Alterations in proximal signaling events and dense granule secretion underlie defective GPVI responses in EXOC3 KO platelets. (A) Representative immunoblots of changes in phosphorylation of early GPVI signaling molecules, Syk and LAT, after CRP treatment (5 µg/mL) for 0.5 minute (i) with densitometric analysis of relative changes in Syk/LAT phosphorylation expressed as a KO:WT ratio after total protein loading adjustment (ii). (B) Washed platelets (1 × 10⁸/mL) from WT and EXOC3 KO platelets were loaded with Fura-2AM before stimulation with increasing concentrations of CRP (0.3-10 µg/mL) and monitored for changes in cytosolic calcium levels. The kinetics of mean changes in calcium responses from 4 independent experiments (i) with quantified area under the curve (AUC) analysis (ii) are presented. The arrow in panel Bi denotes agonist addition. (C) Washed platelets (2 × 10⁷/mL) were treated for 10 minutes with ADP (10 µM), CRP (0.3 µg/mL), U46619 (3 µM) alone, or combined as indicated, fixed, and monitored for changes in integrin α_IIbβ₃ activation (i) and α-granule secretion of P-selectin (CD62P) (ii) by using flow cytometry. Data are presented as the mean ± standard error of the mean. n = 7 (A); n = 4 (B); n = 3 (C). *P < .05, **P < .01, ***P < .001, ****P < .0001 vs indicated sample.