Formation of discoid platelet aggregates is shear dependent and requires platelet and integrin α_IIbβ₃ activation. Washed platelets (150 × 10⁶/mL) reconstituted with RBCs were perfused through microcapillary tubes coated with VWF (10 μg/mL) and fibrinogen (50 μg/mL) at wall shear rates of 600, 1800, or 5000 s⁻¹. Aggregate formation was quantified by determining the number (A) and size (B) of aggregates within a 70 × 90 μm visual field at the indicated time points over a 240-second period. (A) An aggregate composed primarily of reversibly adherent platelets was classified as “unstable” (○, broken line), while a “stable” aggregate was classified as one composed of irreversibly adherent platelets (•, solid line). Each point represents the mean ± SEM from at least 5 independent experiments. (B) The number of platelets within individual aggregates was counted at the indicated time points, and results show data combined from 5 independent experiments. The bar represents the median. *ND, quantitation was not performed because multiple small aggregates merge together into larger single aggregates. (C) (i) Washed platelets reconstituted with RBCs were perfused over a mixed VWF/fibrinogen matrix for 15 seconds to allow a small number of platelets to adhere. Washed platelets, untreated (control) or pretreated with c7E3 Fab (7E3), were reconstituted with RBCs and then perfused over the adherent platelets. The time that translocating platelets interacted with the preadhered stationary platelets was analyzed (control n = 30, 7E3 n = 16). (ii) Blood was collected from healthy donors (NBL) or individuals with Glanzmann thrombasthenia (GBL) (less than 1% α_IIbβ₃ by immunoblot) and perfused over confluent platelet monolayers made from normal platelets (NML) or Glanzmann platelets (GML). The interaction of flowing platelets (prelabeled with the fluorescent dye DiOC₆) with platelet monolayers was viewed in real time using fluorescence microscopy. The duration of adhesion contacts made by platelets tethering to the surface of monolayers was analyzed as described in “Platelet adhesion to monolayers.” Results show data combined from 3 independent experiments; n = 60; bar represents the mean (***P < .005). (D) Washed platelets (150 × 10⁶/mL) reconstituted with RBCs were perfused through microcapillary tubes coated with VWF (10 μg/mL) and fibrinogen (50 μg/mL) at 1800 s⁻¹ in the presence of the integrin α_IIbβ₃ antagonist c7E3 Fab, the activation inhibitor PGE₁, or a combination of the P2Y₁ and P2Y₁₂ receptor antagonists, A3P5PS and ARC69931MX (A3/ARC). The DIC images shown were taken at 120 seconds of perfusion (scale bar = 10 μm), highlighting the complete inhibition of aggregate formation in the presence of PGE₁ and the integrin α_IIbβ₃ antagonist. In contrast, when ADP receptors were inhibited, reversible aggregates formed but remained unstable (schematic diagrams indicate nonaggregated platelets, white; unstable aggregates, gray; and stable aggregates, black; scale bar = 10 μm). Each point represents the mean ± SEM from at least 4 independent experiments.