Analysis of agonist induced activation of IIbβ₃ on β₃-transduced thrombasthenic megakaryocytes.

Cultured cells were harvested for physiologic studies of αIIbβ₃ function at 9 days after transduction with −889Pl^A2β₃. Untransduced and β₃-transduced cells (1.5 × 10⁶/mL) were incubated with PE-GPIbα and FITC-PAC1 antibodies in modified Tyrode buffer containing TRAP, ADP, and epinephrine agonists. Binding of the αIIbβ₃ activation-sensitive antibody, FITC-PAC1, was monitored in the FL1 channel of the flow cytometer on the gated subset of megakaryocytes that expressed GPIbα (FL2). In each panel, FITC-PAC1 binding was measured in the absence (shaded histograms) and presence (unshaded histograms) of an Arg-Gly-Asp–containing peptide (GRGDW) that blocks FITC-PAC1 binding specifically to activated αIIbβ₃. The β₃-transduced megakaryocytes from patients R.S. and E.A. bound FITC-PAC1 at a fluorescence intensity peak value of 13 and 7, respectively, which is on average 10-fold higher than the FITC-PAC1 peak value of 1 for untransduced megakaryocytes from R.S. and E.A. (shaded). The β₃-transduced samples from R.S. and E.A. bound FITC-PAC1 at a fluorescence intensity peak value that was approximately 9% of the peak value of 110 for normal nonthrombasthenic megakaryocytes (shaded top). In the presence of the GRGDW peptide, FITC-PAC1 did not bind to megakaryocytes from β₃-transduced, untransduced, or normal samples as demonstrated with fluorescence intensity peak value of 1 for each sample (unshaded) and β₃-transduced megakaryocytes from R.S. that had a peak value of 2.