Impaired Nbeal2^−/− mouse platelet function in vitro and in vivo. Measurements of activation of platelets from WT (solid symbols) and Nbeal2^−/− mice (open symbols). (A-C) Washed platelets were assessed by flow cytometry for activation in response to varying concentrations of agonists (X-axis) by measuring antibody binding (JON/A) to activated α_IIbβ₃ (A-B) or exposed P-selectin (C). Graphs show mean ± standard error of the mean (SEM) (n = 6 mice per group) for mean fluorescence intensity (MFI) after activation by thrombin (A,C) or ADP (B). (D-F) Optical aggregometry in physiological buffer of washed platelets exposed to varying concentrations of CRP (D), thrombin (E), or the thromboxane analog U46619 (F). The agonist concentration is plotted against the percentage of aggregation (see the “Methods” section). (G) Impedance aggregometry measurement of platelets in citrated blood exposed to collagen (7 μg/mL, n = 6 mice per group); horizontal lines represent mean ± SEM for area under the curve of Multiplate aggregation. (H) Nbeal2^−/− mice showed greater cumulative blood loss in relation to WT in a tail transection bleeding assay; mean ± SEM is shown for WT (solid squares) and Nbeal2^−/− mice (open circles; n = 11 for each). (I-M) Results of intravital videomicroscopy monitoring of platelet accumulation and activation in thrombi formed in response to laser injury of cremaster muscle arterioles. (I-J) Sum and maximal platelet accumulation in thrombi formed in response to injury were determined using a fluorescent anti-mouse GPIbβ antibody (×488); values shown are mean ± SEM for 170 thrombi in 12 WT mice and 155 thrombi in 12 Nbeal2^−/− mice (*P < .05, unpaired t test). (K-L) The time to half-maximal activation ratio for CD41 was determined using an anti-mouse CD41 Fab fragment (K) and for P-selectin (L) using an anti-mouse P-selectin antibody. Values shown are mean ± SEM; for CD41, n = 119 thrombi in 6 WT mice and 93 thrombi in 6 Nbeal2^−/− mice; for P-selectin, n = 69 thrombi in 6 WT mice and 60 thrombi in 6 Nbeal2^−/− mice. (M) The time course of P-selectin activation after injury. Mean ± SEM for WT mice (solid squares) and Nbeal2^−/− mice (open circles) are shown.