Delayed integrin closure accounts for the hyperresponsiveness of Twf2a^−/− platelets. (A) Washed platelets were simultaneously stimulated with thrombin (Thr/T) and collagen-related peptide (CRP/C) for the indicated periods of time. Activation of αIIbβ3-integrins (JON/A-PE) and phosphatidylserine exposure (Anxa5-FITC) on the outer leaflet of the platelet membrane was determined by flow cytometry. Flow cytometry plots are representative of at least 6 individuals. (B) Percentage of cells per quadrant. Q1, JON/A⁺ Anxa5⁻ (top left); Q2, JON/A⁺ Anxa5⁺ (top right); Q3, JON/A⁻ Anxa5⁺ (bottom right); Q4, JON/A⁻ Anxa5⁻ (bottom left). Values are mean (n = 6). (C-F) Platelets were left untreated or preincubated for 10 minutes in the presence of the calpain inhibitor MDL-28170 (MDL/M, 200 μM). Subsequently samples were stimulated with the calcium ionophore A23187 (A23, 10 μM) or thrombin (0.1 U/mL) and CRP (10 μg/mL), lysed, and processed for immunoblotting. Full-length (Ab762) and calpain-cleaved (AbΔ747) β3-integrin (C), as well as filamin A (E), talin (F), and α-tubulin, were probed with the respective antibodies and analyzed by densitometry. Values are mean ± SD (n = at least 4). ***P < .001, **P < .01, and *P < .05, unpaired Student t test.