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，或与等摩尔抗人-Fc IgG或抗人-Fc Fab2抗体预混以完成交联的GPVI-Fc（浓度均为50 μg/ml；333 nM），加入到添加了DiOC6以实现血小板可视化的血液中，并以600/s的剪切速率将血液灌注至斑块匀浆表面。<b>(A)</b> 代表性显微照片展示了血流开始后2、5和9分钟时斑块表面的血小板覆盖情况。<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沉积至斑块与胶原表面的动力学影响。检测频次为每秒1次。结果以均值（实线）±标准差（阴影区域）表示；n = 4~6。针对2、5、9分钟三个时间点，采用重复测量方差分析进行组间比较；若数据不满足方差分析前提，则采用秩和重复测量方差分析（仅针对2分钟时间点）。经Tukey法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结合胶原的动力学过程。（补充细节见视频3与视频4）。将GPVI-Fc分别与PE标记的抗人-Fc Fab2按20:1摩尔比预孵育（GPVI-Fc*Fab2），或与PE标记及未标记抗人-Fc Fab2按1:10的等摩尔混合物预孵育以进行交联（GPVI-Fc*Fab2-XL），随后加入至含阿昔单抗（abciximab）的血液中（最终GPVI-Fc浓度为333 nM），并以600/s的剪切速率灌注至胶原表面。采用10×物镜通过荧光显微镜以每秒1次的频率定量检测PE标记的GPVI-Fc（交联或未交联）与胶原的结合情况。<b>上图：</b> GPVI-Fc*Fab2与GPVI-Fc*Fab2-XL结合胶原的时间进程（面积覆盖度），结果以均值（实线）±标准差（阴影区域）表示；n = 4。<b>下图：</b> 血流开始后3分钟时，结合于胶原表面的GPVI-Fc*Fab2与GPVI-Fc*Fab2-XL的荧光强度表面热图，颜色梯度代表荧光强度的递增。缩写含义同图1与图2。