Assessment of VASP Phosphorylation by FC

The FC assay was performed within 48 hours of sample collection following the instructions of the test kit and as described in previous studies.^8,9 Whole blood samples (10 μL) were first incubated with prostaglandin E1 (PGE1) or PGE1 + adenosine diphosphate (ADP) at RT for 10 minutes and then fixed and permeabilized. Samples were immunolabeled by incubation with a serine 239-phosphorylated VASP-specific mouse monoclonal antibody (clone 16C2) followed by staining with a fluorescein isothiocyanate-labeled anti-mouse polyclonal antibody. Flow cytometric analysis was performed using an FACSCalibur (BD Biosciences, San Jose, California). The platelet population was identified from its forward and side scatter distribution, and 5000 platelet events were gated and analyzed for mean fluorescence intensity (MFI) using BD CellQuest software (BD Biosciences). The PRI was calculated from the MFI of samples incubated with PGE1 or PGE1 and ADP (PGE1 + ADP) according to the formula: