Figure 2.
Figure 2. Soluble fibrinogen binding to activated platelets. (A) Washed platelets incubated with soluble FITC-fibrinogen were activated with ADP, PAR4 receptor-activating peptide (AYPGFK), or convulxin, and bound fibrinogen was detected by FACS analysis. Agonist-induced FITC-fibrinogen bound to the platelets is indicated on the y-axis in fluorescence units. Agonist concentration is provided on the x-axis. Data are the means ± SEM for 3 or 4 independent experiments. Caspase-12–/– platelets bound significantly less soluble fibrinogen following stimulation with ADP (P values range from .02 to .003 for the 3 concentrations shown) and PAR4 receptor-activating peptide (P values range from .01 to .005 for the 3 concentrations shown). There was no significant difference in soluble fibrinogen binding at any of the convulxin concentrations. (B) The expression level of surface integrin αIIb (CD41) as measured by FACS analysis and FITC-conjugated anti-CD41 antibody is indicated on the y-axis in fluorescence units for wild-type (white bars) and caspase-12–/– (dotted bars) platelets at rest and following agonist stimulation. (C) Histograms demonstrate the relative PAR4 receptor expression levels as determined by FACS analysis using FITC-conjugated anti-PAR4 receptor antibody. Wild-type (WT), caspase-12–/– (C12–/–), and PAR4–/– platelets were analyzed. The isotype control is indistinguishable from the PAR4–/– platelets, and the wild-type platelets do not differ from the caspase-12–/– platelets. (D) A Western blot shows no difference in Gαq expression between wild-type (+/+) and caspase-12–/– (–/–) platelets. The same membrane was probed for ERK as a loading control. (E) Surface P-selectin (CD62-P) expression was determined using FACS analysis and FITC-conjugated anti–CD62-P antibody. The expression level is indicated on the y-axis in fluorescence units for wild-type (white bars) and caspase-12–/– (dotted bars) platelets at rest and following agonist stimulation. Data in panels B and E are the means ± SEM for 3 independent experiments, and there is no statistical difference between wild-type and caspase-12–/– platelets in either panel.

Soluble fibrinogen binding to activated platelets. (A) Washed platelets incubated with soluble FITC-fibrinogen were activated with ADP, PAR4 receptor-activating peptide (AYPGFK), or convulxin, and bound fibrinogen was detected by FACS analysis. Agonist-induced FITC-fibrinogen bound to the platelets is indicated on the y-axis in fluorescence units. Agonist concentration is provided on the x-axis. Data are the means ± SEM for 3 or 4 independent experiments. Caspase-12–/– platelets bound significantly less soluble fibrinogen following stimulation with ADP (P values range from .02 to .003 for the 3 concentrations shown) and PAR4 receptor-activating peptide (P values range from .01 to .005 for the 3 concentrations shown). There was no significant difference in soluble fibrinogen binding at any of the convulxin concentrations. (B) The expression level of surface integrin αIIb (CD41) as measured by FACS analysis and FITC-conjugated anti-CD41 antibody is indicated on the y-axis in fluorescence units for wild-type (white bars) and caspase-12–/– (dotted bars) platelets at rest and following agonist stimulation. (C) Histograms demonstrate the relative PAR4 receptor expression levels as determined by FACS analysis using FITC-conjugated anti-PAR4 receptor antibody. Wild-type (WT), caspase-12–/– (C12–/–), and PAR4–/– platelets were analyzed. The isotype control is indistinguishable from the PAR4–/– platelets, and the wild-type platelets do not differ from the caspase-12–/– platelets. (D) A Western blot shows no difference in Gαq expression between wild-type (+/+) and caspase-12–/– (–/–) platelets. The same membrane was probed for ERK as a loading control. (E) Surface P-selectin (CD62-P) expression was determined using FACS analysis and FITC-conjugated anti–CD62-P antibody. The expression level is indicated on the y-axis in fluorescence units for wild-type (white bars) and caspase-12–/– (dotted bars) platelets at rest and following agonist stimulation. Data in panels B and E are the means ± SEM for 3 independent experiments, and there is no statistical difference between wild-type and caspase-12–/– platelets in either panel.

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