Fig. 3: PDI enhances the neutralization of heparan sulfate and inhibition of FXIIa by HRG.

a Purified human HRG was pre-treated with vehicle or recombinant human PDI variants at indicated concentrations and incubated on heparin-coated surface with or without Zn²⁺ (1 μM). The binding of HRG was determined by ELISA (n = 3 independent samples). b Human plasma pre-treated with recombinant human PDI variants was incubated on HUVECs with Zn²⁺ (10 μM). The binding of HRG and antithrombin on the cell surface were determined by immunofluorescence (scale bar: 20 μm). Human plasma pre-treated with recombinant human PDI variants was incubated on HUVECs with or without Zn²⁺ (10 μM). The binding of HRG (c) and antithrombin (d) on the cell surface were determined by cell-based ELISA (n = 6 independent samples). e Purified human HRG was pre-treated with vehicle or recombinant human PDI variants and incubated on FXIIa-coated surface with or without Zn²⁺ (1 μM). The binding of HRG was determined by ELISA (n = 6 independent samples). f Purified human HRG was pre-treated with vehicle or recombinant human PDI variants, and mixed with FXIIa (10 nM) with or without Zn²⁺ (1 μM). The activity of FXIIa was determined using chromogenic substrate S2302 (n = 6 independent samples). g Purified human HRG was pre-treated with vehicle or recombinant human PDI variants, mixed with FXIIa, and then added into DKO mouse plasma to initiate thrombin generation (TGA). The lag time and peak time (h), and the peak height and area under the curve (AUC) (i) from individual TGA curve were calculated (n = 6 independent samples). j Recombinant mouse HRG was pre-treated with vehicle or recombinant mouse PDI variants, and then added into Hrg^−/− mouse plasma. TGA was initiated by addition of aPTT reagent. The lag time and peak time (k), and the peak height and AUC (l) from individual TGA curve were calculated (n = 6 independent samples). Veh vehicle. Data are presented as mean values ± SEM and analyzed by Welch’s ANOVA test (c–f, h, I, k and l). Source data are provided as a Source Data file.