Figure 1: Dynamics of GPVI-Fc Binding and Platelet Adhesion to Collagen Under Flow

Glycoprotein VI–fragment crystallizable (GPVI-Fc) (50 μg/ml final concentration; 333 nM) labeled with phycoerythrin (PE)-conjugated anti–human-Fc antibody (red) was added to blood containing abciximab (to inhibit platelet aggregation and allow only platelet adhesion) before perfusion over collagen (550/s). Differential interference contrast (DIC) and fluorescence images were taken by video microscopy (1 frame/2 s) using a 100× NA1.4 oil objective. Images are representative for 6 individual experiments. Upper rows: overlay of DIC (collagen/platelets) and fluorescence (PE-labeled GPVI-Fc) images. Bottom rows: fluorescence images of PE-labeled GPVI-Fc. See also Video 1. (A) A single platelet (thick arrow) rolls over PE-labeled GPVI-Fc (thin arrows in row below) bound to collagen. Two platelets (asterisks) adhere to segments of collagen fibers that contain less fluorescent GPVI-Fc. (B) A single platelet (arrow, top row) attaching downstream of the fiber and moving back against the blood flow displaces GPVI-Fc-PE from collagen (arrows in bottom row). (C) Structured illumination microscopy imaging. Platelets adhere to collagen segments carrying little GPVI-Fc. Collagen coated onto glass coverslips was stained with anti–collagen type I and type III antibody (Ab) and AlexaFluor405-conjugated secondary Ab (blue). PE-labeled GPVI-Fc (50 μg/ml) was added to blood containing Arg-Gly-Asp-Ser (to inhibit platelet aggregation and allow only platelet adhesion) before the start of perfusion (shear rate 550/s). After 4 min of flow, platelets were fixed and stained with anti-CD41 Ab and DyLight 488-conjugated secondary Ab (green). The fluorescence micrograph shows a maximum intensity projection of 0.15 μm z sections (total z 2.5 μm) of a structured illumination microscopy image (ELYRA PS.1; Carl Zeiss MicroImaging GmbH, Jena, Germany). i and ii, Identical pictures. i, Binding of GPVI-Fc (red) onto collagen fibers (blue). Green channel (platelets) was omitted. ii, Picture as in i but with platelets (green). Image is representative of 5 others from different experiments. Video 2 presents a 3-dimensional representation.