Increased integrin activation in Twf2a−/− platelets. (A-B) Platelet αIIbβ3-integrin (JON/A-PE) activation (A) and P-selectin (anti-CD62P-FITC) exposure (B), serving as a measure for α-granule release, were determined by flow cytometry. (C-D) Platelet adhesion (C) and thrombus formation (D) under flow (1000 s−1) were analyzed in a flow chamber system. Scale bars, 25 μm. Images were acquired with a Zeiss Axiovert 200 inverted microscope (40×/0.6 oil objective). (E) Washed platelets were allowed to spread (15 min) on fibrinogen (100 μg/mL), and phase abundance was determined. (F) Platelet clot retraction in response to stimulation with thrombin (5 U/mL) over time. Residual plasma volume was determined 120 minutes after the addition of thrombin. Images are representative of 4 individuals. (G) Tln recruitment to β3-integrin tails was assessed by immunostaining (talin, magenta; Itgb3, cyan) and confocal microscopy. Scale bars, 3 μm. Images were acquired with a TCS SP8 confocal microscope (100×/1.4 STED WHITE oil objective, Leica Microsystems). (H) Quantification of the peripheral zone (talin and β3-integrin co-localization) width. Values are mean ± SD (n = 6). Images are representative of at least 6 individuals. ***P < .001, **P < .01, and *P < .05, unpaired Student t test (A-G) and Wilcoxon-Mann-Whitney test (H). CRP, collagen-related peptide; CVX, convulxin; rest, resting; Rhd, rhodocytin; U46, U46619, a stable thromboxane A2 analog.
