Fig. 8: A gain-of-function mutant of HRG reduces thrombus formation.

a The disulfide pairs were predicted on the domain structure of mouse HRG based on that of human HRG, with red color indicating the targets of PDI. The gain-of-function mutant of mouse HRG (gof-HRG) was generated by replacing 6 Cys of the 3 target disulfide bonds of PDI: C246-C261, C270–C467 and C315–C320 to Ala, which locked the variant in a reduced form. The binding of wt-HRG and gof-HRG on heparin (b) (n = 12 independent samples) and FXIIa (c) (n = 9 independent samples) was determined by ELISA. d, e The accumulation of HRG following laser injury in the cremaster arterioles of Hrg^−/− mice infused with different HRG variants were visualized using Alexa-488-conjugated anti-HRG. The median fluorescence intensity of HRG (d) was plotted over time. The area under the curve (AUC) for HRG (e) was analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. f–i Platelet accumulation and fibrin generation following laser injury in the cremaster arterioles of Hrg^−/− mice infused with different HRG variants were visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively. The median fluorescence intensity of platelets (f) and fibrin (g) were calculated and plotted over time. The AUC for platelets (h) and fibrin (i) were analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. Data are presented as mean values ± SEM and analyzed by two-tailed Welch’s t-test (b and c) or two-tailed Mann–Whitney U-test (e, h and i). Source data are provided as a Source Data file.