Increased PDE5A phosphorylation in PTPN22-deficient platelets. WT or PTPN22^−/− platelets were treated with CRP (5 μg/mL) (A) for 5, 15, 30, or 60 seconds or thrombin (1 U/mL) (B) for 1, 3, or 5 minutes followed by measurement of PDE5A phosphorylation (Ser92) and expression by western blot (mean ± standard deviation [SD], n = 4 independent experiments; two-way analysis of variance [ANOVA]). (C) WT or PTPN22^−/− platelets were isolated and stimulated with CRP (5 μg/mL) for 1 minute and then the level of cGMP and cyclic adenosine monophosphate (cAMP) was measured by using an enzyme-linked immunosorbent assay kit (mean ± standard error [SE], n = 5-7; t test). (D) After stimulation with CRP (5 μg/mL), the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) (Ser157 and Ser239) was measured (mean ± SD, n = 4 independent experiments; two-way ANOVA). PTPN22^−/− platelets were pretreated with vehicle or 10 μM sildenafil (PDE5A inhibitor) for 1 hour at 37°C followed by measurement of platelet aggregation in response to CRP (0.25 μg/mL) (mean ± SE, n = 4; one-way ANOVA) (E), P-selectin expression in response to CRP (0.5 μg/mL) (mean ± SE, n = 3; one-way ANOVA) (F), or spreading on collagen (mean ± SD, n = 3; one-way ANOVA) (scale bar = 10 μm) (G). *P < .05, **P < .01, ***P < .001. ns, no significance.