Localization of tissue factor in actin-filament-rich membrane areas of epithelial cells

Tissue factor (TF), the cellular receptor and cofactor for clotting factor VII/VIIa (FVII/VIIa), is known mainly as the initiator of the coagulation p rotease cascade. Recently, it was shown that inactivation of the murine TF gene (TF-/-) results in embryonic lethality which is most likely due to som e failure of vascular integrity. On the other hand, gene disruption in mice of coagulation proteins like FVII, prothrombin, and fibrinogen results in phenotypes of embryonic development that contrast with that of TF-/-, sugge sting a role for TF beyond fibrin formation in embryogenesis. In addition, there is a growing body of evidence that cellular TF may be involved in non hemostatic functions. To determine the microtopography of membrane TF with regard to the cytoskeleton organization, we examined the expression pattern s of TF and cytoskeletal proteins in various cell lines by means of double immunofluorescence and electron microscopy (EM). In spreading cells, a gran ular membrane TF expression of the cell cortex and a pronounced granular TF staining of microspikes, lamellipodes, and ruffled membrane areas were obs erved. Especially. actin and alpha-actinin were in close proximity to TF in these regions. Colocalization of TF and nonmuscle filamin (ABP-280) at the leading edge of spreading cells indicated an association of TF with the ac tin filament system, too. Using scanning EM we found gold-labeled TF at lon g processes and actin-filament-containing microspikes of neighboring cells in both branching and contact sites. By the means of immunogold EM we obser ved that TF is localized at the cell surface in a spotty pattern, at the ba se and at the top of budding processes. The observed staining pattern point s to a connection of TF with elements of the cytoskeleton in these highly d ynamic membrane regions, a fact which is underlined by the recently describ ed molecular interaction of TF's cytoplasmic domain with ABP-280. In cells undergoing cytokinesis, we detected also strong TF expression in dynamic me mbrane areas and protrusions of the midbodies, indicating an accumulation o f TF in actin-rich membrane areas with high contractile activity. In additi on, we were able to demonstrate that immobilized ligands for TF, both catal ytically active and inactive FVIIa or anti-TF mAbs, accelerated adhesion an d spreading of TF-expressing cancer cells. Thus, our findings support the c ontention that ligation of cellular TF may be involved in morphogenic proce sses such as adhesion and spreading by an association to cytoskeletal struc tures. On the other hand, incubation of these cells with proteolytically ac tive FVIIa but not with covalently inactivated FVIIa (DEGR-FVIIa) or anti-T F mAbs in solution resulted in increased motility of these cells, indicatin g that not only ligation of TF but also the proteolytic activity of TF-FVII a complex is involved in cell migration. (C) 1999 Academic Press.