Binding of tissue factor pathway inhibitor to cultured endothelial cells-influence of glycosaminoglycans.
Tissue factor pathway inhibitor (TFPI) is mainly bound to the vessel wall and is released to circulating blood after injections of heparin. It has been suggested that the highly positively charged carboxy terminal end of heparin releasable TFPI is bound to negatively charged binding molecule(s), presumably glycosaminoglycans (GAGs), on the luminal surface of endothelial cells. The aim of the present study was to characterize this binding. Confluent monolayers of human umbilical vein endothelial cells (HUVECs) and Ea.hy926 cells were incubated with 125I-labelled recombinant TFPI (rTFPI). Two different rTFPI preparations were used in the experiments; one preparation was full-length rTFPI and one preparation was truncated at the C-terminal end (rTFPI1-161). Binding of 125I-rTFPI reached equilibrium conditions after 2 hours incubation at room temperature. Scatchard plots indicated a single class of binding sites with a mean Kd value of 164 +/- 16 nmol/L for HUVECs and a Kd value of 296 +/- 10 nmol/L for Ea.hy926 cells. The number of rTFPI binding sites per cell were approximately 1.10(7). Binding of 125I-rTFPI1-161 was non-specific. GAGs reduced binding of 125I-rTFPI in a dose-dependent manner by 50-75%. The potency of different GAGs to displace bound rTFPI was in the following order: Unfractionated heparin (UF) > low-molecular weight (LMW) heparin > hexadecasaccharides/octasaccharides/dodecasaccharides > heparan sulfate > dermatan sulfate. Treatment of the cells with heparinase III, with chondroitinase ABC lyase, or with sodium chlorate (to prevent sulfation) did not influence the binding of TFPI. We conclude that the C-terminal end is necessary for binding of TFPI to endothelial cells, but the binding is weak and does not involve GAGs.
Iversen
N
N
Haematological Research Laboratory, Aker University Hospital, Oslo, Norway.
Sandset
P M
PM
Abildgaard
U
U
Torjesen
P A
PA
D000925:Anticoagulants / Q000378:metabolism*; D002704:Chlorates / Q000494:pharmacology; D003871:Dermatan Sulfate / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism; D065427:Factor Xa Inhibitors; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism*; D013997:Time Factors
D000925:Anticoagulants; D002704:Chlorates; D003871:Dermatan Sulfate; D065427:Factor Xa Inhibitors; D005343:Fibrinolytic Agents; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; C032706:sodium chlorate