Platelet PDI does not regulate the β3–talin1 interaction, protein phosphorylation, or platelet spreading on immobilized FG. (A) WT and PDI-null platelets (3 × 107 platelets in 0.3 mL) were treated with or without 0.05 U/mL thrombin (Thr) for 45 seconds in an aggregometer and lysed with ice-cold lysis buffer. Lysates were immunoprecipitated and immunoblotted as described in “Materials and methods.” Data represent mean ± SD (n = 4). (B) Protein phosphorylation levels were determined by immunoblotting with lysates of thrombin-activated WT and PDI-null (CKO) platelets using mouse IgG2b (mIgG2b) or an anti-phosphotyrosine antibody (4G10). The representative blot was obtained from 3 independent experiments. (C-E) Mouse platelets (8 × 106 platelets in 0.4 mL) were incubated on FG-coated surfaces for 2 h at 37°C in the presence of 0.025-0.05 U/mL thrombin. Adherent and spread platelets were stained with rhodamine-conjugated phalloidin. (C) Representative images. Bar = 10 μm. (D) Number of adherent (but not spread, black bars) and fully spread (white and gray bars) platelets. **P < .01 vs WT platelets (total number of adherent and spread platelets) after Student t test. (E) Platelet spreading was analyzed by the surface area, which was measured by the number of pixels divided by the number of platelets in the field. Data represent mean ± SD (n = 3).
