Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by phosphatidylserine.
The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation. Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside. The vesicles were characterized with respect to size and tissue factor content and orientation. Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration. In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation. This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles. With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 13.2 +/- 0.72 nM for factor VII and 4.54 +/- 1.37 nM for factor VIIa. Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme. Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules. Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively. With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased. In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme. An explicit two-site cooperative binding model is presented which fits these complex data. In this model, tissue factor is at least a dimer with two interacting enzyme binding sites.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005167:Factor VII / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D010718:Phosphatidylserines / Q000494:pharmacology*; D011485:Protein Binding; D013381:Subtilisins / Q000378:metabolism; D013925:Thromboplastin / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D008081:Liposomes; D010718:Phosphatidylserines; D014316:Tritium; D005167:Factor VII; D013925:Thromboplastin; D013381:Subtilisins