Demystifying Tissue Factor.

Abstract

Tissue factor (TF) is an integral membrane protein, which is the key initiator of blood coagulation in vivo. Due to the limited availability of natural TF, recombinant proteins of various lengths and origins have been extensively used in research and clinical laboratories worldwide. Experimental results acquired with recombinant TF proteins are frequently used for the understanding of the coagulation processes occurring in vivo, although there is a lack of data confirming the structural and functional identity of natural TF proteins from various sources and recombinant ones. In the current study, human TF from cultured monocytes and purified from placenta were compared with three different species of recombinant TF: 1–218 (extracellular domain only), 1–242 (lacking cytoplasmic domain) and 1–263 (full-length). Anti-TF mAbs gave 93–98% inhibition of TF activity for all TF species tested, in both natural and relipidated preparations. It was established that purified placental TF has a higher affinity for factor VIIa (Kd 0.13 nM) than recombinant counterparts 1–242 and 1–263 (Kd 0.50–0.80 nM). Similarly, placental TF is more efficient in factor X activation by the extrinsic Xase than recombinant TF 1–242 (the second order rate constants are 3.0x107 and 0.7x107 M−1s−1, respectively). We explored the use of these TF species as well as monocyte TF (purified/relipidated and present on LPS-stimulated monocytes) for the initiation of thrombin generation in two in vitro models of blood coagulation. At equimolar concentrations (5 pM; determined by immunoassay), when evaluated in synthetic plasma reconstituted with 2x108/ml platelets, recombinant TF 1-263 provided an initiation phase of ~4 min. Placental TF and relipidated monocyte TF had similar profiles of thrombin generation with an initiation phase of ~3 min. In contrast, 0.5 pM TF on LPS-stimulated monocytes gave an initiation phase of ~1 min. Even at 0.05 pM concentration, monocyte TF was as active as any relipidated protein at 5.0 pM. A similar pattern of relative TF activity was observed in whole blood and plasma PT clotting assays. TF on stimulated monocytes gave the highest activity, exceeding that of any relipidated protein by 100–200-fold. Recombinant TF 1–242 was more active than recombinant TF 1–263 and placental TF in the PT assay but less active in synthetic plasma and whole blood. The lowest overall activity was observed for relipidated monocyte TF. Our data suggest that TF proteins from different sources are different with respect to their functional properties.