Hypercoagulability is often associated with a variety of disease states, leading to cardiovascular complications. Polyethylenimine (PEI) prolonged prothrombin time, demonstrating its anticoagulant potential. In vitro, PEI at low concentration (nM) significantly blocked thrombin-catalyzed fibrin formation, accounting for its mode of anticoagulation. The uncompetitive inhibition by PEI of fibrin formation was independent of the concentration of fibrinogen (FBG), thrombin, or NaCl. PEI showed no effect on thrombin amidolytic activity, suggesting that the blockade of thrombin interaction with FBG could account for the inhibition on fibrin formation. PEI drastically depressed rabbit brain thromboplastin procoagulation monitored by a single-stage clotting assay using human plasma. In a THP-1 monocytic hypercoagulation model, a 4-h exposure to bacterial endotoxin or Ca(2+) ionophore A23187, respectively, resulted in a 5- or 10-fold enhancement in monocytic tissue factor (mTF) procoagulation. mTF hypercoagulation was offset by PEI included in the assay mixture. PEI showed the potential to arrest mTF hypercoagulation with IC(50) around 1.2 nM. Using a chromogenic assay to dissect the extrinsic pathway, we further assessed whether PEI has any effect on other clotting factors. PEI was not an inhibitor for either FVIIa or FXa, having no effect on not only the amidolytic but also their corresponding functionally catalytic activities. Although PEI upregulated TF-dependent FVII activation under the low-salt condition, the effective downstream inhibition of fibrin formation readily abolished and overrode the upstream enhancement, demonstrating the overall anticoagulation. PEI could present a new class of anticoagulant.