Fig 5. Platelet-derived short-chain polyphosphates accelerate the inhibition of platelet TFPIα by FXIa.

(A) FXa generation following initiation with TF was determined in the absence or presence of supernatant from activated platelets. In selected experiments, the supernatant from 2×10⁸ platelets/ml was pretreated with 2 nM FXIa^WT or FXIa^ABS for 60 min in the presence or absence of polybrene (16 μM). In separate experiments, platelet supernatant from activated platelets was incubated with blocking anti-TFPI K1 and K2 antibodies (50 μM). Aprotinin (50 μM) was added to all samples to stop the reaction. Data are mean ± SE (n = 4). * P < 0.05 with respect to vehicle in the presence of FXIa^WT. ** P < 0.05 with respect to vehicle in the presence of FXIa^WT. Mann-Whitney U test was used for statistical comparisons. (B) FXa generation following initiation with TF was determined in the presence of supernatant from activated platelets pretreated with 2 nM FXIa in the presence or absence of PPxbd (250 μg/ml) for 0, 15, 30 or 60 min. Aprotinin (50 μM) was added to all samples to stop the reaction. Data are mean ± SE (n = 3). (C) Supernatant from activated platelets (2.5×10⁸) was incubated with 5 nM FXIa for 0, 30, 60 or 120 min in the absence or presence of PPxbd (250 μg/ml) or aprotinin. The extent of TFPI present in the supernatant was analyzed by western blotting with a polyclonal anti-TFPI antibody. The bands were quantified by densitometry. Data are mean ± SE (n = 3)