Role of PI3Kβ and GSK3 in thrombus stability at a high shear rate. (A) p110β^flox/flox control (WT) or PF4-Cre/p110β^flox/flox (p110β^null) thrombi formed at a physiologic shear rate of 500 seconds⁻¹ during 2 minutes were lysed, and phosphorylation of Akt (Ser473 and Thr308) and GSK3α and β were analyzed by western blot with indicated antibodies. Actin represents the control loading. Results shown are mean ± SEM of 7 independent experiments. Student t test ***P < .001. (B) Whole blood from p110β^flox/flox control (WT) or PF4-Cre/p110β^flox/flox (p110β^null) mice was preincubated with the GSK3 inhibitor (CHIR99021, 1 µM) and was perfused through a collagen-coated microcapillary at a physiological shear rate of 500 seconds⁻¹ and then at a high shear of 4000 seconds⁻¹ (top). Whole blood from humans was preincubated with the PI3Kβ inhibitor (AZD6482, 5 µM) and/or the GSK3 inhibitor (CHIR99021, 1 µM) as indicated and was perfused through a collagen-coated microcapillary at a physiological shear rate of 1500 seconds⁻¹ and then at a high shear rate of 4000 seconds⁻¹ (bottom). Thrombi volumes (µm³) were analyzed in both conditions using ImageJ software. Results shown are mean ± SEM of 3 to 5 experiments. Statistical analysis is 2-way ANOVA, **P < .01, ***P < .001. (C) PRP from p110β^flox/flox control (WT) or PF4-Cre/p110β^flox/flox (p110β^null) mice was preincubated or not with the GSK3 inhibitor (CHIR99021, 1 µM) as indicated and treated with thrombin. Representative time course of fibrin clot contraction (top) and average of the weight of serum extruded (mg) at each time point (bottom) are shown. Graphs represent the averages of 4 independent experiments and are mean ± SEM; 2-way ANOVA. *P < .05; **P < .01; ***P < .001.