Interaction of HRG, FXII, and FXIIa with polyP and the effects of HRG on polyP-induced clotting. (A) b-polyP was immobilized in wells of a streptavidin-coated multi-well plate, and binding of BSA, HRG, FXII, and FXIIa was quantified in the absence or presence of 6 μM ZnCl2. Absorbance values for the associated ligand concentrations are plotted; the data were analyzed by nonlinear regression of a rectangular hyperbola (lines). Symbols represent mean ± standard deviation (SD) of 5 determinations. (B) Clotting after recalcification of control (closed circles with solid red line) or HRG-depleted human plasma (open circles with dashed blue line) in the absence or presence of polyP at the indicated concentrations was monitored by absorbance, and the clot time was calculated as the time to achieve half-maximum absorbance. Lines represent nonlinear regression analysis of the data. Symbols represent mean ± SD of 5 determinations. (C) polyP-induced clotting times in control (red bar) and HRG-depleted human plasma (blue bars) containing human HRG at the indicated concentrations were determined. Bars represent mean ± SD of 4 determinations. *P < .05, **P < .01 compared with normal pooled control plasma. (D) Control and HRG-depleted plasma were incubated with or without 1 μM CTI for 15 minutes followed by the addition of either aPTT-SP reagent, RecombiPlasTin reagent (Instrumentation Laboratory, Bedford, MA), or 40 μg/mL of polyP. Clotting was initiated with 26 mM CaCl2, and the clot time was calculated as the time to half maximum increase in absorbance. Bars represent mean ± SD of 4 determinations each done in duplicate. *P < .05, ****P < .001 comparison between control and HRG-depleted plasma as indicated by the lines. NS, not significant (analysis of variance, Holm-Šídákmethod); PT, prothrombin time.