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Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

Yunjie Huang, Smita Joshi, Binggang Xiang, Yasunori Kanaho, Zhenyu Li, Beth A. Bouchard, Carole L. Moncman and Sidney W. Whiteheart

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Arf6 KO platelets are defective in Fg storage but not in other cargo. (A) Comparison of proteins levels by western blotting between wild-type (WT) and KO platelets. Washed platelet extracts (1 × 107/lane) were loaded, and the indicated proteins were probed for by western blotting using the corresponding antibodies. β-Actin was used as a loading control. The data are representative of at least 3 independent experiments. (B) Comparison of endogenous Fg levels between WT and KO platelets. (i) Washed platelet extracts (1 × 107/lane) were loaded, and the indicated proteins were probed for by western blotting using corresponding antibodies. Each lane represents platelets from a single mouse. RabGDI was used as a loading control. (ii) Quantification of Fg levels in panel Bi was performed using ImageQuantTL and analyzed by SigmaPlot 12.0. (C) Comparison of granule cargo levels between WT and KO platelets. Washed platelet extracts (1 × 107/lane) were loaded, and the indicated proteins were probed for by western blotting using corresponding antibodies. β-Actin was used as a loading control. Quantification was performed using ImageQuantTL, and ratio of KO to WT was calculated. The dash line represents ratio of 1 (KO/WT). The data are representative of at least 2 independent experiments.

  • Figure 2

    Arf6 deletion does not alter the surface levels of total or activated integrin αIIbβ3 or the binding of Fg to platelets. (A) Comparison of surface levels of total integrin αIIbβ3 between WT (light gray) and KO (dark gray) platelets. Using FITC-anti-CD41/61 antibody, levels of total integrin αIIbβ3 were measured by flow cytometry, in resting (i) and thrombin-stimulated (0.1 U/mL; ii) platelets. Unlabeled platelets (black) were used as the background control. (iii) Quantification of flow cytometry data in panels Ai and Aii expressed as GMFI. The data shown are representative of at least 2 independent experiments with triplicates. (B) Comparison of surface levels of activated integrin αIIbβ3 between WT (light gray) and KO (dark gray) platelets. Using phycoerythrin (PE)–Jon/A antibody, levels of activated integrin αIIbβ3 were measured by flow cytometry, in resting (i) and thrombin-stimulated (0.1 U/mL; ii) platelets. Unlabeled platelets (black) were used as the background control. (iii) Quantification of flow cytometry data in panels Bi and Bii expressed as GMFI. Data shown are representative of at least 2 independent experiments with triplicates. (C) Comparison of Fg binding to platelet surface between WT and KO platelets. Washed platelets (5.0 × 108/mL) were incubated, on ice, with FITC-Fg at different concentrations for 20 minutes and then fixed with 2% paraformaldehyde overnight at 4°C. Platelets were analyzed by flow cytometry, and quantification is shown as GMFI. The data shown are representative of 2 independent experiments with triplicates.

  • Figure 3

    Arf6 KO platelets are defective in Fg uptake in vivo and ex vivo. (A) Arf6 deletion impairs platelet uptake of Fg in vivo. (i) WT and KO mice were injected with biotin-Fg via the retro-orbital sinus. Platelets were harvested 24 hours postinjection. Platelet extracts (1 × 107/lane) were loaded, and the indicated proteins were probed for by western blotting using corresponding antibodies. Each lane represents platelets from a single mouse. A mouse without injection (No Inj) was included as a negative control. β-Actin was used as a loading control. (ii) Quantification of biotin-Fg levels in panel Ai was performed using ImageQuantTL and analyzed by SigmaPlot 12.0. (B) Arf6 deletion impairs Fg uptake by platelets ex vivo. (i) Washed platelets (5 × 108/mL) from WT and KO mice were incubated with FITC-Fg (0.05 mg/mL) for 1 hour at 37°C. After removing the extracellular FITC-Fg, platelets were incubated with HEPES Tyrode buffer (pH = 7.4) for another 2 hours. Platelets were fixed with 2% paraformaldehyde overnight at 4°C and subjected to epifluorescence microscopy. The extracellular signal was quenched with 0.1% trypan blue, and representative images, from 4 experiments, are presented. (ii) The number of FITC-positive puncta per platelet was manually counted, and platelets were grouped according to that number. The percentage platelets in each group relative to total was calculated and plotted as a bar graph. Statistical significance was determined using rank sum test. (C) Overexposure of biotin blot in panel Ai, to probe for fragments of biotin-Fg. Starting biotin-Fg (Pre Inj) was included as a comparison. (D) Arf6 deletion impedes uptake of FITC-Fg ex vivo. Washed platelets (1.0 × 109/mL) from 3 WT (black) and 3 KO (open) mice were separately incubated with FITC-Fg (0.15 mg/mL) at 37°C for the indicated times and then fixed with 2% paraformaldehyde overnight at 4°C. Intracellular FITC-Fg levels were measured by flow cytometry, after addition of trypan blue, and expressed as GMFI (mean ± standard deviation). Statistical significance was determined by Student t test. (E) Megakaryocyte uptake of FITC-Fg is below detection under the conditions used. WT and KO mice were injected with FITC-Fg (0.75 mg/mouse) via the retro-orbital sinus. Each group included 2 mice. Bone marrow cells and platelets were harvested 24 hours postinjection and fixed with 2% paraformaldehyde. DIC images and fluorescence images are presented. Megakaryocytes were identified by CD41 staining. (i) The exposure time for FITC-Fg and CD41 images is 2 seconds and 0.5 seconds, respectively. The scale bar is 10 µm. (ii) The exposure time for FITC-Fg is 1 second. The scale bar represents 5 µm. Representative images were selected from >10 different fields of each group.

