55th Annual Meeting Society of Thrombosis and Haemostasis Research – [MP-TF]

Aim: Measurement of Tissue Factor (TF) in plasma is of growing interest in various pathological states (tumor growth and metastasis, atherosclerosis, inflammation). TF can be in 3 different forms: anchored on cell membrane (monocytes, endothelial cells), in a cryptic or activated state; on microparticles´surface (MP-TF); as an alternatively spliced form (asTF), soluble in plasma. We have developed various assays for these different TF forms : 2 ELISA assays for the measurement of full length TF:Ag (FL-TF) and “Total” TF:Ag (FL-TF and asTF); a bio-immunoassay for quantitating MP-TF activity.

Methods: FL-TF ELISA uses a capture murine MoAb specific for an extracellular TF epitope, unexposed on asTF and a second murine MoAb reactive with an extra cytoplasmic TF epitope. Tested specimen are in a diluent which enhances the assay reactivity and suppress non specific interactions (heterophilic antibodies). Total TF ELISA is a similar assay but the capture MoAb targets an extracytoplasmic epitope available on all TF forms. Assay ranges are from 0 to 500 pg/ml. This same MoAb, which does not inhibit TF activity, is used for capture in the MP-TF assay, which is revealed with Factor VIIa, Factor X, Ca++ and the Factor Xa specific substrate CS 11(65). Calibration uses relipidated TF anchored to synthetic phospholipids (0 to 5 pg/ml of TF and 0.1 nM PS/1 pg TF) .

Results: In normal plasmas: FL-TF is below the detection limit (< 10 pg/ml); Total TF has a mean value of 50 pg/ml . Truncated recombinant human TF (1-219) has 1 % reactivity in the FL-TF (1-263) Elisa and 60 % in the Total-TF. Normals were < 0.2 pg/ml (TF equivalent) with the MP-TF assay but some patients were tested > 5 pg/ml. Incubation of human blood with LPS increased FL-TF, asTF and MP-TF.