Use of an Oriented Transmembrane Protein to Probe the Assembly of a Supported Phospholipid Bilayer

Planar-supported phopholipid bilayers formed by te adsorpton of vesicles are increasingly used in the investigation of lipK-ependent reactis. We have studied the way in which these bilayers are forned with phopholipid vesicles coaining the btranembrane protein Tssue Factor (TF). TF complexed with te senne protease, factor Vlla, is the primary initiator of bklod coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the exracellular domain of TF is located on the inner leaflet These vesicles show no cofactor activity for factor Vila as a result of the inability of the extacellular domain of TF to bind Vila. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorpion of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholpid bilayer. The microcapillaries were perftsed with a solution of factors Vlla and X, and the effluent was monitored for factor Xa production, a sensifive measure of the activity of the TF-Vlla complex. For coatings produced with the digested vesicles, minimal TF-Vlla acfivity was observed, showing that the supported bilayer preserves the oientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer