Inhibitory effects of LDL-associated tissue factor pathway inhibitor |
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Authors: | Cecilia Augustsson Ida HildenLars C Petersen |
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Institution: | Haemophilia, in vitro Biology, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark |
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Abstract: | IntroductionTissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI1–161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays.Materials and MethodsLDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4 μM phospholipids or 0.5 nM factor Xa (FXa) and 4 μM phospholipids, respectively.ResultsTFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI1–161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height.ConclusionsOur results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI1–161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements. |
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Keywords: | BSA bovine serum albumin CAT calibrated automated thrombogram dPT diluted prothrombin time FVIIa activated factor VII FX factor X FXa activated factor X GPI glycosyl phosphatidyl inositol HBS hepes buffered saline IP immuno pull down KI inhibitor constant kobs apparent rate constant KPI Kunitz-type protease inhibitor PL phospholipids TBS tris buffered saline TFPI tissue factor pathway inhibitor LDL low-density lipoprotein |
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