Two independent binding sites on monolayers of human endothelial cells are responsible for interaction with coagulation factor VII and factor VIIa.
The interaction of radiolabeled factor VII (FVII) and factor VIIa (FVIIa) with endotoxin-stimulated endothelial cells (EC), known to express tissue factor (TF), and unstimulated EC was studied. FVII/FVIIa binding to EC-monolayers was saturable within 4.5-6 h, reversible, temperature and calcium dependent on both, endotoxin-stimulated and on unstimulated EC. Upon 2 h of incubation on EC, FVII was partially converted to FVIIa in the absence of protease inhibitors. The affinity of this binding was Kd = 45.4 +/- 18.7 nM with a calculated number of binding sites Bmax = 3.75 +/- 0.31 x 10(6) molecules/cell. In addition to unlabeled FVII and FVIIa, other vitamin K-dependent proteins reduced binding of [125I]-FVII/FVIIa to about 60-70%, and this type of common binding site for vitamin K-dependent proteins revealed a Kd = 32.2 +/- 5.6 nM and a Bmax = 3.03 +/- 0.14 x 10(6) molecules/cell. Moreover, in the presence of 1 microM prothrombin to suppress common binding sites, only on endotoxin-stimulated EC additional inhibition of FVII/FVIIa binding was achieved by anti-TF antibodies. The characteristics of the FVII/FVIIa-TF interaction with a Kd = 17.2 +/- 5.2 nM and a Bmax = 342,000 +/- 1,100 binding sites/cell revealed a similar saturation kinetics in radioligand binding and in functional factor X activation within 90-120 min. These data indicate the presence of at least two independent binding sites for FVII/FVIIa on stimulated EC of which about 10% are TF specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Reuning
U
U
Haemostasis Research Unit, Kerckhoff-Klinik, Max-Planck-Institut, Bad Nauheim, Germany.
Preissner
K T
KT
Müller-Berghaus
G
G
D001665:Binding Sites; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology; D005167:Factor VII / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D015951:Factor Xa / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D008050:Lipid A / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism
D004731:Endotoxins; D008050:Lipid A; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa