Presence Of Tissue Factor Research Articles

Coagulation activation in autoimmune bullous diseases

The main autoimmune blistering skin disorders are pemphigus vulgaris (PV) and bullous pemphigoid (BP). They differ in the inflammatory infiltrate, which is more intense in BP. Inflammation is known to activate coagulation in several disorders. Local and systemic activation of coagulation was evaluated in BP and PV. We studied 20 BP patients (10 active and 10 remittent), 23 PV patients (13 active and 10 remittent) and 10 healthy subjects. The coagulation markers prothrombin fragment F1+2 and D-dimer were measured by enzyme-immunoassays in plasma. The presence of tissue factor (TF), the main initiator of blood coagulation, was evaluated immunohistochemically in skin specimens from 10 patients with active PV, 10 patients with active BP and 10 controls. Plasma F1+2 and D-dimer levels were significantly high in active BP (P = 0.001), whereas in active PV the levels were normal. During remission, F1+2 and D-dimer plasma levels were normal in both BP and PV. TF immunoreactivity was found in active BP but neither in active PV nor in normal skin. TF reactivity scores were higher in active BP than in controls or active PV (P = 0.0001). No difference in TF scores was found between active PV and controls. BP is associated with coagulation activation, which is lacking in PV. This suggests that BP but not PV patients have an increased thrombotic risk. The observation that thrombotic complications occur more frequently in BP than in PV further supports this view.

Marqueurs des pathologies thrombotiques: Les microparticules procoagulantes

Thrombosis remains one of the leading causes of mortality and morbidity in developed countries. Relevant markers of the primary thrombotic risk however remain of limited accessibility, and clinicians are left with markers of essentially etiological nature. Fortunately, new entities, testifying to cellular activation or damage within the vascular compartment, have been recently described and are in the validation process. Microparticles (MP) are plasma membrane fragments released by stimulated or apoptotic cells. In the vascular compartment, they constitute a disseminated storage pool of bioactive effectors involved in inflammation, thrombosis, vascular tone, angiogenesis. Their biological characteristics are predetermined by the cytosolic and membraneous components hijacked from the activated cells. Their procoagulant properties are based on, (i) the accessibility of phosphatidylserine, a procoagulant aminophospholipid exposed after stimulation and necessary for the assembly of the blood clotting enzyme complexes, and (ii) the possible presence of tissue factor, the major initiator of the coagulation cascade. The incidence of MP in haemostatic processes has been demonstrated in physiology and pathology. They are now considered true pathogenic markers of the thrombotic risk.

Microparticules circulantes au cours des traumatismes graves et des sepsis : un élément du couplage inflammation–thrombose

Sepsis and trauma lead to a sustained activation of monocytes and endothelium. In the vascular compartment, stimulated cells release microparticles. Circulating MP provide an additionnal procoagulant phospholipid surface enabling the assembly of the clotting enzymes complexes and thrombin generation. Their procoagulant properties rely on the exposition of phosphatidylserine, made accessible after cell stimulation and on the possible presence of tissue factor, the main cellular initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor activity. At sites of endothelium injury, enhanced release or recruitment of procoagulant MP through P-selectin–PSGL-1 pathway could concentrate TF activity above a threshold allowing blood coagulation to be triggered. Converging evidences from experimental or clinical data highlight a role for MP harboring tissue factor in the initiation of disseminated intravascular coagulopathy. In these settings, the pharmacological modulation of MP levels or biological functions through activated protein C or factor VIIa allows challenging issues.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells.

That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Thrombin generation in vascular tissue.

Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.

Co-localization of von Willebrand factor with platelet thrombi, tissue factor and platelets with fibrin, and consistent presence of inflammatory cells in coronary thrombi obtained by an aspiration device from patients with acute myocardial infarction.

Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation. Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16-, CD45- and CD34-positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain. In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet-thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.

The Glycoprotein VI-Phospholipase Cγ2 Signaling Pathway Controls Thrombus Formation Induced by Collagen and Tissue Factor In Vitro and In Vivo

Both collagen and tissue factor can be initiating factors in thrombus formation. We investigated the signaling pathway of collagen-induced platelet activation in interaction with tissue factor-triggered coagulation during the thrombus-forming process. In murine blood flowing over collagen, platelet exposure of phosphatidylserine and procoagulant activity, but not adhesion, completely relied on each of the following signaling modules: glycoprotein VI (GPVI), FcR gamma-chain, Src kinases, adaptor protein LAT, and phospholipase Cgamma2 (PLCgamma2). On flow in the presence of tissue factor, these signaling components were essential for platelet aggregation and greatly enhanced fibrin clot formation. Collagen-stimulated thrombin generation relied on the presence and activity of GPVI, FcR gamma-chain, Src kinase, LAT, and PLCgamma2. The physiological importance of this GPVI pathway was shown in a FeCl3-induced in vivo murine thrombosis model. In both venules and arterioles, signaling through GPVI, FcR gamma-chain, and Src kinases enhanced the formation of phosphatidylserine-exposing and fibrin-rich thrombi. The GPVI-PLCgamma2 activation pathway regulates collagen-dependent coagulation in venous and arterial thrombus formation.

