Platelet aggregation assay

Adult or neonatal citrated whole blood (150μL) was treated with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK; 40μM) and split evenly into two aliquots. Each aliquot was centrifuged (1000g, 5-min), and plasma was removed and stored at 25°C. Each sample was re-suspended with 75μL of HEPES/Tyrode’s solution and incubated with CD45-APC (1:100; BD Biosciences) and either CD31-eFluor 450 or CD31-FITC (1:100, 15-min, 25°C; eBioscience, San Diego, CA), followed by centrifugation (1000g, 5-min). The supernatants were discarded and the pelleted samples were re-suspended in reserved plasma in order to maintain donor plasma fibrinogen levels. The aliquots were combined and treated with a GPIIbIIIa inhibitor (eptifibatide; 20μg/mL), a monoclonal antibody against GPIb (6D1; 20μg/mL), or vehicle for 10-min at 25°C. Samples (10μL) was then added to tubes containing TRAP-6 (10μM), ADP (10μM), U46619 (10μM), CRP (10μg/mL), epinephrine (10μM), ristocetin (1mg/mL), rhodocytin (300nM), calcium ionophore A23187 (10μM), or vehicle in a BioShake iQ shaker plate (1000rpm, 37°C; Quantifoil Instruments, Jena, Germany). At 0, 0.5, 1, 2, and 5-min after agonist treatment, 1μL of the sample was removed and diluted 1:200 with 12.5% BD Cytofix Buffer in PBS. Samples were measured by FACS and analyzed using Flowing software. The rate and degree of platelet aggregation was determined by quantifying double-colored CD31 events at each time point, as adapted from the method used by De Cuyper et al [17]. In order to distinguish platelet-platelet interactions from platelet-WBC interactions, CD45 positive events were excluded from the platelet aggregation analysis. This assay required 150μL of whole blood.