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 <b>(red)</b> 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. <b>Upper rows:</b> overlay of DIC (collagen/platelets) and fluorescence (PE-labeled GPVI-Fc) images. <b>Bottom rows:</b> fluorescence images of PE-labeled GPVI-Fc. See also Video 1. <b>(A)</b> A single platelet <b>(thick arrow)</b> rolls over PE-labeled GPVI-Fc <b>(thin arrows in row below)</b> bound to collagen. Two platelets <b>(asterisks)</b> adhere to segments of collagen fibers that contain less fluorescent GPVI-Fc. <b>(B)</b> A single platelet <b>(arrow, top row)</b> attaching downstream of the fiber and moving back against the blood flow displaces GPVI-Fc-PE from collagen <b>(arrows in bottom row)</b>. <b>(C)</b> 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 <b>(blue)</b>. 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 <b>(green)</b>. 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). <b>i</b> and <b>ii</b>, Identical pictures. <b>i,</b> Binding of GPVI-Fc <b>(red)</b> onto collagen fibers <b>(blue)</b>. Green channel (platelets) was omitted. <b>ii,</b> Picture as in <b>i</b> but with platelets <b>(green)</b>. Image is representative of 5 others from different experiments. Video 2 presents a 3-dimensional representation.