Summary
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 K
d = 45.4 ± 18.7 nM with a calculated number of binding sites B
max = 3.75 ± 0.31 × 106 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 K
d = 32.2 ± 5.6 nM and a B
max = 3.03 ± 0.14 × 106 molecules/cell. Moreover, in the presence of 1 μM 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 K
d = 17.2 ± 5.2 nM and a B
max = 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. The existence of binding sites common for vitamin K-dependent proteins on both types of EC may improve the availability of FVII/FVIIa once EC become stimulated and express TF on their surface.
