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.

将经藻红蛋白（phycoerythrin, PE）标记的抗人-Fc抗体<b>（红色）</b>标记的糖蛋白VI-可结晶片段（Glycoprotein VI–fragment crystallizable, GPVI-Fc）（终浓度50 μg/ml；333 nM）加入含有阿昔单抗（abciximab）（用于抑制血小板聚集，仅允许血小板黏附）的血液中，随后以550/s的切变率将该血液灌流至胶原表面。采用100×数值孔径1.4的油镜，通过视频显微镜（每2秒采集1帧）拍摄微分干涉差（Differential interference contrast, DIC）图像与荧光图像。本图像为6次独立实验的代表性结果。
<b>上排：</b>DIC（胶原/血小板）与荧光（PE标记的GPVI-Fc）图像的叠加图。<b>下排：</b>PE标记的GPVI-Fc的荧光图像。另见视频1。
<b>(A)</b>单个血小板<b>（粗箭头）</b>在结合于胶原的PE标记GPVI-Fc<b>（下排细箭头）</b>表面滚动；两个血小板<b>（星号）</b>黏附于荧光GPVI-Fc含量较低的胶原纤维节段。
<b>(B)</b>单个血小板<b>（上排箭头）</b>在纤维下游附着并逆血流方向移动，使GPVI-Fc-PE从胶原表面脱离（<b>下排箭头</b>所示区域）。
<b>(C)</b>结构照明显微镜（structured illumination microscopy）成像：血小板黏附于GPVI-Fc含量较低的胶原节段。将包被于玻璃盖玻片上的胶原用抗I型和III型胶原抗体（Ab）以及AlexaFluor405标记的二抗<b>（蓝色）</b>染色。将终浓度50 μg/ml的PE标记GPVI-Fc加入含有精氨酸-甘氨酸-天冬氨酸-丝氨酸（Arg-Gly-Asp-Ser, RGDS）（用于抑制血小板聚集，仅允许血小板黏附）的血液中，随后以550/s的切变率启动灌流。灌流4分钟后，固定血小板，并用抗CD41抗体以及DyLight 488标记的二抗<b>（绿色）</b>染色。该荧光显微图像为结构照明显微镜系统（ELYRA PS.1；德国耶拿卡尔蔡司微成像有限公司（Carl Zeiss MicroImaging GmbH））采集的0.15 μm z轴切片（总z轴范围2.5 μm）的最大强度投影图。<b>i</b>与<b>ii</b>为同一图像。<b>i,</b>展示GPVI-Fc<b>（红色）</b>结合于胶原纤维<b>（蓝色）</b>，省略绿色通道（血小板）。<b>ii,</b>与<b>i</b>图像一致，但包含血小板<b>（绿色）</b>。本图像为另外5次独立实验的代表性结果。视频2展示了其三维可视化形式。