Engagement of PECAM-1 Ig-domain 6 leads to conformational changes in the integrin α_IIbβ₃. Washed human platelets were preincubated with the FcγRIIa-specific antibody, IV.3 (to prevent possible Fc-receptor activation), before the addition of 10 μg/mL of normal mouse IgG (NM IgG), PECAM-1.1, PECAM-1.2, or PECAM-1.3. Buffer or RGDW peptide was used as negative and positive controls, respectively. After the addition of FITC-conjugated D3 for 30 minutes at room temperature, platelets were washed and transferred to 450 μL of RCD buffer, pH 7.4, and analyzed by flow cytometry. Note that PECAM-1.2 binding resulted in the exposure of the D3 epitope to almost the same extent as that induced by RGD peptide, a known modulator of integrin conformational change.62 The ability of PECAM-1.2 to induce conformational changes in α_IIbβ₃ varied somewhat from experiment to experiment; sometimes PECAM-1.2 was more effective than RGDW in inducing D3 binding (see Fig 4).