Effects of platelet SHARPIN deletion on platelet fibrinogen binding, aggregation, and tail bleeding times. (A-B) Flow cytometry of fibrinogen binding to agonist-stimulated platelets. Washed platelets were prepared from blood drawn from SHARPIN^fl/fl Pf4-Cre⁻ and SHARPIN^fl/fl Pf4-Cre⁺ mice and incubated for 30 minutes with Alexa-647–conjugated fibrinogen and indicated concentrations of platelet agonists (A) ADP or (B) PAR4 agonist peptide. Results are expressed as specific fibrinogen binding, as described in Methods. Data are from 5 experiments with ADP and 4 with PAR4 agonist peptide. Asterisks indicate statistical significance determined by Student t tests comparing fibrinogen bound at specific concentrations. Analysis of variance (ANOVA) analysis comparing bound fibrinogen at all input PAR4 concentrations attained statistical significance (*P < .05). (C) Representative platelet aggregation tracings from light transmission platelet aggregometry. SHARPIN^fl/fl GPIbα-Cre mouse platelet-rich plasma was diluted 1:2 with Walsh buffer in a glass cuvette warmed to 37°C in a Chronolog aggregometer (Havertown, PA), and the platelets were stimulated with the agonists indicated. Results are representative of at least 3 experiments. (D) αIIb surface expression was determined on platelets prepared (as in A-B) and compared with nonspecific IgG binding (n = 8), n.s., not significant. (E) FITC anti-CD62P binding to unstimulated or PAR 4 agonist peptide-stimulated platelets (n = 3). *P < .05; **P < .01). (F) Tail bleeding times were performed as described in Methods. Data represent results from SHARPIN^fl/fl Pf4-Cre⁻ mice (n = 19) and SHARPIN^fl/fl Cre⁺ mice (n = 21). The time to arrest of bleeding is shown. **P = .01.