Figure 3: Cross-Linking of GPVI-Fc With Anti–Human-Fc IgG or Anti–Human-Fc Fab2 Increases Its Inhibition of Platelet Aggregation Stimulated by Collagen or Plaque Under Flow

Buffer (control), GPVI-Fc, or GPVI-Fc (50 μg/ml; 333 nM) premixed with equimolar anti–human-Fc IgG or anti–human-Fc Fab2 antibodies for crosslinking was added to blood containing DiOC6 for platelet visualization and perfused over plaque homogenate at a shear rate of 600/s. <b>(A)</b> Representative micrographs display platelet coverage of plaque at 2, 5, and 9 min after start of blood flow. <b>(B)</b> Effect of buffer, GPVI-Fc, GPVI-Fc*IgG-XL, or GPVI-Fc*Fab2-XL on the kinetics of platelet deposition from flowing blood onto plaque and collagen. Measurements are each second. Mean <b>(solid line)</b> ± SD <b>(shaded area)</b>; n = 4 to 6. Comparison at 2, 5, and 9 min by using repeated measures analysis of variance or if inappropriate by repeated measures analysis of variance on ranks (only 2 min). Significance of secondary pair-wise comparisons by Tukey correction is indicated by bars. ∗p &lt; 0.05. <b>(C)</b> Kinetics of not cross-linked and cross-linked GPVI-Fc binding to collagen. (Videos 3 and 4 present additional details.) GPVI-Fc pre-incubated with PE-labeled anti–human-Fc Fab2 (20:1 mol/mol) (GPVI-Fc*Fab2) or with an equimolar mixture of PE-labeled and unlabeled anti–human-Fc Fab2 (1:10) for cross-linking (GPVI-Fc*Fab2-XL), was added to blood (333 nM GPVI-Fc final concentration) containing abciximab and perfused over collagen at a shear rate of 600/s. Binding of PE-labeled GPVI-Fc (cross-linked or not cross-linked) to collagen was quantified every second by fluorescence microscopy using a 10× objective. <b>Top:</b> Time course of GPVI-Fc*Fab2 and GPVI-Fc*Fab2-XL binding to collagen (area coverage). Mean <b>(solid line)</b> ± SD <b>(shaded area)</b>; n = 4. <b>Bottom:</b> Fluorescence intensity surface plots of GPVI-Fc*Fab2 and GPVI-Fc*Fab2-XL bound to collagen at 3 min after start of blood flow. Color gradients indicate increase of fluorescence intensity. Abbreviations as in Figures 1 and 2.

将缓冲液（对照组）、GPVI-Fc，或与等摩尔量抗人-Fc IgG或抗人-Fc Fab2抗体预混合以进行交联的GPVI-Fc（50 μg/ml；333 nM），添加至含有用于血小板可视化的DiOC6的血液中，并以600/s的剪切率将血液灌流至斑块匀浆表面。<b>(A)</b> 代表性显微照片展示了血流启动后2、5和9分钟时斑块的血小板覆盖情况。<b>(B)</b> 缓冲液、GPVI-Fc、GPVI-Fc*IgG-XL及GPVI-Fc*Fab2-XL对流动血液中血小板向斑块及胶原蛋白表面沉积动力学的影响。每秒钟采集一次测量数据。数据以均值（实线）±标准差（阴影区域）表示；n=4~6。在2、5和9分钟时间点采用重复测量方差分析进行组间比较，若不满足方差分析前提条件则采用秩和重复测量方差分析（仅针对2分钟组）。经Tukey校正后的次级两两比较显著性以误差棒标注，*p < 0.05。<b>(C)</b> 交联与未交联GPVI-Fc与胶原蛋白的结合动力学过程。（补充实验细节详见视频3与视频4。）将GPVI-Fc与PE标记的抗人-Fc Fab2以20:1的摩尔比预孵育（GPVI-Fc*Fab2），或与比例为1:10的PE标记及未标记抗人-Fc Fab2混合体系进行交联（GPVI-Fc*Fab2-XL），随后添加至含有阿昔单抗的血液中（最终GPVI-Fc浓度为333 nM），并以600/s的剪切率将血液灌流至胶原蛋白表面。采用配备10倍物镜的荧光显微镜，每秒定量记录PE标记的GPVI-Fc（交联或未交联）与胶原蛋白的结合情况。<b>上图：</b> GPVI-Fc*Fab2与GPVI-Fc*Fab2-XL与胶原蛋白结合的时间进程（面积覆盖度）。数据以均值（实线）±标准差（阴影区域）表示；n=4。<b>下图：</b> 血流启动后3分钟时，结合于胶原蛋白表面的GPVI-Fc*Fab2与GPVI-Fc*Fab2-XL的荧光强度表面图。颜色梯度代表荧光强度的递增。缩写同图1与图2。