Platelet Function Assays in Blood

Citrated venous whole blood collected from each participant was used to quantify PAC-1 mAb binding and P-selectin expression, platelet-platelet aggregation, static adhesion on collagen and VWF, and adhesion and thrombus formation under low and high shear flow as previously described (19 (link)). For the clinical investigations, up to 25 mL of peripheral venous blood was obtained; the experimental assay panel used less than 500 μL in all. The activation, aggregation, and static adhesion assays were performed with seven agonists: 100 μM thrombin receptor activator 6 (TRAP-6; Tocris Bioscience, Bristol, UK); 100 μM adenosine diphosphate (ADP; Sigma-Aldrich, St. Louis, MO); 100 μM thromboxane A₂ receptor agonist (U46619; Tocris Bioscience); 100 μM epinephrine (Chrono-Log, Havertown, PA); 100 μg/mL collagen related peptide (CRP; R. Farndale, Cambridge University, Cambridge, UK); 100 μM ristocetin (Chrono-Log); and 10 mg/mL calcium ionophore (Invitrogen, Carlsbad, CA). All antibodies were from BD Biosciences (San Jose, CA) with the exception of eFluor450-CD31 (ThermoFisher, Waltham, MA); FITC-CD31 (ThermoFisher); and FITC-CD62 (OriGene Technologies, Rockville, MD). Other material sources include bovine serum albumin, BSA (Sigma-Aldrich); human VWF (Haematologic Technologies, Essex Junction, VT); fibrillary equine type I collagen (Chrono-Log); and cytofix (BD Biosciences).