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First published online October 30, 2006
doi: 10.1242/10.1242/jcs.03195

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Disabled-2 is a novel IIb-integrin-binding protein that negatively regulates platelet-fibrinogen interactions and platelet aggregation

¹ Graduate Institute of Basic Medical Sciences, Chang Gung University, Taoyuan 333, Taiwan, Republic of China
² School of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan, Republic of China
³ Department of Pharmacology, New York University School of Medicine, New York, NY 10016, USA
⁴ Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9110, USA
⁵ Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan, Republic of China
⁶ Graduate Institute of Medical Biotechnology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shen, Taoyuan 333, Taiwan, Republic of China

View larger version (53K): [in a new window]   Fig. 1. Subcellular localization of DAB2 in human platelets. (A) Association of DAB2 with platelet -granules. Platelet homogenates (2x109) were separated by sucrose-density-gradient (30-60%) centrifugation. Eighteen fractions (700 µl each) were collected from the top and aliquots were subjected to western blot analysis using the anti-p96 (DAB2), anti-P-selectin and anti-PF4 antibodies. (B) Colocalization of DAB2 with PF4. Immunofluorescence staining of DAB2 (green) and PF4 (red) was performed with platelets cytospun on a glass slide and was observed by confocal microscopy. The image of individual platelets was obtained by using confocal microscopy analytical software to enlarge and focus the indicated platelet. (C) Immunoelectron microscopy of resting platelets revealed the presence of DAB2 in -granules. Washed platelets were immunostained with anti-DAB2 antibody. After labeling with 10-nm colloidal-gold-conjuagated protein G, the subcellular distribution of DAB2 was observed by electron microscopy. Arrows indicate positive staining of the immunogold particle. Original magnification 15,000x.

View larger version (45K): [in a new window]   Fig. 2. DAB2 is released during agonist-induced platelet aggregation in a PKC-dependent manner. (A) Release of DAB2 during agonist-induced platelet aggregation. Washed platelets (1.4x108) were stimulated with the indicated concentration of agonists. The platelet pellet and the suspension medium into which protein is secreted were separated for western blot analysis with anti-p96, anti-PF4 and anti-PKC antibodies. The lysates of platelet pellets were also used as a control to demonstrate the presence of PKC in the platelet pellet but not the suspension medium. (B) PKC-dependent DAB2 release during platelet aggregation. Washed platelets with or without pretreatment of the pan-PKC inhibitor Ro-31-8220 (20 µM) were stimulated by thrombin (0.1 U/ml) or TPA (1 µg/ml). The suspension medium was collected for western blot analysis with anti-96 (DAB2) and anti-PF4 antibodies. The suspension medium from resting platelets was used as a control (C) for the experiment.

View larger version (22K): [in a new window]   Fig. 3. DAB2 binds to platelets and megakaryocytic differentiating K562 cells. (A) DAB2 binds to the surface of activated platelets. Washed platelets (1.5x108) were either untreated (R, resting platelets) or were stimulated with thrombin (Th, 0.1 U/ml), TPA (1 µg/ml), collagen (Col, 10 µg/ml), U46619 (U, 1 µM) or TRAP (10 µM) for 10 minutes. Platelets were analyzed by flow cytometry after incubation with anti-DAB2 (1:100 dilution) and FITC-conjugated goat anti-mouse secondary antibody. The percent of platelets with specific DAB2 binding is presented. (B) The PTB domain of DAB2 is crucial for the interaction with platelets. Resting or TRAP-stimulated (10 µM) platelets (1.5x108/well) were added for 3 hours to 24-well plates pre-coated with DAB2-PTB, DAB-M or control GST proteins (100 µg/ml). Platelet adhesion was then quantified by Crystal Violet assay. (C) TPA-treated (T, 10 ng/ml) or vehicle control ethanol-treated (E, 0.01%) K562 cells (5x105 cells/well) were added to plates pre-coated with DAB2-PTB or control GST protein (100 µg/ml). Cell adhesion was quantified by Crystal Violet assay. (D,E) TPA- and ethanol-treated K562 cells (5x105) were incubated with DAB2-PTB for 3 hours. Binding of DAB2 was analyzed by incubating K562 cells with FITC-conjugated anti-GST antibody, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and observed by confocal microscopy (D). The percent of K562 cells with DAB2-PTB binding was determined by flow cytometry (E). The data represent the mean ± s.d. of three to six experiments. **P<0.001 when compared with thrombin-treated platelets (A), TRAP-stimulated platelet adhesion to GST (B) and ethanol-treated K562 cell adhesion (C), or binding to DAB2-PTB (E).

