Quantitation of Natural, Recombinant, Cell-Surface and Plasma Tissue Factor.

Abstract

Although it is well established that membrane-anchored cell-surface tissue factor (TF) initiates coagulation when blood contacts damaged tissue, several investigators have reported the existence of a circulating form of TF or TF-like antigen in blood. One report describes the molecular nature of circulating plasma TF as lacking the transmembrane domain, however, the TF activity of this material is a matter of debate. Therefore detection, quantitation and characterization of plasma TF steps towards defining the functional role of circulating TF and its potential contribution to thrombosis and hemostasis. We have developed highly specific and sensitive enzyme-linked (ELISA) and fluorescence-based immunoassays by which all known forms of TF including recombinant (r) truncated (1–218 and 1–242) and full-length (1–263), natural placenta, monocyte in situ as well as plasma TF were detected and quantitated. Immunoblotting confirmed the crossreactivity of anti-TF mAbs with TF from various sources and also revealed that there are significant differences between rTF and TF isolated from natural sources. This could suggest differences in procoagulant activity of rTF and natural TF that has to be investigated. The enzymatic immunoassay of TF was suitable for quantitative analyses of rTF and TF from commercial sources and placenta TF with detection level in the 80 pM range. Since the concentrations of TF on cell surface and in plasma are significantly lower than that found in commercial products, we integrated the double mAb immunoassay into Luminex Multi-Analyte Platform (LMAP) technology and developed a fluorescence-based immunoassay. This new assay was 400-fold more sensitive than ELISA and concentrations as low as 0.2 pM TF could be detected reliably. In addition to higher sensitivity, another advantage of fluorescence-based immunoassay of TF over the existing assays is the ability to determine specific and non-specific binding simultaneously in a single reaction well and such approach enhances assay specificity and reliability. TF on the surface of unstimulated and Lipopolysaccharide-stimulated immortalized human monocytic cells (TIB-202 cell line, ATCC) was quantitated. Unstimulated monocytes displayed between 55-110 TF molecules/cell (3–6 pg TF or 0.1–0.2 pM for 106cells/ml) while stimulated monocytes expressed 8–13 fold more TF as compared to unstimulated cells (730–840 TF molecules/cell, 40–46 pg, 1.2–1.4 pM for 106cells/ml). We also report here that the concentration of TF in plasma of "healthy" individuals (n=92) is in the range of 0.5±1.04 pM (16.2±31.2 pg/ml) and this concentration is substantially lower than that reported by other groups. There are significant variations in the antigen concentration among individuals; in 30% there was no detectable TF antigen (<0.2 pM). Assays of TF set the platform for further analyses of the concentrations of plasma TF in order to establish ranges and variations that occur during specific treatment regimens or due to the presence of certain pathological conditions.