Discovery and biochemical characterization of POmAb. (A) Human prothrombin (Uniprot entry: P00734) is made of 4 domains, which are connected by 3 linkers. Fragment-1, comprising residues 1-155, was injected into BALB/c mice for immunization experiments. (B) Conformational equilibrium of prothrombin (ProT), showing large relocation of fragment-1 (red) relative to the protease domain (yellow) as prothrombin transitions from closed to open forms and strategic location of residues S91 and Y93 in kringle-1 (cyan). (C) Closed-up view of the interface between kringle-1 (red) and protease domain (yellow) that forms in closed prothrombin, highlighting key interactions (yellow lines) of residues S91 and Y93 in kringle-1 (cyan) with residues W468, W547, and D552 (green) of the protease domain. (D) Characterization of POmAb by ELISA. POmAb was incubated with specified proteins, and binding was detected with an anti-mouse γ-specific IgG antibody conjugated with horseradish peroxidase and TMB (3,3′,5,5′-tetramethylbenzidine) substrate. Each binding experiment was independently repeated a minimum of 3 times. (E-I) Surface plasmon resonance experiments. Shown are reference-subtracted real-time association and dissociation profiles resulting from flowing kringle-1 (0-1000 nM) (E), kringle-2 (0-1000 nM) (F), prothrombin (0-1000 nM) (G), prothrombin (0-1000 nM) with 200 μM argatroban (H), and prothrombin variant S91A/Y93A (0-2000 nM) (I) onto immobilized POmAb. Fit is shown in black. Rate constants obtained by fitting the sensograms are reported in supplemental Table 1. (J) FRET histograms of prothrombin labeled at residues 101 in kringle-1 and 478 in the serine protease domain in the absence (orange; top) and presence of 500 nM POmAb (purple; bottom). β₂GPI, β₂-glycoprotein I; FXII, factor XII; RU, resonance units; tPA, tissue-type plasminogen activator.