  • Figure 4

    Arf6 KO platelets have enhanced spreading but normal static adhesion. (A) Quantification of static platelet adhesion on Fg- and BSA-coated surface. Washed calcein-labeled platelets (2.5 × 108/mL) from WT and KO mouse were incubated on Fg- or BSA-coated surfaces for 30 minutes at 37°C. The number of adherent platelets was measured using a plate reader with excitation/emission at 485/538 nm, and referenced to a standard curve. Each bar represents platelets from a single mouse. Statistical analysis was performed using Student t test. (B) Quantification of platelet surface area when spread on Fg-coated surfaces. Washed platelets from WT and KO mice (2.0 × 107/mL) were supplemented with 1 mM Ca2+ and then incubated on Fg-coated surfaces for the indicated times. They were fixed with 4% paraformaldehyde, and DIC images were taken using Nikon Eclipse E600 microscope (Nikon) with a 100X/1.40 NA, DIC H oil objective lens (Nikon) with a Zeiss camera (AxioCam MR). Images were processed with Zen 2011 (blue edition; Zeiss) and quantified by Image J (v1.47; National Institutes of Health). (C-D) Representative DIC images of WT (C) and KO (D) platelets spread at the 120 minutes point. The data are representative of at least 3 independent experiments.

  • Figure 5

    Arf6 KO platelets have enhanced platelet clot retraction without noticeable defect on myosin light-chain phosphorylation (MLC-P), actin polymerization, or Rac1/RhoA activation. (A) Washed platelets from WT and KO mice (3 × 108/mL) were supplemented with 0.5 mg/mL human Fg and 1 mM Ca2+. Clot retraction was initiated with thrombin (0.1 U/mL), and images were taken at the indicated times (i) and quantified (ii). Clot size in panel A was measured using Image J v1.48 and normalized to clot size at time 0 (clot volume %). The data are representative of at least 5 independent experiments. (B) KO mice showed no defect in MLC-P upon thrombin stimulation. Washed platelets from WT and KO mice were prepared at 4 × 108/mL in HEPES Tyrode buffer (pH 7.4) and kept resting or stimulated with thrombin (0.1 U/mL) for the indicated times. The reaction was stopped by addition of sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer containing both protease inhibitor and phosphatase inhibitor cocktails. The lysates were probed for MLC-P by western blotting. β-Actin was used as a loading control. The blots shown are representative of at least 3 independent experiments. (C) Arf6 deletion did not affect thrombin-induced F-actin formation. Resting and thrombin-stimulated platelets (2 × 107) were fixed and permeabilized with Triton X-100 in the presence of tetramethylrhodamine (TRITC)-phalloidin. The bound TRITC-phalloidin was solubilized and measured using a microplate spectrofluorimeter. The data are representative of 2 independent experiments. (D-E) KO platelets showed no defect in Rac1/RhoA activation. Washed platelets (5 × 108/mL) from WT and KO mice were kept resting or stimulated with thrombin (0.1 U/mL) for the indicated times. The reactions were stopped by addition of 2× GTPases-pulldown lysis buffer containing a protease inhibitor cocktail. Rac1-GTP/RhoA-GTP were recovered as described in “Methods.” (Di and Ei) Western blotting of the pellets (Rac1-GTP or RhoA-GTP) and the supernatant (total Rac1 or total RhoA). (Dii and Eii) Quantification of Rac1-GTP/total Rac1 and RhoA-GTP/total RhoA. Quantification was performed using ImageQuantTL, and the ratio of Rac1-GTP/total Rac1 or RhoA-GTP/total RhoA was plotted in a bar graph. Blots shown are representative of at least 2 independent experiments.

  • Figure 6

    FITC-Fg transits through Rab4- and Rab11-positive compartments after internalization. (A) Rab4- and Rab11-containing compartments are present in human platelets. Washed human platelets (5 × 108/mL) were fixed with 2% paraformaldehyde, washed, and allowed to adhere to poly-d-lysine–coated coverslips. Immunofluorescence staining was done as described in “Methods.” Platelets were incubated with anti-Rab4 mouse monoclonal antibody and anti-Rab11 rabbit polyclonal antibody, and then with Alexa 568 conjugated goat anti-mouse IgG or Alexa 488 conjugated anti-rabbit IgG, respectively. Images were taken with a Nikon A1R confocal microscope (60X/1.49 NA DIC N2 oil) and digitally magnified ×30. Scale bar represents 1 µm. (B) Internalized FITC-Fg goes through Rab4- and Rab11-positive compartments in WT mouse platelets. Washed WT mouse platelets (5 × 108/mL) in HEPES Tyrode buffer containing 1 mM Ca2+ were incubated with FITC-Fg (0.05 mg/mL) for the indicated times and then fixed with 2% paraformaldehyde at room temperature. After washing with PBS, the fixed platelets (5 × 107/mL) were allowed to adhere to poly-d-lysine–coated coverslips. Immunofluorescence staining was performed as described in “Methods” using anti-Rab4 rabbit polyclonal antibody or anti-Rab11 rabbit polyclonal antibody followed by TRITC-conjugated goat anti-rabbit secondary antibody. Images were taken with a Nikon A1R confocal microscope (60X/1.49 NA DIC N2 oil) and digitally magnified ×15. Scale bar represents 1 µm.