![Figure 2. VAMP-3−/− platelets had defective fibrinogen uptake. WT and VAMP-3−/− platelets (1.0 × 109/mL) were kept resting (A) or stimulated with ADP (10µM) (B), then incubated with FITC-Fg (0.06 or 0.12 mg/mL) at 37°C for 30 minutes. The platelets were then put on ice for 20 minutes and fixed with 2% PFA (final concentration), and geometric mean fluorescent intensity measurements were taken by flow cytometry before and after the addition of 0.04% TB. Quantification of data shows both geometric mean fluorescence intensity measurements before addition of TB (WT/ VAMP-3−/−–TB; dark bars), which gives the total fluorescence, and after addition of TB (WT/ VAMP-3−/−+ TB; light bars), which gives the measure of internal fluorescence. As explained in Methods, WT and VAMP-3−/− platelets were added to each well in an opaque 96-well plate and either incubated with FITC-Fg (2 µM) (C) or low-molecular-weight (10 kDa) Oregon Green 488-Dextran (2 µM) (D) for times from 0 to 60 minutes at 37°C. Similarly, WT and VAMP-3−/− platelets were incubated with either FITC-Fg (0.1-5 µM) (E) or low-molecular-weight (10 kDa) Oregon Green 488-Dextran (1-100 µM) (F) for 30 minutes at 37°C. Reactions were stopped at given points, and fluorescence was measured before and after the addition of 0.04% TB. Data were plotted using SigmaPlot software (v13.0) and graphed as numbers of molecules of Fg or dextran endocytosed per platelet. No ADP was added to the plate assays. Statistical analyses were performed using 1-way ANOVA test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ .001; n.s., not significant. (G) Washed WT and VAMP-3−/− platelets (1 × 109/mL) were incubated with FITC-Fg at final concentrations of 1 µM at 37°C for increasing times up to 30 minutes. Platelets were fixed with 2% PFA (final concentration) and mixed with 0.1% TB before imaging. Platelets were visualized as described in the supplemental Methods. Exposure times for DIC were 100 ms, whereas for the FITC, laser were 500 ms. Scale bars, 5 µM. (H) Quantification of the number of FITC+ puncta/platelet in both WT and VAMP-3−/− samples were plotted using SigmaPlot software (v13.0). Statistical significance determined using Mann-Whitney U test; **P ≤ .01. Data are representative of 3 independent experiments (mean ± standard error of the mean [SEM]) for all.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/130/26/10.1182_blood-2017-02-768176/4/blood768176f2.jpeg?Expires=1735814118&Signature=keHHutADOVASMZdGe3s6T~VRGR9PrAE7bPrUQm3KZwhFhtMZpIykVSi0~TCSYC6gVqIQrHSVqg2~VPookKHwZPSz01CcizEGkMyaVQQ5pht5Ex-3apA1OSzXIMUHD1nyetIvJIgE0FRwqomE1EPvAxpjgo3FelHG2PsoWuCO42~gQpChOYLgBRWLt7cLS9CvZ~wzd5I7nd2LG~3c-NvAPy9dnUq-bct7L4Xqza8X7FsGvToggVQThyShM15qxuWUuR0L91lC3iQDTM5shqvDvNCSEtgHdQoQz8yKx6TkoeELTl3IZjjHugGd3y5B5e4NJZHhoyLiW4-fsjHwB9xjgA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
VAMP-3−/−platelets had defective fibrinogen uptake. WT and VAMP-3−/− platelets (1.0 × 109/mL) were kept resting (A) or stimulated with ADP (10µM) (B), then incubated with FITC-Fg (0.06 or 0.12 mg/mL) at 37°C for 30 minutes. The platelets were then put on ice for 20 minutes and fixed with 2% PFA (final concentration), and geometric mean fluorescent intensity measurements were taken by flow cytometry before and after the addition of 0.04% TB. Quantification of data shows both geometric mean fluorescence intensity measurements before addition of TB (WT/ VAMP-3−/−–TB; dark bars), which gives the total fluorescence, and after addition of TB (WT/ VAMP-3−/−+ TB; light bars), which gives the measure of internal fluorescence. As explained in Methods, WT and VAMP-3−/− platelets were added to each well in an opaque 96-well plate and either incubated with FITC-Fg (2 µM) (C) or low-molecular-weight (10 kDa) Oregon Green 488-Dextran (2 µM) (D) for times from 0 to 60 minutes at 37°C. Similarly, WT and VAMP-3−/− platelets were incubated with either FITC-Fg (0.1-5 µM) (E) or low-molecular-weight (10 kDa) Oregon Green 488-Dextran (1-100 µM) (F) for 30 minutes at 37°C. Reactions were stopped at given points, and fluorescence was measured before and after the addition of 0.04% TB. Data were plotted using SigmaPlot software (v13.0) and graphed as numbers of molecules of Fg or dextran endocytosed per platelet. No ADP was added to the plate assays. Statistical analyses were performed using 1-way ANOVA test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ .001; n.s., not significant. (G) Washed WT and VAMP-3−/− platelets (1 × 109/mL) were incubated with FITC-Fg at final concentrations of 1 µM at 37°C for increasing times up to 30 minutes. Platelets were fixed with 2% PFA (final concentration) and mixed with 0.1% TB before imaging. Platelets were visualized as described in the supplemental Methods. Exposure times for DIC were 100 ms, whereas for the FITC, laser were 500 ms. Scale bars, 5 µM. (H) Quantification of the number of FITC+ puncta/platelet in both WT and VAMP-3−/− samples were plotted using SigmaPlot software (v13.0). Statistical significance determined using Mann-Whitney U test; **P ≤ .01. Data are representative of 3 independent experiments (mean ± standard error of the mean [SEM]) for all.
