Inhibition of PDE3 ameliorates APS IgG–induced platelet activation by increasing cAMP. (A-B) Surface CD62P, (C) released ATP, and (D) cellular cAMP were measured in platelets treated with cilostazol (PDE3-i, 10 μM) and BAY 60-7550 (PDE2-i, 10 μM) before stimulation with 100 μg/mL cont IgG or APS IgG. ∗∗P < .01, ∗∗∗P < .001, and ns by a 1-way ANOVA with the Tukey multiple comparisons test (n = 4). (E) Representative immunoblots for (pVASP, Ser 157), total VASP, and GAPDH in platelets treated with A_2AR-i (5μM), A_2AR-a (5 μM), dibutyryl-cAMP (0.5 mM), 8-Br-cAMP (1 mM), forskolin (1 μM), PDE3-i (10 μM), or PDE2-i (10 μM) for 20 minutes, followed by stimulation with 100 μg/mL of either cont IgG or APS IgG for 1 hour (n = 3). (F-G) Flow cytometric evaluation of surface CD62P in platelets pretreated with A_2AR-a (5 μM), forskolin (1 μM), or PDE3-i (10 μM), followed by stimulation with affinity-purified (F) aβ2GPI IgG (20 μg/mL) or (G) aPT IgG (10 μg/mL) for 1 hour. (H-I) Quantification and representative images of platelet adhesion and accumulation in human whole blood stained with DiOC6 and treated with 100 μg/mL cont IgG or APS IgG along with A_2AR-a (5 μM), dibutyryl-cAMP (0.5 mM), 8-Br-cAMP (1 mM), PDE3-i (10 μM) perfused through a collagen-coated chamber at arterial shear (n = 5). Data represent mean ± SEM. Two-way ANOVA. Scale bars represent 100 μm. Data represent mean ± SD for panels A-G. ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, by a 1-way ANOVA with the Tukey multiple comparisons test (n = 5). MFI, Mean fluorescence intensity; UT, Untreated.