Association between platelet hyperactivity and low CD73 activity in APS. (A) Estimation of ectonucleotidase activity by measuring free inorganic phosphate (μM) using the Malachite Green assay kit, after the addition of AMP (100 μM) to purified platelets from the fresh blood of healthy controls (n = 20), patients who are aPL⁺ (n = 38), or patients with aPL-negative VTE disease [VTE (aPL⁻), n = 8]. (B) Spearman correlation of CD73 activity with cAMP level and (C) surface CD62P for aPL⁺ patients as indicated. ∗∗∗∗P < .0001 and ns by a 1-way ANOVA with the Tukey multiple comparisons test. (D) Flow cytometric evaluation of surface expression of CD73 on platelets treated with 100 μg/mL cont IgG or APS IgG. ∗P < .05 by unpaired t test (n = 4). (E) Evaluation of CD73 expression using flow cytometer in platelets treated with aFcγRIIa (1 μg) or PLC-i (1 μM) before inducing 100 μg/mL of APS IgG for 1 hour (n = 4). (F) Schematic representation of healthy platelets treated with affinity-purified antibodies followed by AMP to measure surface CD73 activity. (G-H) Platelets were pretreated with aFcγRIIa (1 μg) or PLC-i (1 μM). Platelets were then stimulated with affinity-purified aβ2GPI IgG (20 μg/mL) (G) or affinity-purified aPT IgG (10 μg/mL) (H) for 1 hour. Platelet surface CD73 activity was measured using the Malachite Green assay kit after adding AMP (100 μM). (I) Schematic representation of the degradation of AMP to adenosine by CD73, including inhibition of CD73 by the selective inhibitor PSB 12379. (J) Platelet CD62P measured on PSB 12379 (20 μM) treated platelets in the presence or absence of APS IgG (100 μg/mL) (n = 5). 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).