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Figure 2 The release of sCD40L from the surface of activated platelets is inhibited by glycoprotein (GP) IIb/IIIa antagonists. The platelet-rich plasm (with platelet count adjusted to 300,000/µl) was incubated (22°C, 20 min) with the indicated concentrations of GP IIb/IIIa antagonist (note log scale). A small aliquot was removed and incubated (22°C, 3 min) with 50 µM isoTRAP, fluorescein isothiocyanate-conjugated activated GP IIb/IIIa-specific monoclonal antibody PAC1, and PerCP-conjugated GP IIIa-specific monoclonal antibody RUU-7F12, then fixed with 1% formalin and analyzed by flow cytometry. The remaining aliquot was then activated (37°C, 1 h) with 50 µM isoTRAP without stirring, centrifuged twice, and the supernatants were tested in duplicate for sCD40L by enzyme-linked immunosorbent assay. Values for "Released sCD40L" were determined by subtracting sCD40L values obtained from samples that were not isoTRAP-activated from values obtained from samples that were iso-TRAP activated. Data are mean ± SEM, n = 3. Asterisks indicate significance as compared with samples with no GP IIb/IIIa antagonist.
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