Concentration-dependent loss of single platelets following agonist stimulation resulting from formation of platelet-platelet and platelet-leukocyte aggregates. Representative dot plots of platelet populations and quantitative gating, as identified by anti-CD41 APC antibody staining, in (A) unmixed blood and (B) vehicle-mixed samples remains stable. (C) Platelet population was depleted in agonist (collagen 1 μg/mL) mixed samples. Platelet loss occurred in a concentration-dependent manner following treatment with increasing concentrations of the common agonists (D) collagen, (E) PAR4-amide AYPGKF, and (F) U46619. Loss was significantly reduced by inclusion of prostacyclin (epoprostenol, 2 μg/mL). Analysis of (Gi) vehicle- and (Hi) collagen-stimulated whole blood acquired using an ImageStreamX Mark II incorporating a ×60 objective lens. Scale bars represent 7 μm and identified platelet-platelet and platelet-leukocyte aggregates. Platelets identified by anti-CD41 (green), leukocytes by anti-CD45 (yellow), and erythrocytes by anti-Ter119 (red). Confocal imaging of (Gii) vehicle-treated and (Hii) collagen-stimulated samples, with subsequent erythrocyte-lysis, confirmed respective absence and presence of stimulated platelet (CD42c, green)-platelet and platelet-neutrophil (MRP-14, red) aggregates. Images were acquired in 3 dimensions by confocal microscopy using a Zeiss LSM 5 PASCAL confocal laser-scanning microscope incorporating a ×63 oil-dipping objective lens (numerical aperture 1.4) and the image acquisition software IMARIS. Scale bars represent 20 μm. Data reported as mean ± SEM (n = 3-11), *P < .05 by paired t test. FSC, forward scatter.
