The influence exerted by a restricted phospholipid microenvironment on theexpression of tissue factor activity at the cell plasma membrane surface

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 ce lls, showing a reduced ability to externalize PhtdSer, were transfected wit h human TF cDNA. PhtdSer exposure and TF activity were examined in transfec ted cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externaliz ation potency. TF expression was evidenced by flow cytometry and its activi ty measured using functional assays. PhtdSer exposure was monitored by enzy matic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noti ceable TF expression level, clone DC9 presented only a weak TF activity eve n after ionophore stimulation. The apparent K-m, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for T HP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 tha n THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts o f available PhtdSer abolished this difference. The DC9 clone appears suitab le for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Su ch cells should enable further assessment of the role of TF as a receptor c oupled to intracellular signaling pathways and its fate during apoptotic ce ll death.