FcγRIIa-mediated platelet activation and lowering of cAMP by IgG purified from APS plasma. (A) Purified platelets obtained from healthy volunteers (n = 4) were treated with various concentrations of control IgG (cont IgG) or APS IgG for 1 hour, and cAMP was measured by assay kit. Platelets pretreated with either a monoclonal anti-FcγRIIa antibody (aFcγRIIa, clone IV.3, 1μg) or U73122 (PLC-i, 1μM), followed by stimulation with 100 μg/mL APS IgG for 1 hour. Platelets were evaluated for surface CD62P (α-granules) using a flow cytometer (B), released ATP (dense granule) using luminescent-based assay (C), and cellular cAMP using an assay kit (D). (E-H) CD62P and cAMP were measured in platelets pretreated with aFcγRIIa (1 μg) or PLC-i (1 μM). After incubation, platelets were stimulated with affinity-purified aβ2GPI IgG (20 μg/mL) or affinity-purified aPT IgG (10 μg/mL) for 1 hour. Data represent mean ± SD. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001, and ns by a 1-way ANOVA with the Tukey multiple comparisons correction (n = 4). PLC-I, phospholipase C-inhibitor.