The Local Phospholipid Environment Modulates the Activation of Blood Clotting

Abstract

Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa⅐tissue factor complex on stable bilayers containing 67 ؎ 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa⅐tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa⅐tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.In both normal hemostasis and many life-threatening thrombotic diseases, blood clotting is triggered when tissue factor (TF), 5 an integral membrane protein, binds the plasma serine protease factor VIIa (fVIIa) (1). The resulting two-subunit membrane-bound enzyme TF⅐VIIa activates the plasma zymogen factors IX (fIX) and X (fX) by limited proteolysis. Erwin Chargaff (of nucleic acids fame) demonstrated in the 1940s that TF procoagulant activity requires it to be associated with phospholipids, and research over the ensuing decades has demonstrated that TF is a membrane-spanning protein that must be incorporated into bilayers containing anionic phospholipids for optimal activity (reviewed by Bach (2)). Despite this extensive history, we still have an indistinct picture of how anionic phospholipids contribute so profoundly to the enzymatic activity of membrane-bound protease complexes involved in blood clotting (3).Membranes composed of mixed phospholipids (and other lipids) can form membrane microdomains with locally different surface properties. A noted example is the formation of cholesterol-and sphingolipid-rich lipid rafts and caveolae on cell surfaces (4, 5). Experiments using giant unilamellar vesicles have shown that even liposomes containing simple binary mixtures of neutral and anionic phospholipids spontaneously form anionic phospholipid-rich me...

Thematic Review Series: Glycerolipids. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

“…The induction of PS exposure on cell surfaces is not restricted to apoptotic cells. For example, the exposure of PS on the surface of activated platelets initiates the bloodclotting cascade (17,(26)(27)(28), as the proteolytic activity of the factor VIIa-tissue factor complex requires very high local concentrations of PS (29). Moreover, PS becomes exposed on the outside of sperm during their maturation (30,31).…”

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

“…In the last decade, the lipid bilayer nanodisc system developed by the Sligar laboratory has become an important tool to characterize membrane proteins (19 -20). Many proteins have been reconstituted in nanodiscs, including human tissue factor (21), bacteriorhodopsin (22)(23), G-protein-coupled receptors (24 -26), chemoreceptor Tar (27)(28), translocon SecYEG (29 -31), and F 0 F 1 -ATPase (32). ABC transporters, such as the maltose system MalEFGK (33-34), the lipid A exporter MsbA (35), and P-glycoprotein (19,36), have also been reconstituted in nanodiscs, albeit generally with a low coupling efficiency.…”

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