Figure 3.

Oral administration of ARQ 092 blocks AKT phosphorylation and activation of neutrophils and platelets isolated from SCD mice ex vivo. Vehicle (0.01 M phosphoric acid) or ARQ 092, 100 mg/kg, was given orally to SCD mice. Blood and bone marrow were collected 30 min after treatment. Isolated (A–C) neutrophils or (D–F) platelets were incubated with or without 10 μM fMLP or 0.025 U/mL thrombin for 2 min, respectively. (A and D) To determine the phosphorylation levels of Src, PI3K, and AKT, equal amounts (50 μg) of cell lysate protein were immunoblotted, followed by densitometry (n = 3). (B and C) The surface amount of αMβ2 and soluble fibrinogen binding were measured by flow cytometry. (E) P-selectin exposure was measured by flow cytometry. (F) Platelet aggregation was induced by 0.025 U/mL thrombin or 0.25 μg/mL cross-liked collagen-felated peptide (CRP). The representative aggregation traces were obtained from two independent experiments. All other data represent the mean ± SD (n = 3). *P<0.05, ***P<0.001, and ****P<0.0001 versus unstimulated vehicle control (or between two groups), ANOVA and Tukey test.