View larger version (29K): [in a new window]   Fig. 4. IIbß3 integrin mediates DAB2 binding to platelets and the surface of CHO-IIbß3-integrin cells. (A) Effect of the functionally blocking mAbs and the RGDS peptide on the interaction of DAB2 with platelets. Resting or TRAP-stimulated (10 µM) platelets (1.5x108) were incubated with the indicated mAbs (20 µg/ml) or RGDS peptide (100 µM) for 30 minutes. The percent of platelets with DAB2 binding was determined by flow cytomery after incubating the platelets with anti-DAB2 (H110) antibody (1:200 dilution) and FITC-conjugated donkey anti-rabbit secondary antibody. (B) Effects of functional blocking mAb and RGDS peptide on the binding of recombinant DAB2-PTB to platelet surface. TRAP-stimulated platelets (1.5x108) were incubated with the mAb or RGDS peptide at room temperature for 30 minutes. DAB2-PTB or control GST protein (100 µg/ml) was added into the assay mixture and incubated for 3 hours. DAB2-PTB binding was detected as described in A except that an anti-GST antibody was used. (C) Expression of IIbß3 integrin on CHO-K1 and CHO-IIbß3-integrin cells. The percent of cells with IIbß3 integrin expression was determined by flow cytometry using 10E5 (20-30 µg/ml) and FITC-conjugated goat anti-mouse secondary antibody. (D) Binding of DAB2 to the surface of CHO-IIbß3-integrin cells. DAB2-PTB or control GST protein (100 µg/ml) were added to the cultured CHO-K1 and CHO-IIbß3-integrin cells for 2 hours. After incubation with FITC-conjugated anti-GST antibody (green) and counterstaining with DAPI (blue), confocal microscopy analysis was performed to visualize DAB2 binding. (E) Effects of function blocking mAbs and RGDS peptide on DAB2 interaction with CHO-IIbß3-integrin. The CHO-IIbß3-integrin cell suspension was incubated with the indicated mAbs or the RGDS peptide for 30 minutes. Then, DAB2-PTB or control GST protein (100 µg/ml) were added into the cell suspension and incubated for 3 hours. DAB2 binding was determined by flow cytometry using FITC-conjugated anti-GST antibody. The data represent the mean ± s.d. of three to six experiments. **P<0.001 compared with TRAP-stimulated platelets treated with mIgG (A and B), CHO-K1 cells (C) or CHO-IIbß3-integrin cells pre-treated with mIgG (E).

View larger version (40K): [in a new window]   Fig. 5. Mapping the interaction of IIbß3 integrin and DAB2. (A) Schematic representation of the recombinant GST-IIb integrin and ß3 integrin mutant proteins. The shaded box of IIb integrin (aa 171-334) represents the fibrinogen-binding site. The shaded region (aa 692-722) of ß3 integrin represents the transmembrane domain. (B) Amino acids 171-464 of IIb integrin interact with DAB2. Cell lysates (1 mg) of DAB2-transfected K562 cells were collected for GST-pull-down assays using the indicated GST-IIb integrin (constructs a-d) and GST-ß3 (construct e) proteins or control GST proteins (25 µg). The pull-down lysates (upper panel) and the aliquots of the input GST-proteins (lower panel) were subjected to western blot analysis with anti-DAB2 and anti-GST antibodies. The expected band for each purified protein is denoted by asterisk. (C) Schematic representation of the RGD motif and its flanking sequences for human, rat, and mouse DAB2. (D) The DAB2-RGD peptide interferes with DAB2–IIb-integrin interaction. Cell lysates (1 mg) of DAB2-transfected K562 cells were subjected to GST-pull-down analyses with GST-IIb-integrin aa 171-464 (construct d) in the absence or presence of DAB2-RGD and DAB2-RGE peptide (100 µg/ml). The pull-down lysates were subjected to western blot analysis using anti-DAB2 antibody. (E) The resting or TRAP-stimulated (10 µM) platelets (1.5x108) were added into the plates pre-coated with GST, DAB2-PTB or DAB2-D66E (100 µg/ml) for 3 hours at 37°C. Platelet adhesion was then quantified by the Crystal Violet assay. The data represent the mean ± s.d. (n=5). **P<0.001 compared with the TRAP-stimulated platelet adhesion to GST control protein.

