A novel immunoassay for the quantification of human tissue factor binding to activated factor VII.
The binding of tissue factor to factors VII and VIIa (VII/VIIa) is the primary step for coagulation activation. Variants of human tissue factor leading to alterations in the binding to factor VII/VIIa have not been reported. We hypothesize that increased or decreased binding of tissue factor to factor VIIa might result in thrombosis and bleeding, respectively. The aim of this study was to establish an enzyme-linked immunosorbent assay to detect abnormalities in the binding of human tissue factor to recombinant factor VIIa (rVIIa). Tissue factor obtained from human monocytes was bound to rVIIa on microtiter wells in the presence of calcium. A murine antibody against human tissue factor and a biotinylated goat anti-mouse immunoglobuline were added. After incubation with streptavidin-horseradish peroxidase, colour development was measured using a chromogenic indicator system. Optimal assay conditions were obtained at tissue factor concentrations of 50-1500 pg/ml and rVIIa concentrations of 2.5 microg/ml. The binding of tissue factor to rVIIa was calcium-dependent and was inhibited by a monoclonal tissue factor antibody directed against the binding sites of tissue factor to rVIIa. The assay was evaluated in 23 healthy volunteers. Intra- and interassay variabilities were 3.9% and 10.2%, respectively. Among 22 subjects with unexplained bleeding and 47 patients with unexplained thrombosis, an individual with a decreased or increased binding of tissue factor to rVIIa could not yet be identified. In conclusion, this novel enzyme-linked immunosorbent assay can be used to detect and quantify an increased or a decreased binding of human tissue factor to rVIIa. Studies in patients indicate that, if such a defect exists, it is not a common cause of thrombosis or bleeding.
Weltermann
A
A
Department of Medicine 1, University of Vienna, Austria.
Eichinger
S
S
Kyrle
P A
PA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002118:Calcium / Q000494:pharmacology; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D015942:Factor VIIa / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012016:Reference Values; D013925:Thromboplastin / Q000378:metabolism*
D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium