Flow cytometric aggregation assay and characterization of integrin activation. (A) Aggregation of control, Glanzmann, and LAD-III platelets upon stimulation with PMA, ristocetin, collagen (Student t test LAD-III vs Glanzmann, P < .001), or CRP. Preincubation with 9O12, mAb LIA, or tirofiban blocks ligand binding to GPVI, integrin α2β1, and αIIbβ3, respectively. Aggregation of the control platelets was set to 100% for each separate experiment (n = 4-6). Combinations of inhibitors did not add to the effects noticed when used as a single blocking agent (data not shown). (B) Characterization of αIIbβ3 integrin by flow cytometry upon stimulation with PMA, ristocetin, collagen, or CRP in control, Glanzmann, and LAD-III platelets. αIIbβ3 total expression was measured with C17 (top panel), and active αIIbβ3 was measured with PAC-1 antibodies (middle panel). The ratio of active integrin versus total integrin expression was calculated from the respective mean fluorescence intensity (MFI) after subtraction of isotype controls and normalizing unstimulated platelets to 1 (bottom panel). (C) Characterization of α2β1 integrin by flow cytometry upon stimulation with PMA, ristocetin, collagen, or CRP in control, Glanzmann, and LAD-III platelets. α2β1 total expression was measured with HUTS-21, and active α2β1 was measured with HUTS-4. The ratio of active integrin versus total integrin expression was calculated from the respective mean fluorescence intensity (MFI) after subtraction of isotype controls and normalizing unstimulated platelets to 1.