CyPA deficiency leads to reduced α_IIbβ₃-integrin activation, degranulation, delayed spreading, and defective thrombus formation under flow. (A) Blood from Cypa^+/+ and Cypa^−/− mice was washed twice and incubated with 1 and 5 μg/mL of CRP, 0.002 and 0.02 U/mL of thrombin, 2 and 10μM ADP, and 1 μg/mL of CVX for 15 minutes in the presence of JON/A-PE, which only binds to the activated form of integrin α_IIbβ₃. Platelets were gated by their forward and side scatter characteristics. For each measurement, the mean fluorescence intensity (MFI) is shown for JON/A-PE (A) and anti–P-selectin-FITC (B; n = 6 per group). *P < .05; **P < .01; ***P < .001. (C) Platelets from the indicated mice were washed and allowed to spread on immobilized human fibrinogen (1 mg/mL) for 20 and 60 minutes after thrombin stimulation. Representative confocal images of 6 individual experiments are shown. Scale bar indicates 10 μm. (D) Quantitative assessment of adherent platelets, filopodia formation, and the number of fully spread platelets at different time points (20 and 60 minutes). Bar graphs depict mean values ± SD (n ≥ 6 mice per group). After 20 minutes, fully spread platelets were 22.3% ± 5.5% of Cypa^+/+ platelets versus 2.2% ± 2.1% of Cypa^−/− platelets; filopodia formation, 39.7% ± 3.9% of Cypa^+/+ platelets versus 22.3% ± 8.6% of Cypa^−/− platelets; platelet adhesion, 38% ± 5.5% of Cypa^+/+ platelets versus 75.5% ± 8.1% of Cypa^−/− platelets. After 60 minutes, fully spread platelets were 48.2% ± 4.5% of Cypa^+/+ platelets versus 55.2% ± 2.6% of Cypa^−/− platelets; filopodia formation, 39.5% ± 2.5% of Cypa^+/+ platelets versus 36.2% ± 2.5% of Cypa^−/− platelets; platelet adhesion, 12.3% ± 3.8% of Cypa^+/+ platelets versus 8.7% ± 1.51% of Cypa^−/− platelets. (E) Release of ATP on platelet stimulation with indicated agonists. Determination of ATP in the supernatant of platelets using a luminometric assay. Results are shown as the mean micromolar ATP concentration ± SD (n = 4 per group). Thr indicates thrombin. (F) Thrombus formation under flow. Whole blood was perfused over a collagen matrix at the indicated shear rates. Bar graphs depict mean values of thrombus surface coverage at a shear rate of 1000/s and 1700/s ± SD (n = 8 per group). U46619 (3μM) was added to whole blood before it entered the flow chamber as indicated. (G) Representative phase-contrast images at a shear rate of 1000/s (left panel) and 1700/s (right panel) are shown. Scale bar indicates 50 μm.