Inhibition of APS IgG–accelerated platelet activation, clot retraction, and procoagulant platelet formation by an A_2AR agonist. (A) Purified platelets were pretreated with A_2AR-a (5 μM) and A_2BR-a (5 μM), followed by stimulation with 100 μg/mL cont IgG or APS IgG for 1 hour, and surface CD62P was assessed using flow cytometry. (B-C) Using assay kits, released ATP in supernatant and cellular cAMP were measured in platelets pretreated with A_2AR-a (5 μM) followed by stimulation with 100 μg/mL of cont IgG or APS IgG for 1 hour. Forskolin (1 μM) was used as a positive cont for cAMP quantification. (D-E) Flow cytometric quantification of PS exposure along with CD62P in platelets treated with either vehicle or various concentrations of A_2AR-a, followed by stimulation with 100 μg/mL cont IgG or APS IgG. (F) The representative images show the clot retraction was measured for 1 hour in platelet-rich plasma, supplemented with red blood cells, incubated with 50 μg/mL of cont IgG or APS IgG in the presence or absence of A_2AR-a (5 μM) before adding thrombin (0.25 U/mL). (G) Quantified clot size over time and the values are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by 2-way ANOVA with Tukey multiple comparisons tests, n = 3 individual donors per group. Data represent mean ± SD. ∗P <.05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001, and ns by a 1-way ANOVA with the Tukey multiple comparisons test (n = 4).