Increased cyclooxygenase expression and thromboxane A2 secretion in Jak2^VF platelets. (A) Immunoblot and quantification of COX-1 and COX-2 expression in resting platelets lysates of VF-Gp1ba mice and control mice. (B) Immunoblot and quantification of COX1 expression in platelets lysates from mice with Jak2^VF CH or respective controls. (C-D) COX 1 (C) and COX2 expression (D) in young platelets of Jak2^VF CH or control mice (assessed by flow cytometry). (E) Immunoblot and quantification of p-cPLA2 expression in resting and thrombin-stimulated (0.5 U/mL; 5 minutes) platelets lysates of VF-Gp1ba mice and control mice. (F) Immunoblot and quantification of p-PLCγ2 and p-Syk expression in resting and thrombin-stimulated (0.5 U/mL; 5 minutes) platelets lysates of VF-Gp1ba mice and control mice. (G) Ca²⁺ in resting and thrombin-stimulated platelets (0.5 U/mL; 5 minutes) was assessed by Fura Red staining and analyzed by flow cytometry as mean fluorescence intensity (MFI). (H) ROS in platelets was assessed by H2DCFDA staining and analyzed by flow cytometry as MFI. (I) Immunoblot and quantification of NOX2 expression in platelets lysates, which was normalized to β-actin. (J) TXB2 (stable metabolic product of TXA2) detected in supernatant of isolated platelets at baseline or after stimulation with 0.5 U/mL. (K) P-selectin surface expression on WT platelets treated with supernatant (SN) from stimulated Ctrl and Jak2^VF platelets with or without preincubation with TXA2 receptor antagonist Daltroban (1 μM). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Student t test or Mann-Whitney U test or 2-way analysis of variance as appropriate.