ANNEXIN-V AS A PROBE OF THE CONTRIBUTION OF ANIONIC PHOSPHOLIPIDS TO THE PROCOAGULANT ACTIVITY OF TUMOR-CELL SURFACES

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of I-125-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (K(d) 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (K(d) 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of I-125-annexin V binding sites remained constant. Annexin V may be useful both as a probe to determine differences in the anionic phospholipid expression and possible differences in the location and exposure of PS between cell lines (observed as the capacity of PS to act as a cofactor of blood coagulation reactions).