Fig 1.
Short-chain polyphosphates accelerate the inhibition of TFPIα by FXIa.
rTFPI (10 nM) was incubated with 2 nM FXIa for 10, 15, or 30 min in the presence or absence of 25 μM Zn2+,5 mM Ca2+, 10 μM SCP, Ca2+ and SCP or Zn2+ and SCP. rTFPI was analyzed by western blotting, with a polyclonal anti-TFPI antibody under non-reducing conditions. The bands were quantified by densitometry. Data are mean ± SE (n = 3).
Fig 2.
Characterization of the interaction between polyphosphate and TFPIα.
(A) 96-well plates were coated with 5 μg/ml TFPIα (○,●) or BSA (◻) and increasing concentrations of biotinylated-polyphosphate (bio-polyP) (◻, ○,●) was added to selected wells. Selected experiments were done in the presence of 100 μM SCP (●). Binding was detected with streptavidin-HRP. Data are mean ± SE (n = 3).
Fig 3.
Short-chain polyphosphates accelerate the inhibition of TFPIα by FXIa.
(A) TFPIα (10 nM) was pretreated with FXIa (2 nM) in the absence or presence of 25 μM Zn2+, 10 μM platelet-size polyphosphate (SCP), or Zn2+ and SCP. After 30 min of incubation with FXIa, aprotinin (50 μM) and polybrene (16 μM) were added to all samples to stop the reaction. FXa generation by the TF-FVIIa complex in the presence of different concentrations of TFPIα was measured. (B) TFPIα (10 nM) was pretreated with different concentrations of FXIa in the presence of 25 μM Zn2+ and 10 μM SCP. After 30 min of incubation with FXIa, aprotinin (50 μM) and polybrene (16 μM) were added to all samples to stop the reaction and FXa generation by the TF-FVIIa complex in the presence of different concentrations of TFPIα was measured. (C) TFPIα (10 nM) was pretreated with 4 nM FXIaABS in the presence of 25 μM Zn2+, 10 μM SCP, or Zn2+ and SCP. After 30 min of incubation with FXIaABS, aprotinin (50 μM) and polybrene (16 μM) were added to all samples to stop the reaction and FXa generation by the TF-FVIIa complex in the presence of different concentrations of TFPIα was measured. Data are mean ± SE (n = 3).
Fig 4.
Short-chain polyphosphates accelerate the inhibition of TFPIα by FXIa.
(A) FXa (0.5 nM)-induced clotting time of FX-depleted plasma was measured in the presence of vehicle or 5 nM TFPIα pretreated with 1 nM FXIa + 25 μM Zn2+ for 30 min in the presence of 0, 0.6, 1.25, 2.5,5 or 10 μM SCP. After 30 min of incubation with FXIa, aprotinin (50 μM) and polybrene (16 μM) were added to stop the reaction. (B) FXa (0.5 nM)-induced clotting time of FX-depleted plasma was measured in the presence of vehicle or 5 nM TFPIα pretreated with 0.06, 0.125, 0.25, 0.5 or 1 nM FXIa for 30 min in the presence of 10 μM SCP and 25 μM Zn2+. After 30 min of incubation with FXIa, aprotinin (50 μM) and polybrene (16 μM) were added to all samples to stop the reaction. Data are mean ± SE (n = 3).
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 FXIaWT or FXIaABS 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 FXIaWT. ** P < 0.05 with respect to vehicle in the presence of FXIaWT. 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)
Fig 6.
FXI is required for short-chain polyphosphates to inhibit the anticoagulant effect of TFPIα in plasma.
(A) TF-induced clotting times were measured in normal plasma in the presence of increasing concentrations of TFPIα in the absence (○, ◻, △) or presence of 5 μM SCP (●, ■, ▲). Plasma was pretreated with vehicle (○,●), 20 μg/ml 1A6 (△, ▲), or 50 μg/ml 10C9 (◻,■). (B) TF-induced clotting times were measured in FIX-depleted plasma (FIX-dep) (■,●) or FXI-depleted plasma (FXI-dep) (◻,○) in the presence of increasing concentrations of TFPIα in the absence (■,◻) or presence of 5 μM SCP (○,●). (C) TF-induced clotting times were measured in FIX-depleted plasma in the presence of increasing concentrations of TFPIα in the absence (○, ◻, △) or presence of 5 μM SCP (●, ■, ▲). Plasma was pretreated with vehicle (○,●), 20 μg/ml 1A6 (△, ▲), or 50 μg/ml 10C9 (◻,■). Data are mean ± SE (n = 3).