Activation of a Proteolytic System by a Membrane Lipoprotein: Mechanism of Action of Tissue Factor

Abstract

One pathway of blood coagulation, the extrinsic system, is initiated by a specific interaction between tissue factor, which is a membrane lipoprotein, and factor VII, one of the plasma coagulation factors. Factor VII was prepared from bovine plasma by adsorption onto and elution from BaSO4. The eluate was chromatographed on DEAE-Sephadex and purified by preparative disc-gel electrophoresis. Factor VII complexed with purified bovine-brain tissue factor and, when eluted from the complex, factor VII had a greater mobility in acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, i.e., it had a reduced molecular weight. Factor VII was also cleaved in the presence of orthophenanthroline, an inhibitor of the peptidase activity of tissue factor. Prior treatment of factor VII with diisopropylphosphorofluoridate, however, completely blocked its cleavage and the development of coagulant activity, although factor VII treated with diisopropylphosphorofluoridates complexed equally well as the native protein with tissue factor. Factor VII in whole bovine plasma was also inhibited by the drug. Factor VII labeled with [32P]diisopropylpliosphorofluoridate and radioautographed after electrophoresis in gels showed two major components, only one of which was labeled. We conclude that tissue factor initiates blood coagulation by facilitating a proteolvtic attack within the factor VII "complex." The most likely mechanism is proteolysis of one form of factor VII by the diisopropylphosphorofluoridatesensitive enzyme, although additional intramolecular proteolysis may be involved.The addition of small amounts of various tissues to blood markedly enhances the rate at which it coagulates. This acceleration is due to an interaction of tissue factor, a specific lipoprotein present in many tissues, and factor VII, a plasma coagulation factor. The protein component of tissue factor was previously purified by conventional techniques after solubilization in deoxycholate (1). In this communication we report the purification of bovine factor VII and preliminary observations on its reaction with tissue factor.We show that the factor VII preparation contains two major molecular species, one of which is sensitive to diisopropylphosphorofluoridate (DFP). During its reaction with tissue factor, factor VII is converted to a lower molecular weight species, i.e., it is hydrolyzed. The data indicate that the protein that catalyzes this proteolysis is a form of factor VII. Further, we show that the peptidase activity associated with tissue factor (2) is not required for its coagulant action. MATERIALS AND METHODSSodium dodecyl sulfate (SDS) the supernatant was diluted with 500 ml of water and 500 ml of 15 mM Tris-HCl (pH 7.5). 7 g of dry DEAE-Sephadex A-50 was added, and the mixture was stirred for 20 min. The Sephadex was then collected on a Btichner funnel and washed with 400-ml quantities of 15 mMI Tris-HCl, 3 times; 100 mMI Tris HCl, 3 times; and finally 3 times with 0.14 MI NaCl in 100 mM Tris-HCl. The slurry was added to ...

Concanavalin A inhibits tissue factor coagulant activity.

“…While the complete complex appears to be necessary for coagulant activity (12,17), the factor X converting site appears to reside in the factor VII molecule (18,19). In the bovine system, diisopropylphosphorofluoridate (DFP), the seryl protease inhibitor, blocks the coagulant activity of the complex and is incorporated into the factor VII (12,18,19); there is no evidence that tissue factor hydrolyzes factor VII during complex formation (19). In native bovine plasma, factor VII incorporates DFP (diisopropylphosphoryl [DIP]-factor VII).…”

Lessons From Sudden Coronary Death

“…Advanced lesions contain smooth muscle cells and macrophages that express tissue factor, and this can be shown to activate the extrinsic pathway of coagulation. 74,75 However, to our knowledge, there have not been careful studies of when this critical initiating factor for the extrinsic pathway is first found during the progress of atherosclerotic lesion formation. Even less is known about the natural expression of tissue factor pathway inhibitor or the presence of annexin V, a cytoplasmic molecule released during cell death that binds phosphatidylserine, a critical cofactor for tissue factor.…”

Regulation of the Initiation of Coagulation by Factor VII

“…This finding suggests that factor VII zymogen itself possesses low activity -in the bovine case, approximately 0.8% that of the enzyme [15]. The existence of an unusu ally active catalytic site in factor VII zy mogen is also indicated by the following facts: (a) Factor VII is the only coagulation protein that is inactivated in plasma by diisopropylfluorophosphate (DFP), an irrevers ible inhibitor of serine enzymes [16], (b) The purified single-chain protein incorporates 3H-DFP stoichiometrically [17], (c) DFP in teracts with the zymogen only about 4 times more slowly than with the enzyme [15]; in contrast, trypsinogen reacts with DFP nearly 7,500 times more slowly than trypsin [18], (d) The Km of a synthetic substrate, benzyloxycarbonyl-arginine-p-nitrobenzyl ester is approximately 17-fold higher for factor VII than for factor VIIa, whereas the kcat is only 2-fold lower [19]. Taken together, the evi dence shows that factor VII is much more active than most known zymogens.…”