Binding of endothelial VWF to platelet GPIb plays a pivotal role in our laser/ROS-induced thrombosis model in both capillaries and arterioles. (A) In vivo imaging of VWF mobilization to the EC surface of small capillaries. Fluorescent anti-VWF antibody (red) was administrated to wild-type mice and visualized before and after laser/ROS-induced injury. VWF is expressed on the EC surface within the affected vessel after laser/ROS-induced injury. The cells were also costained with lectin (blue, vasculature), anti-GPIbβ (green, platelets), and Hoechst (green, nucleus). (B) Relative change in anti-VWF signals after laser/ROS-induced thrombus formation in wild type (WT), TNF-α KO, and TNF-R1 KO mice. Anti-VWF signal intensity was determined in the EC surface regions (white boxes in panel A) before (0 seconds) and 20 seconds after laser/ROS-induced injury. Note that VWF expression on endothelium after laser/ROS-induced injury was significantly reduced in both KO mice. AU denotes arbitrary units (n = 15 vessels from 5 animals). Representative original VWF images are shown in supplemental Figure 7. Asterisks indicate statistical significance (P < .05). (C) Numbers of platelets attached to vessel walls 10 seconds after laser/ROS-induced injury in WT, P-selectin KO (P-sel^−/−), and E-selectin KO (E-sel^−/−) mice, as well as WT mice treated with control IgG (WT + IgG), or neutralizing anti-GPIbα antibody (WT + Ab-GPIbα). Numbers of attached cells were markedly reduced in anti-GPIbα–treated WT mice (n = 30 vessels from 5 animals in each group). (D) Calculated numbers of platelets in developing thrombi 20 seconds after laser/ROS-induced injury (n = 30 vessels from 5 animals for each group). (E) Visualization of VWF mobilization to the EC surface in small arterioles, performed as in A. (F-G) Quantification of initial platelet attachment and thrombus development after laser injury in small arterioles (n = 30 vessels from 5 animals in each group). Asterisks indicate statistical significance (P < .05). The scale bar is 10 μm.