Andexanet alfa and UHRA reverse the anticoagulation activity UFH and enoxaparin. An aPTT assay was performed in heparinized, noncitrated human platelet–poor plasma. (A-B) Andexanet alfa and UHRA neutralized UFH and enoxaparin anticoagulation activity. However, PER977 at all tested concentrations showed no anticoagulation reversal activity. Experiments were performed in triplicate. Results are expressed as the median with interquartile ranges of 15 measurements from 5 independent experiments (n = 5 donors). Black horizontal lines represent median, and error bars represent interquartile range. Asterisks indicate significant differences. Statistical significance was determined by comparing the antidote treated group to the UFH/enoxaparin control using a Kruskal-Wallis test followed by a Dunn post-test (*P < .05, **P < .02, ***P < .0006, ****P < .0001). (C) Microplate whole-blood clotting assay was performed in heparinized and noncitrated human whole blood. Neutralization of UFH activity was verified by measuring changes in absorbance at 510 nm. Neutralization of anticoagulation activity of UFH and subsequent stable clot generation reduces absorbance. Stable clot formation was observed in heparinized blood containing UHRA (50, 100, and 200 µg/mL) or andexanet alfa (200 µg/mL), but not with PER977. UHRA and andexanet alfa can reverse the anticoagulation activity of UFH. However, we observed an increase in absorbance in PER977-treated samples in comparison with UFH control itself, possibly due to hemolysis. Experiments were performed in triplicate. Results are expressed as the mean ± SE of 15 measurements from 5 independent experiments (n = 5 donors).