HRG kills S pyogenes and killing occurs in HRG-containing plasma and HRG-containing clots. (A) S pyogenes in Tris buffer, pH 7.4 (black bars) or MES buffer, pH 5.5 (white bars), was incubated with purified human HRG (Hu.HRG), mouse HRG (Mo.HRG), the HRG-derived peptide GHH20, or albumin, and the percentage killing was calculated. Data are mean ± SEM; n = 3. (B) S pyogenes in MES buffer, pH 5.5, was incubated with 5μM human HRG (squares) or GHH20 (circles) in the presence of an increasing concentration of purified SIC. Data are mean ± SEM; n = 3. (C) S pyogenes in MES buffer, pH 5.5, was added to wild-type or HRG-deficient plasma (final pH 6.5-7.0), and growth was determined over time. The data shown are representative of 5 experiments, which all gave the same profile of results. (D) S pyogenes in MES buffer, pH 5.5, was added to increasing concentrations of plasma diluted in MES buffer: wild-type plasma (black bars), HRG-deficient plasma (white bars), or HRG-deficient plasma reconstituted with purified HRG (gray bars). Growth was determined after 6 hours. Data are representative of 3 experiments, which all gave the same profile of results. (E) Thrombin (1 U/mL) was used to initiate clot formation in wild-type and HRG-deficient plasma. The washed clots were incubated with S pyogenes bacteria in MES buffer, pH 5.5, and the percentage killing was calculated. Data are mean ± SEM; n = 8. ***P < .001 (Student t test). (F-I) S pyogenes in MES buffer, pH 5.5 (F-G) or Tris buffer, pH 7.4 (H-I) was incubated with purified human HRG (5μM) and subjected to negative staining and transmission electron microscopy.