Spontaneous binding of PAC-1 and fibrinogen to resting platelets.

Platelets were washed and resuspended (2 × 10⁶) in Tyrode-HEPES buffer containing 10⁻⁷ M PGE₁ and incubated without stimulation with 20 μg/mL FITC–PAC-1 (A) or 250 nM FITC-fibrinogen (B) for 20 and 40 minutes, respectively, at room temperature (RT). Samples were diluted and fixed in 1% formaldehyde and subjected to flow cytometry. The mean fluorescence intensities (MFIs) of PAC-1 and fibrinogen binding in the presence (dark histogram) or in the absence of 2 mM RGDS peptide (clear histogram) obtained for the patient and control are indicated on the histograms. The fact that the patient's platelets expressed only 20% of the usual levels of GPIIb-IIIa reinforces the difference in ligand binding between N.M. and control platelets.