Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-␤-cyclodextrin-reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding. IntroductionCholesterol is a lipid precursor for steroid hormones and bile salts and is present in cell membranes and circulation. Cholesterol in the membrane regulates flexibility and mechanical stability of the membrane. 1 Further, cholesterol plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Lipid rafts are shown to contribute to the regulation of various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors, and signaling pathways. [2][3][4][5] Tissue factor (TF) is the cellular receptor for clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces triggers the coagulation cascade. 6 Studies suggest that exposure of TF to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture, resulting in acute coronary events and myocardial infarction. [7][8][9][10] Since cholesterol/ oxidatively modified low-density lipoprotein (LDL) present in atherosclerotic plaques is thought to play an important role in the atherogenesis through its biologic effects, including TF expression, many earlier studies were focused on investigating the effect of cholesterol on TF expression. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to suppress plasma LDL cholesterol levels in patients with primary hypercholesterinemia, were shown to inhibit TF expression in both in vitro and in vivo. 11,12 Consistent with this, dietary lipid lowering was found to reduce TF expression in rabbit atheroma. 13 However, in vitro studies on...

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF⅐FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF⅐FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI⅐Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI⅐Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI⅐Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.

Activated protein C (APC) is a serine protease, an effector enzyme of the natural anticoagulant pathway. APC is approved for treatment of severe sepsis characterized by the increased concentrations of H(2)O(2) and hypochlorite. We found that treatment of APC with these oxidants markedly inhibits the cleavage of the APC-specific chromogenic substrate, suggesting that oxidants can induce changes in the structure of the active site of APC. Resistance of oxidant-treated APC to chemical digestion with cyanogen bromide (CNBr) implies that methionine oxidation can at least in part be responsible for inhibition of APC. Since methionine residues, the main targets of oxidants in APC, are not included in the active site, we hypothesize that oxidation induces allosteric changes in the architecture of the catalytic triad of APC. Using molecular dynamics (MD) simulations we found that methionine oxidation alters the distance between cSer195Ogamma and cHis57Nepsilon2 atoms placing them in positions unfavorable for the catalysis. At the same time, neither distances between Calpha atoms of the catalytic triad cAsp102-cHis57-cSer195, nor the overall structure of APC changed significantly after oxidation of the methionine residues. Disruption of the H-bond between Ndelta1 of cHis57 and carboxyl group of cAsp102, which can take place during the hypochlorite-induced modification of cHis57, dramatically changed the architecture of the catalytic triad in oxidized APC. This mechanism could contribute to APC inactivation by hypochlorite concurrently with methionine oxidation. These are novel findings, which describe potentially pathophysiologically relevant changes in the functional stability of APC exposed to the oxidative stress.

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na + to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active-site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na + -binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na + by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K D to partially restore its activity. Both mutants exhibited ∼2-3-fold higher K D for interaction with Na + and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both the Na + -binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na + , but TM has no effect on the conformation of this residue.Thrombin is an allosteric trypsin-like serine protease in plasma that is responsible for clotting fibrinogen and up-regulating the clotting cascade by activating platelets, cofactors V and VIII and factor XIII during injury to blood vessels (1-5). Thrombin also down-regulates its own production by a negative feed back loop mechanism when it binds to endothelial cell surface glycoprotein thrombomodulin (TM) 1 to activate protein C to activated protein C (APC), thereby initiating the anticoagulant pathway (6-9). APC inhibits thrombin generation by proteolytically degrading the activated forms of factors V and VIII, which are essential cofactors for the prothrombinase and intrinsic Xase complexes, respectively (10). The proteolytic activity of thrombin is primarily regulated by the plasma serpin antithrombin (AT) which functions as a pseudo-substrate to trap the protease in the form of an inactive and irreversible acylated complex incapable of interacting with any true substrate (11). Structural and mutagenesis data have indicated that several different ligands bind to distinct loops removed from the active site (exosite) to allosterically modulate the substrate specificity of thrombin, thereby rendering the protease capable of selecting its distinct substrates and cofactors in the opposite procoagulant and anticoagulant pathways (5,12,13). Thus, it has been...

Factor VIIa binding to tissue factor on cell surfaces not only triggers the coagulation cascade but also induces various intracellular responses that may contribute to many pathophysiological processes. Active siteinhibited factor VIIa, similar to factor VIIa, binds to tissue factor on cell surfaces and subsequently gets internalized and degraded. At present, it is unknown whether factor VIIa and active site-inhibited factor VIIa undergo a similar intracellular processing. The data presented herein show that although a fraction of both the internalized factor VIIa and active site-inhibited factor VIIa recycle back to the cell surface, the amount of active site-inhibited factor VIIa recycled back to the cell surface was substantially higher than that of factor VIIa. Furthermore, internalized factor VIIa and not active site-inhibited factor VIIa associates with nuclear fractions. Factor VIIa associated with the nuclear fraction was intact and functionally active. In contrast to factor VIIa, tissue factor is not found in the nuclear fraction. Additional studies show that the internalized factor VIIa specifically associates with cytoskeletal proteins, actin, and tubulin. In summary, the present data reveal that despite the common pathway of tissue factor-mediated processing, considerable differences exist in the trafficking of factor VIIa and active site-inhibited factor VIIa in fibroblasts.