A fraction of total cellular tissue factor procoagulant activity remains masked or "encrypted" in intact cells. Decryption of this activity partly involves the extracellular exposure of anionic phospholipids such as phosphatidylserine. Because of the potential association of tissue factor and phospholipid scramblase activity with lipid rafts, we have explored the role of lipid rafts in regulating factor VIIa/tissue factor activity. In HEK293 cells, tissue factor antigen was not stably associated with lipid rafts, yet disruption of rafts with methyl-␤-cyclodextrin resulted in a 3-fold stimulation of tissue factor procoagulant activity. Treatment with methyl-␤-cyclodextrin was not associated with cytotoxicity and did not result in the exposure of additional tissue factor antigen. Factor VIIa/tissue factor activity decrypted with methyl-␤-cyclodextrin was quantitatively similar to that obtained by using lytic concentrations of octyl glucoside but more sensitive to inhibition by cell surface tissue factor pathway inhibitor and the phospholipid binding protein, annexin V. Partial decryption of tissue factor was achieved with methyl-␤-cyclodextrin prior to complete disruption of lipid rafts, suggesting the role of an enzyme localized to lipid rafts in the transbilayer transport of phosphatidylserine. We conclude that lipid rafts are required for the maintenance of cellular tissue factor in an encrypted state. IntroductionTissue factor (TF) is a 45-kDa glycoprotein anchored to the plasma membrane by way of a single transmembrane spanning ␣-helix. 1,2 The extrinsic coagulation cascade is initiated when TF binds and activates circulating factor VIIa (fVIIa) to form a protease complex (fVIIa/TF) that cleaves and activates the zymogens, factor IX and factor X (fX), generating active proteases, factor IXa and factor Xa (fXa), respectively. TF is expressed primarily on subendothelial tissues, but TF expression may be induced on endothelial cells by inflammatory mediators such as tumor necrosis factor ␣ (TNF-␣). 3,4 TF may also be found circulating on monocytes, 5 on microparticles derived from various cellular sources, [6][7][8] and in a soluble form arising from alternate processing of the TF gene. 9 Although subendothelial TF may be responsible for initiating fibrin formation at sites of vascular injury, bloodborne TF may be an important contributor to propagation of the developing thrombus. 8,10 TF procoagulant activity is regulated by tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor that binds both fVIIa and Xa 11,12 and by availability of anionic phospholipid. Phosphatidylserine (PS), for example, increases the k cat /k m of fVIIa/TF by 3 orders of magnitude. [13][14][15] Cell surface TF is fully accessible to fVIIa and anti-TF antibodies, but a fraction of fVIIa/TF activity is suppressed or encrypted. 15,16 Decryption of TF has been achieved by treating TF-expressing cells with hydrogen peroxide, 17 calcium ionophore, 15,18 or lytic concentrations of detergents such as Triton X-100 or octyl glu...

Objective-3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) exhibit antithrombotic properties that are independent of reductions in circulating LDL cholesterol. We hypothesized that these antithrombotic properties are mediated by membrane alterations secondary to disrupted lipid metabolism. Methods and Results-EA.hy926 cells were incubated in the presence of 1 mol/L atorvastatin supplemented with fetal bovine serum or lipid-depleted serum mixtures. Lipid restriction alone had no effect on cell lipid composition but when atorvastatin was included, phosphatidylserine, sphingomyelin, and cholesterol were reduced by 50% while ceramide content decreased by 70%. These changes in lipid composition did not alter the association of decay accelerating factor or tissue factor with lipid rafts. Atorvastatin in combination with lipid restriction reduced factor VIIa/tissue factor activity by as much as 75% but did not alter tissue factor expression. Prothrombinase activity was reduced to an extent similar to factor VIIa/tissue factor. Mevalonic acid but not LDL reversed the observed changes in lipid content and prothrombinase activity induced by atorvastatin. These findings were confirmed in primary cells. Conclusions-Inhibition

SummaryTissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphatidylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/ tissue factor by cell-surface associated TFPI.

HMGCoA reductase inhibitors (statins) exhibit poorly understood antithrombotic properties that are independent of reductions in circulating LDL cholesterol. The activities of the vitamin K-dependent proteolytic complexes such as factor VIIa/tissue factor (fVIIa/TF) and the prothrombinase complex are sensitive to the membrane environment. The plasma membrane is marked by lateral segregation of cholesterol-rich lipid rafts and by transbilayer segregation of phosphatidylserine to the internal membrane leaflet. We hypothesized that the antithrombotic properties of statins are mediated by their impact on cholesterol and phospholipid metabolism. We utilized the immortal cell line EA.hy926 to clarify the mechanism whereby statins alter thrombosis. The endothelial characteristics of these cells include expression of tissue factor pathway inhibitor, nitric oxide synthase, and von Willebrand factor. EA.hy926 cells were incubated in the presence of physiologic concentrations of atorvastatin (1 μM) supplemented with 10% fetal bovine serum (FBS) or 10% of a serum mixture consisting of FBS and lipid-depleted FBS (1:3 v/v) to restrict cellular access to exogenous sterol. Cells cultured in the presence of 1 μM atorvastatin and lipid depleted serum grew at identical rates over 72 hours to cells that were incubated in the absence of atorvastatin. Following 72 hours of treatment with atorvastatin and lipid restriction, cells were harvested and membrane lipids examined by tandem mass spectrometry. Lipid restriction alone had no effect on cell lipid composition but when atorvastatin was included, phosphatidylserine, sphingomyelin, and cholesterol were reduced by 50% while ceramide content decreased by 70% (normalized to lipid phosphate). The changes in lipid compostion did not alter the association of CD59 with detergent-resistant buoyant membrane domains indicating that lipid rafts remained intact. The functional impact of atorvastatin treatment on EA.hy926 cells was assessed by measuring fVIIa/TF activity. Basal and decrypted fVIIa/TF activities were reduced by 60% and 75%, respectively, compared to cells treated with no atorvastatin, atorvastatin alone, or lipid restriction alone. Expression of TF was not altered by atorvastatin and lipid restriction as assessed by both ELISA and western blot, suggesting that changes in fVIIa/TF were mediated by reduced exposure of phosphatidylserine. To confirm the role of phosphatidylserine in diminished fVIIa/TF activity, the prothrombinase complex was reconstituted in the presence of cells treated identically with atorvastatin and lipid restriction. Prothrombinase activity was reduced by 50% compared to control cells, an extent similar to the observed decrease in basal fVIIa/TF. The inclusion of 200 μM mevalonic acid to the culture media partially reversed the observed changes in lipid content and prothrombinase activity induced by atorvastation. We conclude that atorvastatin combined with a reduction in exogenous sterol limits exposure of phosphatidylserine at the cell surface and restricts the activity of proteolytic enzyme complexes that propagate the coagulation cascade.