Moderately reduced activation of Cyfip1^−/−platelets. (A) Expression of indicated proteins in control and mutant platelets was assessed by western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control (n = 3). (B) Platelet count per microliter and (C) platelet size given as mean forward scatter (FSC) was determined via flow cytometry (n = 4, representative for 4 independent experiments). Values are mean plus or minus standard deviation (SD); ***P < .001. (D) Flow cytometric determination of inside-out activation of the αIIbβ3 integrin (JON/A-phycoerythrin [PE] antibody) and (E) degranulation-dependent P-selectin exposure (fluorescein isothiocyanate [FITC]-labeled anti–P-selectin antibody) in response to the agonists ADP, U46619 (thromboxane analog), thrombin (Thr), CRP, convulxin (CVX), and rhodocytin (RC) (n = 5, representative for 4 independent experiments). Values are mean plus or minus SD; **P < .01, ***P < .001. (F) Washed platelets from Cyfip1^+/+ and Cyfip1^−/− mice were activated with the indicated agonists. Second-wave inhibitors: indomethacin 10 µM, EDTA, apyrase 2 U/mL (representative curve of at least 6 independent samples). (G) Determination of tyrosine phosphorylation after stimulation with 1 μg/mL CRP under stirring conditions (1000 rpm) at 37°C. Fifty-microliter aliquots were taken at the indicated time points. Samples were blotted on a polyvinylidene difluoride (PVDF) membrane and stained using the 4G10 antibody. GAPDH served as loading control (n = 3). MFI, mean fluorescence intensity.