Disinhibition of actin assembly prevents integrin closure in Twf2a^−/− platelets. (A-B) Resting (A) or fibrinogen-spread (B) platelets were stained for F-actin (red) and α-tubulin (green) after the indicated time periods. Images were acquired with a TCS SP8 confocal microscope (100×/1.4 STED WHITE oil objective, Leica Microsystems). Scale bars, 3 μm. Images are representative of at least 6 individuals. (C) Relative F-actin content of resting and activated platelets was determined by flow cytometry. (D) Control (dimethyl sulfoxide) or toxin-treated (5 μM jasplakinolide, 2.5 μM latrunculin A) platelets remained resting or were activated with the indicated agonists and concentrations. αIIbβ3-integrin activation (JON/A-PE) was determined by flow cytometry. (E-F) Latrunculin A–treated (2.5 μM for 10 minutes) platelets were left untreated (rest) or preincubated for 10 minutes in the presence of the calpain inhibitor MDL-28170 (200 μM). Subsequently, samples were stimulated with the Ca²⁺ ionophore A23187 (10 μM) or thrombin (0.1 U/mL) and collagen-related peptide (10 μg/mL), lysed, and processed for immunoblotting. Full-length (Ab762) and calpain-cleaved (AbΔ747) β3-integrin, as well as α-tubulin, were probed with the respective antibodies and analyzed by densitometry. (G-H) Platelets were left untreated or stimulated for the indicated time points with 0.1 U/mL thrombin, lysed, and processed for immunoblotting. Total Pfn1 (aa 126-137), phospho-Y¹²⁹ Pfn1 (G), total n-cofilin, phospho-cofilin (H), and α-tubulin were probed with the respective antibodies and analyzed by densitometry. Values are mean ± SD (n = at least 4). A23, A23187 (Ca²⁺ ionophore); CRP/C, collagen-related peptide; MDL/M, MDL28170 (calpain inhibitor); Rhd, rhodocytin; Thr/T, thrombin; U46, U46619 (synthetic thromboxane A₂ analog). ***^,###P < .001, **^,##P < .01, and ^#,$P < .05, unpaired Student t test.