3K3A-APC with reduced anticoagulant activity but normal cytoprotective activity is safe in humans when given as a high-dose bolus regimen. (A) The polypeptide ribbon model of APC depicts the N-terminal Gla domain at the bottom, which binds EPCR and phospholipid membranes; the EGF-1 and EGF-2 domains in the middle; and the protease domain at the top, with the “active site” triad residues (His211, Asp257, Ser360) labeled in red. Light blue coloring highlights 3 Lys residues (KKK191-193) on top of the protease domain, which form a positively charged exosite that recognizes factor Va. (B) Mutation of these Lys residues to Ala residues in the human APC variant 3K3A-APC (area outlined in yellow, middle image) deletes a factor Va binding site and consequently reduces anticoagulant activity by >90% (left graph) but does not affect 3K3A-APC’s antiapoptotic activity when tested in endothelial cell apoptosis assays (right graph). This 3K3A-APC variant is designated to be “signaling-selective” because it lacks most anticoagulant activity but retains normal cell-signaling actions. (C) In a phase 1 clinical study of 3K3A-APC, high-dose boluses were safe when administered to healthy adults and formed the basis for US Food and Drug Administration approval of a phase 2 study of 3K3A-APC in ischemic stroke patients (http://www.neuronext.org/neuronext-pleased-announce-funding-our-fourth-approved-trial-safety-evaluation-3k3a-apc-ischemic). Panel C reprinted from Lyden et al²³  with permission.