Impact of FPR2/ALX in platelet spreading, hemostasis, and cyclic adenosine monophosphate (cAMP)–mediated signaling. (A) Platelet adhesion and spreading on fibrinogen-coated glass surface was analyzed in the absence and presence of WRW4 (1.25, 2.5, 5, and 20 µM) by confocal microscopy (magnification ×60; bar indicates 10 µm). The number of adhered (Ai) and spread (Aii) platelets and the relative surface area of spread platelets (Aiii) were determined by analyzing the images using ImageJ. Ten random fields of view were recorded for each sample. Data represent mean ± SEM (n = 3). (B) The impact of FPR2/ALX in the modulation of hemostasis was analyzed using tail bleeding assay in control or Fpr2/3−/− mice. Data represent mean ± SEM (n = 8 in each group). (C) The level of cAMP in human isolated platelets in the presence or absence of WRW4 (i) and control and Fpr2/3−/− mouse platelets (ii) was analyzed using a cAMP assay kit. Data represent mean ± SEM (n = 4). (D) The phosphorylation of VASP at Ser-157 was analyzed in the presence of WRW4 (i) and in platelets obtained from Fpr2/3−/− mice (ii) by immunoblot analysis using selective antibodies. The blots are representative of 3 separate experiments. P values shown are as calculated by 1-way ANOVA followed by Bonferroni's correction in most of the experiments except for the data shown in panels B-C, where a nonparametric Mann-Whitney U test and a 2-tailed unpaired Student t test were used, respectively (*P < .05; **P < .01; ***P < .001).