Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer

Abstract

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood 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 extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.

Modular chemical mechanism predicts spatiotemporal dynamics of initiation in the complex network of hemostasis

“…We used patterned supported phospholipid bilayers (25,26) to present patches of the clotting stimulus, a lipid surface containing phosphatidylserine with reconstituted human tissue factor (TF) (27, 28). TF was incorporated into bilayers as previously described (27,28). TF is an integral membrane protein that is exposed at sites of vascular damage and atherosclerotic plaque rupture (15,16).…”

Role of the Activation Peptide Domain in Human Factor X Activation by the Extrinsic Xase Complex

“…The recovery of TF in the reconstituted mixture was assessed using known amounts of 125 I-labeled TF added as a tracer to the initial reaction mixture. The final effective concentration of TF following dialysis was considered to be one-half the total concentration due to the two possible orientations of the protein (33)(34)(35) Most experiments were also repeated using TF/PCPS preparations that were reconstituted by preincubation of TF with small unilamellar PCPS vesicles in the presence of 0.05% (v/v) Tween 20 as described previously (28,32). In these experiments, the effective TF concentration was considered equivalent to the total concentration (34).…”

Supported Bilayer Lipid Membranes as Ion and Molecular Probes

“…Thus, it is not surprising that today BLMs and liposomes are the most used model systems for biomembrane studies. 37 Electrically speaking, both BLMs and biomembranes possess very high capacitance (~1 µF cm -2 ) and dielectric breakdown strength (>200000 V/cm). 7,37,38 By virtue of its molecular organization, a BLM possesses a very high electrical resistance which is important in minimizing the high background noise associated with an ion-conducting aqueous environment.…”

“…37 Electrically speaking, both BLMs and biomembranes possess very high capacitance (~1 µF cm -2 ) and dielectric breakdown strength (>200000 V/cm). 7,37,38 By virtue of its molecular organization, a BLM possesses a very high electrical resistance which is important in minimizing the high background noise associated with an ion-conducting aqueous environment. Since an ion or a charged species is usually not able to penetrate a metallic surface, a redox reaction must take place in order to observe a current.…”

“…In a sense, the lipid bilayer as existing in BLMs and liposomes may be considered as a solvent for a number of hydrophobic materials. 37,38 In this connection it should be mentioned that ion-channels and electrical excitability are closely related phenomena that have been extensively investigated. 6,[33][34][35] For example, Kihara and colleagues 23 have reported ion transport through BLMs and liquid membranes.…”