Flow cytometry analysis of VASP-P

To measure ADP-induced platelet activation, the VASP-239 phosphorylation state was determined in citrate-anticoagulated blood samples using the PLT VASP/P2Y₁₂ kit, as described previously²⁰. Samples were treated in accordance with the manufacturer's protocol and were analyzed within 4 h of collection. In brief, whole blood was incubated for 10 min at room temperature with either the solutions of PGE₁ or PGE₁ plus 10 μmol/L ADP that were provided with the kit. These blood samples were then treated with fixative containing paraformaldehyde. Platelets were permeabilized and incubated with either an anti-VASP-P mouse monoclonal antibody (16C2) or the negative isotype control provided with the kit. Samples were then incubated with a polyclonal anti-mouse IgG antibody conjugated to fluorescein isothiocyanate and a platelet counterstaining reagent-PE prior to immediate analysis by flow cytometry using a FACS Calibur (Becton Dickinson, San Jose, CA, USA). The platelet population was identified using the forward and side scatter distributions, and 5000 platelets were gated and collected. Data from the negative isotype control was used to correct the mean fluorescent intensities (MFIcs) of PGE₁ (MFIc_PGE) and PGE₁ plus ADP (MFIc_PGE+ADP) samples that were incubated with anti-VASP-P. The platelet reactivity index (PRI) was calculated as outlined in the kit using the mean corrected fluorescence intensities and the following formula: PRI=[(MFIc_PGE - MFIc_PGE+ADP)/MFIc_PGE] ×100.