Effect of IP polyP on plasma clotting and thrombin generation in wild-type (WT) and HRG-deficient mice. (A) Clotting times were determined by recalcification of plasma obtained from WT or HRG^−/− mice administered IP saline or polyP. (B and C) Thrombin generation was determined in plasma from saline- or polyP-treated WT or HRG^−/− mice collected at the indicated time points. Thrombin generation was initiated with 15 mM CaCl₂ in the presence of 15 µM of phosphatidylcholine-phosphatidylserine vesicles and was quantified by monitoring hydrolysis of 1 mM Z-Gly-Gly-Arg-AMC thrombin substrate. (D) Functional polyP concentrations in plasma at various time points after IP injections of polyP were quantified in a thrombin generation assay by comparing peak thrombin concentrations at the sampling times shown with a standard curve of peak thrombin concentrations generated by adding known amounts of polyP to plasma from WT or HRG^−/− mice. N = 9 mice per group; bars represent mean ± standard deviation. **P < .01, ***P < .001, ****P < .0001 compared with saline-treated WT mice; ^##P < .01, ^###P < .001, ####P < .0001 compared with saline treated HRG^−/− mice (analysis of variance, Holm-Šídákmethod).