Fig. 4.

Functional characterization of Sca1/EGFP^+/− platelets. Blood cells collected from Sca1/EGFP Tg mice 3 d after GDQ or CON injection were permeabilized, fixed, and stained with antibodies against (A) PE-Cy7-labeled anti-CD41; or (B) AF647-labeled anti-filamin A. The median fluorescence intensity (MFI) of bound antibody was taken to represent levels of the cognate antigen. (C) Blood from mice treated with GDQ or CON was diluted in Tyrode’s buffer (1.5–100 µL final), to which AF647-fibrinogen (15 μg/mL) was added with 0.2 mM Ca²⁺ or 2 mM EDTA (negative control) and 10 µM ADP to activate platelets at room temperature. After 10 min, AF405-anti-mouse GPIbα MoAb (5A7) was added (to identify platelets) and fibrinogen binding to Sca1/EGFP + or − platelets was separately evaluated. (D, Left panel) Bound fibrinogen calculated as the ratio of AF647 MFI values on platelets with added Ca²⁺ or EDTA; ND indicates no Sca1/EGFP⁺ platelets detected in untreated mice. (D, Right panel) Fibrinogen bound to all platelets without gating for EGFP fluorescence. (E) Evaluation of AF647-Annexin V binding to blood platelets in the presence of 3 mM Ca²⁺ and 200 nM apixaban (FXa inhibitor to prevent coagulation) with or without the addition of the platelet GPVI agonist, Alborhagin (2.5 μg/mL). For additional details, see panel (C). (F, Left and Right) AF647-Annexin V binding to platelets measured with the same method as fibrinogen in (D, Left and Right). Data in (A, B, D, and F) are shown as 25th–75th percentile boxes with min-to-max-whiskers and individual values (n = 4 and 6, respectively). Statistical analysis was performed by RM one-way ANOVA with Geisser–Greenhouse correction and Tukey’s posttest for paired comparisons (black asterisks) or nonparametric Friedman test followed by Dunn’s posttest (red asterisks), except for data in D, F, Right panel, analyzed by two-tailed paired t test. *P <0.05, **P <0.01, ***P <0.001.