Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions

Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.     

Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation

We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.     

Cooperative interaction between factor VII and cell surface-expressed tissue factor

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.     

Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages

Activation of coagulation factor X via the intrinsic pathway requires the assembly of factors IXa and VIII on lipid membranes. It is known that the platelet expresses membrane sites for assembly of factors IXa/VIII and promotes efficient factor X activation. We now show that human blood monocytes, but not lymphocytes or polymorphonuclear leukocytes, also express appropriate sites for factors IXa/VIII assembly. The maximal rate of factor X activation by factors IXa (0.75 nM) and VIII (1 unit/ml) assembled on monocytes is similar to the maximal rate on platelets. This rate, adjusted per micromole of lipid phosphorus, is 1636 +/- 358 nM factor Xa/min on monocyte, and 1569 +/- 54 nM factor Xa/min on platelets. At physiologic concentrations of factors X and VIII, the activation rate increases with factor IXa concentration asymptotically approaching a maximum. Half-maximal rate is achieved with 1.0 +/- 0.16 nM factor IXa. Monocytes and macrophages, but not platelets, can express membrane tissue factor and thus promote simultaneous assembly of two distinct factor X-activating protease complexes. In these studies, blood monocytes and alveolar macrophages are used as membrane sources in kinetic experiments comparing factor X activation by intrinsic (factor IXa/VIII) versus extrinsic (factor VII/tissue factor) protease complexes. At plasma concentration of factors VIII and VII, apparent Km on the monocyte is 14.6 +/- 1.4 nM for intrinsic and 117.0 +/- 10.1 nM for extrinsic activation. The apparent Km on alveolar macrophages is 12.1 +/- 1.9 and 90.6 +/- 10.2 nM for intrinsic and extrinsic activation, respectively. Maximal rates on monocytes at saturating concentration of factors IXa, VIII, and VII are 48.0 +/- 11.2 nM factor Xa/min, for intrinsic activation, and 16.5 +/- 5.5 nM factor Xa/min, for extrinsic activation. These data show that the monocyte/macrophage is the only blood-derived cell type with membrane sites for both intrinsic and extrinsic pathway assembly. We have exploited this characteristic of the monocyte/macrophage membrane to demonstrate that factor X activation by the intrinsic pathway protease is more efficient than activation via the extrinsic pathway protease complex.     

The interaction of human factor VIIa with tissue factor.

The interaction of factor VIIa with tissue factor (TF) results in an increase in the catalytic efficiency for the hydrolysis of several synthetic peptidyl p-nitroanilide substrates by factor VIIa. The binding of human recombinant factor VIIa to recombinant human TF incorporated into vesicles containing phosphatidylcholine (TF/PC) or phosphatidylcholine/phosphatidylserine (TF/PCPS) was studied using the increased rate of H-D-phenylalanyl L-pipecoyl L-arginine p-nitroanilide (S2238) hydrolysis as a signal for the interaction. The saturable dependence of rate on increasing concentrations of factor VIIa or TF/PCPS yielded no obvious evidence for cooperativity and could be analyzed according to the interaction of factor VIIa with independent noninteracting sites (Kd = 259 +/- 60 pM, n = 1.05 +/- 0.12 mol of factor VIIa/mol of TF at saturation). Identical titration curves and equilibrium parameters were derived from titrations using TF/PC or TF in the absence of phospholipids, indicating that possible protein-membrane interactions do not further stabilize the extrinsic Xase complex. The dissociation constant for the interaction of factor VIIa with TF/PCPS inferred from measurements of factor X activation (Kd = 197 +/- 38 pM) was comparable with the values obtained from measurements of S2238 hydrolysis. In contrast to the membrane-independent nature of the enzyme-cofactor interaction, the rate of factor X activation was reduced by approximately 50-fold when the enzyme complex was assembled using solution-phase TF. Collectively, the result indicate that the membrane dependence of extrinsic Xase function primarily results from an influence of the membrane surface on factor X utilization.     

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.     

Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Binding of alpha-2-antiplasmin with fibrinogen/fibrin and their fragments independent of factor XIII

Possible interaction of alpha-2-antiplasmin with fibrinogen, fibrin and their fragments independent of factor XIII as well as the inhibitor effect on the Glu-plasminogen activation by tissue activator were studied. It was shown that alpha-2-antiplasmin is adsorbed on desAA- and desAABBfibrin films (Kd 69.0 +/- 1.0 nM 68.6 +/- 5.3 nM, respectively). Glu-Plasminogen has no effect on the inhibitor binding with desAABBfibrin. Alpha-2-antiplasmin shows strong affinity for fibrin D-dimer (Kd 65.0 +/- 4.0 nM) and D-fragment of fibrinogen (Kd 119.0 +/- 21.0 nM), but it does not interact with E-fragment. The inhibitor inside the fibrin clot decreases 10 times the activation rate of Glu-plasminogen by the tissue activator both is the presence and without factor XIII at physiological ratio of Glu-plasminogen, tissue activator, fibrin and alpha-2-antiplasmin. Thus we have shown that fibrinogen/fibrin binds alpha-2-antiplasmin independent of the factor XIII. Binding sites of the inhibitor are localized in D-fragment of fibrinogen and/or fibrin D-dimer. Alpha-2-antiplasmin inhibits the Glu-plasminogen activation by tissue activator on fibrin.     

Structural requirements for expression of factor Va activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions

Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.     

Tissue factor pathway inhibitor binds to platelet thrombospondin-1

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that down-regulates tissue factor-initiated blood coagulation. The most biologically active pool of TFPI is associated with the vascular endothelium, however, the biochemical mechanisms responsible for its cellular binding are not entirely defined. Proposed cellular binding sites for TFPI include nonspecific association with cell surface glycosaminoglycans and binding to glycosyl phosphatidylinositol-anchored proteins. Here, we report that TFPI binds specifically and saturably to thrombospondin-1 (TSP-1) purified from platelet alpha-granules with an apparent K(D) of approximately 7.5 nm. Binding is inhibited by polyclonal antibodies against TFPI and partially inhibited by the B-7 monoclonal anti-TSP-1 antibody. TFPI bound to immobilized TSP-1 remains an active proteinase inhibitor. Additionally, in solution phase assays measuring TFPI inhibition of factor VIIa/tissue factor catalytic activity, the rate of factor Xa generation was decreased 55% in the presence of TSP-1 compared with TFPI alone. Binding experiments done in the presence of heparin and with altered forms of TFPI suggest that the basic C-terminal region of TFPI is required for TSP-1 binding. The data provide a mechanism for the recruitment and localization of TFPI to extravascular surfaces within a bleeding wound, where it can efficiently down-regulate the procoagulant activity of tissue factor and allow subsequent aspects of platelet-mediated healing to proceed.     

Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

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 TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa 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. TFPI. Xa-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 TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-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.     

The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor.

The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51-88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the beta-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.     

The evaluation of complex-dependent alterations in human factor VIIa.

Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.     

Tissue factor is not involved in the mitogenic activity of factor VIIa

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.