Knockdown of α-actinin augmented PAR1-AP–induced PAC-1 binding in CMK. Lentiviral particles encoding a shRNA for α-actinin or a control shRNA were transduced to CMK cells. (A) Intracellular α-actinin and surface αIIbβ3 were determined by flow cytometry. (B) CMK cells were stimulated with PAR1-AP (50μM) under nonstirring conditions with FITC–PAC-1 for 3 minutes. In contour plots, PAC-1 binding is shown on the x-axis, and transduction of the lentiviral particles is estimated by DsRed2 expression on the y-axis. (C) Bar charts represent PAC-1 binding to highly transduced cells (DsRed2 fluorescence > 250) and to less transduced cells (DsRed2 fluorescence < 50). (D) α-Actinin shRNA–transduced CMK cells were transiently transfected with plasmid encoding for wild-type or mutant α-actinin. Cells were stimulated with PAR1-AP (50μM) under nonstirring conditions with FITC–PAC-1 for 3 minutes. Error bars represent SEMs of 3 experiments in PAR1-AP and EDTA and of 8 experiments in no additive condition. *P < .05.