title: Role of exosite binding modulators in the inhibition of Fxa by TFPI.
abstract: Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.
authors.forename: Sameera
authors.initials: S
authors.lastname: Peraramelli
authors.affiliation: Stella Thomassen, Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, 6200 MD Maastricht, the Netherlands, Tel.: +31 43 388 4160, Fax: +31 43 388 4159, E-mail: s.thomassen@maastrichtuniversity.nl.
authors.forename: Stella
authors.initials: S
authors.lastname: Thomassen
authors.forename: Alexandra
authors.initials: A
authors.lastname: Heinzmann
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: Rudolf
authors.initials: R
authors.lastname: Hartmann
authors.forename: Friedrich
authors.initials: F
authors.lastname: Scheiflinger
authors.forename: Michael
authors.initials: M
authors.lastname: Dockal
authors.forename: Jan
authors.initials: J
authors.lastname: Rosing
mesh_terms: D001777:Blood Coagulation; D002384:Catalysis; D005165:Factor V / Q000737:chemistry; D015943:Factor Va / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry*; D065427:Factor Xa Inhibitors / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008074:Lipoproteins / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry; D011516:Prothrombin / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry
chemical_list: D065427:Factor Xa Inhibitors; D006495:Heparin, Low-Molecular-Weight; D008024:Ligands; D008074:Lipoproteins; D010743:Phospholipids; D011134:Polysaccharides; D017293:Protein S; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa
keywords: Exosites; TFPI; factor V/Va; factor Xa; prothrombin