Figure 5. Activation of FXII by long polyP promotes FIX activation in absence of FXI.

(A) Plasma clotting time was measured in recalcified FXI-deficient plasma in the presence of vehicle (−), 150 nM α-FXIIa or 50 μM long polyP. In selected experiments, FXI-depleted plasma was pretreated with 20 μg/ml 1A6, 20 μg/ml 14E11, 20 μg/ml anti-FIX mAb, 50 μg/ml CTI or 20 μg/ml rivaroxaban for 10 minutes. The clotting time was recorded, with a maximum observation period of 1000 seconds. (B) Plasma clotting time was measured in recalcified FIX-depleted plasma in the presence of vehicle (−) or 150 nM FXIIa, 50 μM long polyP, or 10 nM FXIa. In selected experiments, FIX-depleted plasma was pretreated with 20 μg/ml 1A6, 20 μg/ml 14E11, 20 μg/ml anti-FIX mAb, 50 μg/ml CTI or 20 μg/ml rivaroxaban for 10 minutes. The clotting time was recorded, with a maximum observation period of 2500 seconds. Data are mean ± SE (n = 3). (C) FIX activation in a purified system following the addition of 100 nM FIX, 100 nM FXII, 50 nM PK and 50 nM HK in the presence of vehicle or 10 μM long polyP. FXIa generation was measured by western blot. (D) Prothrombin activation was measured in a purified system following the addition of 100 nM FXII, 50 nM PK and 50 nM HK to 1 μM prothrombin in the presence of vehicle (◇, ◆) or 10 μM long polyP (△, ▲), in the presence (◆, ▲, ●) or absence of Ca²⁺ (◇, △, ○). Long polyP plus FXII and PK in the absence of prothrombin resulted in no hydrolysis of the thrombin substrate (●, ○). Thrombin generation was measured with a chromogenic substrate. (E) Long polyP induced FIX activation in FXI-deficient plasma. Plasma was incubated with 50 μM long polyP in the absence of calcium in the presence or absence of CTI (50 μg/ml), then subjected to SDS-PAGE and western blotting with an antibody against FIX.