PolyP plus β-thrombin accelerates plasma clotting and enhances thrombin generation. (A) PolyP shortens plasma clotting times triggered by β-thrombin (measured using a mechanical coagulometer). Citrated FXII- or FXI-deficient plasmas containing 100 μg/mL corn trypsin inhibitor and 20μM PCPSPE were incubated for 1 minute at 37°C with 12nM β-thrombin and varying polyP concentrations, after which CaCl₂ was added and the time to clot formation was measured. PolyP tested with FXII-deficient plasma included 22mer (●), 65mer (▿), 101mer (♦), 211mer (○), or 445mer (▴). FXI-deficient plasma was tested with 445mer polyP (▵). Data are mean ± SE (n = 5). Panels B and C are mean thrombin generation (CAT) curves for FXII-deficient plasmas containing 100 μg/mL corn trypsin inhibitor, 20μM PCPSPE, and 20nM FVa (4 experiments of triplicate wells). (B) Concentration dependence of polyP's ability to enhance thrombin generation in the presence of 20nM β-thrombin and varying polyP (101mer at 0-50μM phosphate). (C) Effect of polyP polymer length on thrombin generation in the presence of 20nM β-thrombin and with or without 50μM polyP (65mer, 101mer, or 445mer). (D-H) Mean peak thrombin levels from experiments represented in panels B and C (± SE; n = 4) obtained with 20nM β-thrombin and varying polyP. (D) Peak thrombin levels in FXII-deficient plasma at the indicated concentrations of polyP 65mer (▿), 101mer (♦), or 445mer (▵). In control experiments, polyP 445mer was preincubated with 250 μg/mL EcPPXc (◊) or predigested with 40 μg/mL rPPX1 (□). (E) Peak thrombin levels in FXII-deficient plasma preincubated with anti-FXI antibody ± 50μM polyP 101mer. (F) Peak thrombin levels in FXI-deficient plasma ± 50μM polyP 101mer. (G) Peak thrombin levels in FXI-deficient plasma to which 4 μg/mL FXI had been added ± 50μM polyP 101mer. (H) Peak thrombin levels in FXI-deficient plasma to which 50pM FXIa had been added ± 50μM polyP 101mer.