Reduced integrin activation in platelets expressing CalDAG-GEFIΔC1. (A) Representative flow cytometric analysis of the GFP expression in platelets from chimeric mice expressing intact CalDAG-GEFI (WT/GFP, solid black line) or CalDAG-GEFIΔC1 (ΔC1/GFP, light gray shaded area) in comparison to WT control platelets (dark gray shaded area). (B) Western blot analysis of CalDAG-GEFI expression in CalDAG-GEFI^−/− (KO), WT, and CalDAG-GEFIΔC1 (ΔC1) platelets. Mutant CalDAG-GEFI was detected at a lower molecular weight due to the deletion of the C1 domain (49 amino acids≈5.5 kDa). (C) Immunofluorescence staining for CalDAG-GEFI in WT and CalDAG-GEFIΔC1 (ΔC1) platelets. Results shown in panels A-C are representative of 3 independent experiments. (D) αIIbβ3 integrin activation (JON/A-PE binding²³ ) was determined in WT (solid bars), CalDAG-GEFI^−/− (KO, checkered), CalDAG-GEFIΔC1 (ΔC1, vertically striped), or CalDAG-GEFI+/− (HET, horizontally striped) platelets activated with PAR4 peptide (1mM, top) or convulxin (Cvx, 750 ng/mL, bottom) in the presence of the P2Y12 inhibitor, 2-MesAMP. n = 6, 3 independent experiments.