STUDIES OF THE MECHANISM FOR ENHANCED CELL-SURFACE FACTOR VIIA TISSUEFACTOR ACTIVATION OF FACTOR-X ON FIBROBLAST MONOLAYERS AFTER THEIR EXPOSURE TO N-ETHYLMALEIMIDE

Fibroblast monolayers constitutively expressing surface membrane tissu e factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 mi n to inhibit aminophospholipid translocase activity without inducing g eneral cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the bin ding of I-125-annexin V and by the ability of the monolayers to suppor t the generation of prothrombinase. Specific binding of I-125-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to (125)l-rVIIa, but this differe nce progressively diminished with longer exposure times. A brief expos ure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold e nhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fol d difference persisted despite increasing time of exposure to rVIIa. A dding prothrombin fragment 1 failed to diminish the enhanced VIla/TF a ctivation of factor X of NEM-treated monolayers. Moreover, adding anne xin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the e nhanced VIIa/TF activation of factor X. These data provide new evidenc e for a possible mechanism by which availability of anionic phospholip id in the outer layer of the cell membrane limits formation of functio nal VIIa/TF complexes on cell surfaces.