Background: Procoagulant platelets expose phosphatidylserine (PS) on their surface, where it supports thrombin generation, through activation of a scramblase protein (TMEM16F). Inhibiting platelet PS exposure could be a novel anti-thrombotic approach, though currently there are no known selective inhibitors of platelet PS exposure. Platelet PS exposure is commonly quantified by the % of platelets that bind annexin V (AV %+ve).  This detection and anlaysis method, though convenient, may not be the most sensitive assay for screening novel inhibitors of platelet PS exposure.

Aims: Characterise the sensitivities of different PS exposure assays.

Methods: Washed human platelets were incubated with R5421 or DMSO. Scramblase and flippase activity were measured by flow cytometry using NBD-PS. PS expsoure following stimulation with 10μM A23187 was measured using multiple assays: a plate-based luminesence AV-binding assay, end-point and real-time flow cytometry assays using AV-FITC, lactadherin-FITC, or FRET pair AV-eGFP/AV-Alexa594. Liposomes containing different %PS were detected using each assay.

Results: Liposomes containing different %PS demonstrated that end-point AV binding by flow cytometry was the least sensitive measure of membrane PS composition. Decreased PS exposure following treatment with R5421 was not detectable using single colour AV %+ve analysis but could be detected using NBD-PS-, lactadherin-, AV FRET- and luminesence-based assays. Alternative analysis of single colour AV binding could convey the inhibition of PS exposure by R5421.

Conclusions: Anlaysis of platelet PS exposure by  AV %+ve measures the commitment of platelets to becoming procoagulant but is not sensitive to differences in the extent of PS exposure.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/annexin-v-binding-detects-platelet-procoagulant-commitment-but-is-not-sensitive-to-reductions-in-the-level-of-platelet-ps-exposure/