title: Regulation of thrombin generation by TFPI in plasma without and with heparin.
abstract: The purpose of this study was to investigate the impact of recombinant glycosylated TFPI (rg-TFPI) from BHK cells, nonglycosylated TFPI (r-TFPI) from Escherichia coli, and truncated TFPI (1-161) on thrombin generation (TG) in plasma treated with and without heparin in vitro and ex vivo. Fasting plasma samples were collected from 6 healthy persons. TG was assessed by the calibrated automated thrombography (CAT) method. The addition of increasing concentrations (0-200 ng/mL) of different TFPI caused a 5% to 30% prolongation of lag time for TF (3.0 pM) induced TG, with the most pronounced effect for rg-TFPI and the least pronounced effect for truncated TFPI, but without affecting endogenous thrombin potential (ETP) in TF-induced coagulation. Removal of native TFPI from plasma by anti-TFPI IgG treatment shortened lag time by 35 +/- 4% without affecting ETP. Increasing concentrations (0-200 ng/mL) of various TFPI in the presence of low heparin concentrations (0.1 IU/mL) prolonged lag time and decreased ETP by 25% to 75% with the most prominent effect promoted by glycosylated full-length TFPI. The effect of neutralizing antibodies against TFPI and antithrombin (AT) was studied in plasma in the presence of heparin administered in vitro or ex vivo. The results revealed that TFPI and AT acted in synergy as inhibitors of coagulation in terms of the effect on both initiation (lag time) and propagation (ETP). Our data demonstrated that the CAT assay appropriately assessed the impact of TFPI on initiation and propagation of TG in a physiological plasma milieu with and without heparin. TFPI contributed significantly to regulation of coagulation initiation (lag time). The C-terminal region and, to a lesser extent, glycosylation of the TFPI molecule were essential for its anticoagulant function in the absence and presence of heparin.
authors.affiliation: Center for Atherothrombotic Research (CART), Department of Medicine, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway. ellenb@fagmed.uit.no
authors.forename: Ellen
authors.initials: E
authors.lastname: Brodin
authors.forename: Hege
authors.initials: H
authors.lastname: Appelbom
authors.forename: Bjarne
authors.initials: B
authors.lastname: Osterud
authors.forename: Ida
authors.initials: I
authors.lastname: Hilden
authors.forename: Lars C
authors.initials: LC
authors.lastname: Petersen
authors.forename: John-Bjarne
authors.initials: JB
authors.lastname: Hansen
mesh_terms: D000328:Adult; D000991:Antithrombins / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D008074:Lipoproteins / Q000097:blood* / Q000494:pharmacology*; D008297:Male; D010446:Peptide Fragments / Q000097:blood* / Q000494:pharmacology*; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D014019:Tissue Donors
chemical_list: D000991:Antithrombins; D008074:Lipoproteins; D010446:Peptide Fragments; C081249:tissue factor pathway inhibitor (1-161); D006493:Heparin; D013917:Thrombin