PolyP attenuates the binding of fibrinolytic enzymes and the cofactor activity of fibrin. (A) Fibrin clots containing plasminogen (0.55μM) were formed with purified fibrinogen (2.4μM) in the absence (gray line) and presence (black line) of polyP₆₅ (325μM) by adding thrombin (1 U/mL) and CaCl₂ (5mM). Clots were lysed by overlaying with tPA (5nM) in the presence of S2251 (0.12mM), and readings were recorded at 405 nm. Plasmin generation at 405 nm over time is shown, and results are expressed as mean ± SEM, n = 3. (B) Fibrinogen was captured on the surface of a CM5 chip and converted to fibrin by thrombin (1 U/mL) in the absence and presence of polyP₆₅ (325μM). Binding of 125nM (black), 250nM (orange), 500nM (blue), and 1000nM (green) plasminogen was analyzed (dashed lines). The surface was then treated with plasmin (30nM), and binding of plasminogen, at the concentrations described above, was repeated (solid lines). (C) As described for panel B with 31.25nM (black), 62.5nM (orange), 125nM (blue), and 250nM (green) tPA instead of plasminogen. (B-C) Experiments performed in triplicate and are expressed as mean ± SEM. (D) Schematic representation of polyP-induced down-regulation of fibrinolysis. (1) Fibrin formed in the presence of polyP has a heterogeneous structure composed of tightly knotted regions interspersed with large pores. (2) Plasmin (yellow) is less efficient at cleaving this heterogeneous fibrin structure, thereby reducing the exposure of C-terminal lysines (K) on the fibrin surface. (3) The capacity of fibrin to sequester tPA and plasminogen on its surface is subsequently reduced, and the cofactor role of fibrin in tPA-mediated plasminogen activation is diminished. (4) This leads to less plasmin formation. (5) As a result fibrinolysis is down-regulated in clots formed in the presence of polyP.