Effect of alkaline phosphatase (AP) on clotting times and thrombin generation in plasma from wild-type (WT) or HRG-deficient mice. (A and B) AP was incubated for 15 minutes at 37°C with plasma from WT or HRG^−/− mice collected 60 minutes after IP polyP injection. Thrombin generation was initiated by adding 15 mM CaCl₂ and was quantified by monitoring the hydrolysis of 1 mM Z-GGR-AMC thrombin substrate. N = 6 mice per group. (C) Plasma recalcification times were determined in plasma collected from WT or HRG^−/− mice 60 minutes after IP polyP administration with or without preincubation with AP. Absorbance was measured at 405 nm for 1 hour, and the clot time was determined as the time to half maximal absorbance. N = 6 mice per group; bars represent mean ± standard deviation. (D) Plasma samples from mice injected with polyP before and after AP treatment were subjected to electrophoresis on a TBE-urea gel, and polyP was imaged by negative staining with 4′,6-diamidino-2-phenylindole. PolyP composed of either 70 or 1000 phosphate units (polyP-70 and polyP-1000, respectively) was used as standards. Representative images from 6 gels per treatment group are shown. **P < .01, ***P < .001 compared with plasma from WT mice without AP; ^##P < .01, ^###P < .001 compared with plasma from HRG^−/− mice without AP (analysis of variance, Holm-Šídákmethod).