PolyP and fibrin augment αFXIIa-mediated plasminogen activation. (A) Plasminogen activation was analyzed by incubating 200 nM αFXIIa and 200 nM Glu-plasminogen (dotted line) in the presence of either 3.8 μM soluble fibrin (SF; light gray), 70 μM polyP70 (dark gray), or both SF and polyP70 (black). Plasmin activity was detected using the chromogenic substrate S2251 at 405 nm. Data represent mean ± SEM (n = 3; P < .001). (B-C) The plasminogen activator function of αFXIIa (200 nM) was analyzed in the presence of different surfaces, including polyP70 (70 μM), RNA (10 μg/mL), and collagen (5 μg/mL). (B) Plasmin activity, generated from 200 nM plasminogen, was detected using S2251. (C) Fibrinolysis was analyzed by forming clots from fibrinogen (3.8 μM), αFXIIa (200 nM), Glu-plasminogen (200 nM), thrombin (0.25 U/mL), and CaCl2 (10 mM). Lysis was monitored at 405 nm, and mean ± SEM are shown as the time from maximal absorbance of the clot to 50% lysis (n = 3; ***P < .001). (D) Fibrin clots were formed and monitored as described in panel C, with 70 μM polyP70 (black), 1 pM tPA (gray), or both polyP70 and tPA (dark gray). The clots formed with tPA (dashed line) and both tPA and polyP (light gray) were formed in the absence of αFXIIa. Mean data ± SEM are expressed as percentage turbidity (n = 4; P < .0001).
