been thoroughly studied. Here, we characterised the effects of R5421 on platelet function and its suitability for use as a scramblase inhibitor.Experimental ApproachHuman platelet activation was measured following pretreatment with R5421 and stimulation with a range of agonists. Phosphatidylserine exposure was measured using annexin V binding. Integrin αIIbβ3 activation and α‐granule release were measured by flow cytometry. Cytosolic Ca2+ signals were measured using Cal520 fluorescence. An in silico ligand‐based screen identified 16 compounds which were tested in these assays.Key ResultsR5421 inhibited A23187‐induced phosphatidylserine exposure in a time‐ and temperature‐dependent manner. R5421 inhibited Ca2+ signalling from the PAR1, PAR4 and glycoprotein VI receptors as well as platelet αIIbβ3 integrin activation and α‐granule release. R5421 is therefore not a selective inhibitor of platelet scramblase activity. An in silico screen identified the pesticide thiodicarb as similar to R5421. It also inhibited platelet phosphatidylserine exposure, Ca2+ signalling from the PAR1 and glycoprotein VI, αIIbβ3 activation and α‐granule release. Thiodicarb additionally disrupted Ca2+ homeostasis in unstimulated platelets.Conclusion and ImplicationsR5421 is not a selective inhibitor of platelet scramblase activity. We have identified the pesticide thiodicarb, which had similar effects on platelet function to R5421 as well as additional disruption of Ca2+ signalling which may underlie some of thiodicarb's toxicity.
Highlights
Background and Purposeethaninidothioic acid (R5421) has been used as a scramblase inhibitor to determine the role of phospholipid scrambling across a range of systems including platelet procoagulant activity
Platelets play a central role in arterial thrombosis, the main proximal cause of acute coronary syndrome (Libby, 2013)
Platelets are activated at sites of atherosclerotic plaque rupture, leading to extensive platelet aggregation mediated by integrin αIIbβ3 (Huang et al, 2019)
Summary
R5421 has been used as a scramblase inhibitor to determine the role of phospholipid scrambling across a range of systems including platelet procoagulant activity. The selectivity of R5421 has not been thoroughly studied. We characterised the effects of R5421 on platelet function and its suitability for use as a scramblase inhibitor. Experimental approach: Human platelet activation was measured following pre-treatment with R5421 and stimulation with a range of agonists. Phosphatidylserine exposure was measured using annexin V binding. Integrin αIIbβ activation and α-granule release were measured by flow cytometry. Cytosolic Ca2+ signals were measured using Cal520 fluorescence. An in silico ligand-based screen identified 16 compounds which were tested in these assays
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