View larger version (36K): [in a new window]   Fig. 6. The RGD motif is involved in DAB2-mediated inhibition of agonist-induced platelet aggregation. (A) DAB2 selectively inhibits agonist-induced platelet aggregation. Platelet aggregation was induced by the indicated concentration of agonists in the presence of GST, DAB2-PTB or DAB2-D66E (100 µg/ml). The platelet aggregation curves (left panel) and the percent of aggregation (right panel) were obtained and quantified by using a platelet aggregometer. (B) Schematic representation of the putative thrombin-cleavage sites in human DAB2-PTB and rat B. The putative thrombin-cleavage sites and the size of the cleavage product for human DAB2-PTB and rat B were shown. (C) Thrombin is a DAB2 protease. The recombinant human DAB2-PTB and in vitro transcription- and translation-derived [S35]-methionine-labeled rat B were incubated in the absence (C) or presence (T) of thrombin (10 U/ml). Aliquots of the reaction mixtures were fractionated on SDS-PAGE and were visualized by Commassie Blue staining (DAB2-PTB) or autoradiography (B). (D) The DAB2-RGD peptide inhibits agonist-induced platelet aggregation. Platelet aggregation was induced by agonists at indicated concentration in the presence of DAB2-RGD or DAB2-RGE peptides (100 µg/ml). The platelet aggregation curves (left panel) and the percent of aggregation (right panel) was obtained and quantified by using a platelet aggregometer. The data represent the mean ± s.d. of three to four experiments. *P<0.05 and **P<0.001, compared with platelet aggregation induced by the same agonist in the presence of control GST protein (A) or DAB2-RGE (D). NS, not significant.

View larger version (26K): [in a new window]   Fig. 7. DAB2 inhibits the interaction between platelet and/or megakaryocytic cells and fibrinogen. (A,B) The RGD motif is involved in DAB2-mediated inhibition of TRAP-stimulated platelet adhesion to fibrinogen. Resting and TRAP-stimulated (10 µM) platelets were added to plates pre-coated with fibrinogen (10 µg) in the absence (control) or presence of soluble GST, DAB2-PTB and DAB2-D66E (100 µg/ml) (A), or DAB2-RGD and DAB2-RGE peptides (100 µg/ml) (B) for 3 hours at 37°C. Platelet adhesion with fibrinogen was quantified in Crystal Violet assays. (C,D) Dose-dependent inhibition of K562 cell adhesion to fibrinogen by DAB2-RGD peptide. Ethanol-treated (E) or TPA-treated (T, 10 ng/ml) K562 cells were added to plates pre-coated with fibrinogen (10 µg) in the presence of the indicated concentration of DAB2-RGD or DAB2-RGE peptides. K562-cell adhesion was observed by inverted microscopy (C, 100x). Cell adhesion of TPA-treated cells was also quantified in Crystal Violet assays (D). Data represent the mean ± s.d. (n=4). **P<0.001 compared with platelets stimulated with TRAP only (A and B) or TPA-treated cells in the absence of DAB2 peptide (D).

View larger version (25K): [in a new window]   Fig. 8. Model for the effects of platelet DAB2 on platelet aggregation. DAB2 is secreted from platelets upon agonist-induced platelet aggregation. Secreted DAB2 binds to the extracellular region of IIb integrin and interferes with the IIbß3-integrin–fibrinogen interaction that results in the inhibition of platelet aggregation. Whether or not Ser24 phosphorylation of platelet DAB2 plays a role during platelet activation and aggregation still needs to be established.

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