Reversible aggregation of discoid platelets in vitro is promoted by increasing matrix reactivity. Washed platelets (150 × 106/mL) reconstituted with RBCs were perfused through microcapillary tubes coated with VWF (10 μg/mL) and varying concentrations of fibrinogen (5, 10, 20, or 50 μg/mL) at 1800 s−1. (A) DIC images were taken at the indicated time points on a VWF (10 μg/mL) or VWF/fibrinogen matrix (10 μg/mL / 50 μg/mL, respectively). Schematic diagrams highlight nonaggregated platelets (white), reversible aggregates (gray), and stable aggregates (black). Scale bar = 10 μm. On VWF, aggregates remained reversible, while on VWF/fibrinogen reversible aggregates (60 seconds) eventually became stable (90 to 240 seconds). (B) Aggregate formation was quantified by determining the number (i) and size (ii) of aggregates within a visual field. (i) Graph showing the number of unstable (○, broken line) and stable (•, solid line) aggregates. Each point represents the mean ± SEM from at least 4 independent experiments. (ii) Each dot represents the number of platelets within an aggregate, showing data combined from 4 independent experiments (bar represents the median). *ND, quantitation was not performed because multiple small aggregates merged together into larger single aggregates. Scale bar = 10 μm. (C) SEM image demonstrating a typical reversible discoid platelet aggregate forming on a mixed VWF/fibrinogen matrix. Note that all platelets cluster around a central activated platelet through the development of thin membrane tethers. Platelets were fixed at 60 seconds. Scale bar = 2 μm. (D) SEM images demonstrating the sequential platelet morphologic changes associated with shear-dependent platelet aggregation. Platelets were perfused through VWF/fibrinogen-coated microcapillary tubes (10 μg/mL / 50 μg/mL, respectively) at 1800 s−1. These representative images demonstrate discoid platelets forming membrane tethers during surface translocation on the mixed matrix (30 seconds), a typical unstable aggregate composed of several discoid platelets clustered around a central partially spread platelet (60 seconds) and platelets that have undergone classical shape change (sphering and extension of multiple filopodia extensions) during stable aggregate formation (90 seconds). Scale bar = 2 μm.
