Fig. 4: HRG accumulates on the vessel wall during thrombus formation.

a The accumulation of platelets and HRG during laser injury-induced thrombus formation in murine cremaster arterioles were visualized using Dylight-649-congugated anti-CD42c (Red) and Alexa-488-conjugated anti-HRG (Green), respectively, with irrelevant IgG as background control (488-IgG) (scale bar: 30 μm) (Supplementary Movie 1). The median fluorescence intensity of HRG (b) and platelets (c) were calculated and plotted over time from mice treated or untreated with eptifibatide (10 μg/g body weight, repeated every 15~20 min). The area under the curve (AUC) for platelets (d) and HRG (e) were analyzed from each individual thrombus in different treatment groups as indicated. The number of thrombi (n value) analyzed in each group was indicated above the bars. f A representative focal plane of HRG accumulation and vascular endothelium, as detected by Alexa-488-conjugated anti-HRG (Green) and Alexa-647-conjugated anti-CD31 (Red), respectively, approximately 5 min after laser-induced vessel injury, was extracted from in vivo two-photon scanning of the cremaster arterioles. The areas of the cremaster arteriole (ROI1, cyan) and the thrombus (ROI2, magenta) were manually defined (scale bar: 20 μm). g The intensities of green (HRG) and red (CD31) fluorescence were plotted spatially along the white solid line as indicated in f. h The scatter plot of pixel intensities of green (HRG) and red (CD31) fluorescence in ROI1 and ROI2 as indicated in f were presented. The Pearson’s correlation coefficients (r) for pixels with intensity above the threshold (indicated by the dashed lines) in the thrombus (ROI2) and in the area without the thrombus (ROI1-ROI2) were inserted. Veh vehicle, Epti eptifibatide. Data are presented as mean values ± SEM and analyzed by Kruskal–Wallis test with Dunn’s multiple comparisons (d and e). Source data are provided as a Source Data file.