Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets.

GPVI was located by use of fluorophore‐conjugated GPVI dimer‐specific Fab (antigen‐binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III , GPVI ‐specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real‐time interactions, and direct stochastic optical reconstruction microscopy ( dSTORM ), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein I b, integrin α 2 β 1 , and phosphotyrosine.

Materials and methods

Non‐inhibitory, recombinant dimer‐specific 204‐11 Fab 10 was derived from clone 204‐11 21 by Kaketsuken (Kumamoto, Japan). Antibodies were fluorescently labeled by use of a Microscale Protein Labelling kit (Molecular Probes, Eugene, OR, USA); degree of fluorescent labeling = 2–5 dye molecules per protein molecule. The antibodies used were: 4G10 (anti‐phosphotyrosine; Millipore Merck, Billerica, MA, USA); anti‐human CD42b/GPIb, clone 486805 (R&D Systems, Abingdon, UK); 16B4 (mouse anti‐human CD49b/integrin α 2 chain; Bio‐Rad, Hercules, CA, USA); Alexa Fluor 647‐conjugated AffiniPure F(ab′)2 fragment goat anti‐mouse IgG, Fcγ‐specific (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA); Gi9 (anti‐integrin α 2 ; Abcam, Cambridge, UK); and Alexa Fluor 647‐conjugated anti‐human CD62P (Bio‐Rad). Other materials, of reagent grade or better, were obtained from commercial sources.

Platelet preparation

Washed platelets were prepared from acid–citrate–dextrose‐anticoagulated blood from healthy volunteers 22 , and resuspended at 3–5 × 10 7 platelets mL −1 in HEPES–Tyrodes buffer (HT) (134 m m NaCl, 0.34 m m Na 2 HPO 4 , 2.9 m m KCl, 12 m m NaHCO 3 , 20 m m HEPES, 5 m m glucose, pH 7.3).

Preparation of collagenous substrate‐coated glass dishes for imaging

Thirty‐five‐millimeter glass (0.7 mm)‐bottomed MatTek dishes (MatTek, Ashland, MA, USA) were coated with 10 μg mL −1 cross‐linked CRP (CRP‐XL), III‐30, or collagen III (Col III), in phosphate‐buffered saline (PBS) (0.01 m phosphate buffer, 0.0027 m KCl, 0.137 m NaCl, pH 7.4) and Horm collagen (Horm), in manufacturer‐supplied diluent, overnight at 4 °C. The dishes were washed with PBS, blocked with 1% bovine serum albumin/PBS (heat‐denatured, filtered) for 1 h, and then washed with PBS to make them ready for platelet spreading. TIRFM Adhesion of Alexa Fluor 488–204‐11 Fab‐labeled washed platelets (3 × 10 7 platelets mL −1 in HT containing 2 m m MgCl 2 ) to immobilized collagenous substrate was imaged with TIRFM (Nikon TIRF system mounted on a Nikon Eclipse Ti inverted microscope, with a Nikon × 60 numerical aperture [NA] 1.49 TIRF objective; Nikon UK, Ltd., Surrey, UK). Images were obtained at 5‐s intervals for 20–30 min at 37 °C, and this was followed by fixation in formalin and confocal imaging. Integrin α 2 β 1 blockade was achieved by preincubating platelets with 10 μg mL −1 Gi9 blocking antibody. Platelet morphology was followed with differential interference contrast (DIC) microscopy. dSTORM Alexa Fluor 647‐204‐11 Fab‐labeled washed platelets were allowed to adhere to collagenous substrate‐coated MatTek dishes. Adherent platelets were fixed, permeabilized, and stained with phalloidin–Alexa Fluor 488. Samples were imaged in switching buffer as described previously 22 on a Nikon Eclipse Ti‐E N‐STORM system in dSTORM mode using Perfect Focus, with a CFl SR Apochromat TIRF × 100 oil, 1.49‐NA objective lens and an N‐STORM filter cube with excitation from the Agilent Ultra High Power Dual Output Laser bed (170‐mW, 647‐nm laser), and image capture with an Andor IXON Ultra 897 EMCCD camera. Thirty thousand frames were captured with Nikon nis elements v4.2, and reconstructed by the use of storm analysis module v3.2, with drift correction and Gaussian rendering of data points. Detected points with a photon count of < 500 were discarded from reconstructed data before further processing. Points in the reconstructed images represent individually identified fluorescent blinking events, which are referred to as molecules.

Confocal imaging

Platelets were preincubated with Alexa Fluor 488‐conjugated or Alexa Fluor 647‐conjugated 204‐11 Fab (4 μg mL −1 ) alone or with another fluorescently labeled antibody. In some experiments, platelets were also treated with an inhibitor or inhibitory antibody. The platelets were allowed to adhere to a collagenous substrate, fixed, and imaged with an FV300 IX81 laser‐scanning confocal microscope with a × 60 oil immersion objective (Olympus UK, Southend‐on‐Sea, UK). Where indicated, platelets were permeabilized (0.1% Triton/PBS) following fixation, and stained for actin (Alexa Fluor 647–phalloidin) or phosphotyrosine (4G10, 5 μg mL −1 ).

Flow cytometry to measure GPVI dimer formation

Washed platelets were preincubated with dimethylsulfoxide (DMSO) (0.25% final concentration) or actin antagonist (cytochalasin D, latrunculin A, or jasplakinolide; 10 μ m ; 0.25% DMSO, final concentration), and GPVI dimer formation was measured by flow cytometry 10 .

Data analyses

Data analyses, performed with prism v7 (GraphPad, San Diego, CA, USA), are described in the figure legends. Differences among treatment groups in the flow cytometry experiments were calculated with paired t ‐tests. dSTORM cluster analysis was performed within matlab for each 3 × 3‐μm region of interest (ROI), as described by Owen et al . 23 , with modifications described in Pollitt et al . 18 . Platelets were identified by phalloidin staining of the actin cytoskeleton. ROIs were positioned within the platelet, ensuring that the edges of the ROI fell within the platelet boundary. Clusters of < 3 points were discounted. The numerical data, processed with Microsoft Excel, were analyzed as detailed in the figure legends. The degree of colocalization between the GPVI and phophotyrosine confocal images was quantified with image‐pro premiere 9.2 (Media Cybernetics, Rockville, MD, USA).

Supporting information Movie S1. GPVI forms clusters when platelets spread on immobilized collagenous substrates. Click here for additional data file. Fig. S1. DIC images corresponding to the fluorescence images in Fig. 6 . Click here for additional data file. Click here for additional data file.