Blood Coagulation and the Risk of Atherothrombosis

Atherosclerosis is a multifactorial disease that is caused by many years of exposure to atherogenic stimuli that already at a young age lead to early lesions, the so-called "fatty streaks". One of the mechanisms involved in atherogenesis is the activated blood coagulation system, but its primary role in this process has not been established. From the fatty streak onwards, evidence of activated coagulation is histologically present in the form of fibrin/fibrinogen molecules. With advancing lesions, the presence of tissue factor protein, introduced by invading macrophages or locally expressed by vascular smooth muscle cells (VSMC) becomes more prominent. Tissue factor has been shown to be an important agonist of arterial thrombosis upon erosion of the luminal plaque surface or frank rupture of plaques. Recent data indicate the production of factor VII, probably expressed by VSMC in plaque lesions, which raises the possibility that formation of a catalytic tissue factor/factor VIIa complex occurs in the atherosclerotic vessel wall. Theoretically, such a complex could then induce local generation of thrombin and fibrin molecules, but its precise role is still unknown. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor - factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. Evidence is accumulating that the tissue factor/factor VII complex is involved in important coagulation-independent biological processes (inflammation, cell migration, apoptosis), either via direct signal transduction through tissue factor or by (in)direct activation of protease activated receptors. The presence of localized vascular "mini-cascades" of coagulation proteins may target a new role of coagulation in vessel wall remodeling and plaque vulnerability. Keywords: Coagulation, atherothrombosis, atherosclerosis, thrombin, fibrinogen

Of four mutations in the factor VII gene in Tunisian patients, one novel mutation (Ser339Phe) in three unrelated families abrogates factor X activation

Hereditary factor VII (FVII) deficiency is a rare bleeding disorder. Dysfunctional FVII variants characterized by normal or reduced levels of FVII antigen and discordantly low FVII activity have been described. In this study, seven unrelated Tunisian patients with FVII deficiency were examined. Molecular analysis revealed that three probands harbored a novel Ser339Phe mutation, one proband was inferred to have a novel splice site mutation in intron 2, c.226-2 A>G and three probands had two previously described mutations, Arg304Gln and Cys310Phe. Expression of Ser339Phe in baby hamster kidney cells yielded secretion of FVII antigen at a concentration of 225+/-50 ng/ml, compared with 181+/-47 ng/ml in cells transfected with wild-type FVII but with no demonstrable FVII activity. FVII Ser339Phe bound to tissue factor similarly to the binding of commercial recombinant activated FVII or recombinant wild-type FVII and was normally activated by activated factor X. The major defect of FVII Ser339Phe was its inability to activate factor X in the presence of tissue factor. Modeling predicted that the substitution of Ser339 by Phe abrogated substrate docking.

Zymogenic and enzymatic properties of the 70-80 loop mutants of factor X/Xa.

The Ca(2+) binding 70-80 loop of factor X (fX) contains one basic (Arg(71)) and three acidic (Glu(74), Glu(76), and Glu(77)) residues whose contributions to the zymogenic and enzymatic properties of the protein have not been evaluated. We prepared four Ala substitution mutants of fX (R71A, E74A, E76A, and E77A) and characterized their activation kinetics by the factor VIIa and factor IXa in both the absence and presence of cofactors. Factor VIIa exhibited normal activity toward E74A and E76A and less than a twofold impaired activity toward R71A and E77A in both the absence and presence of tissue factor. Similarly, factor IXa in the absence of factor VIIIa exhibited normal activity toward both E74A and E76A; however, its activity toward R71A and E77A was impaired approximately two- to threefold. In the presence of factor VIIIa, factor IX activated all mutants with approximately two- to fivefold impaired catalytic efficiency. In contrast to changes in their zymogenic properties, all mutant enzymes exhibited normal affinities for factor Va, and catalyzed the conversion of prothrombin to thrombin with normal catalytic efficiencies. However, further studies revealed that the affinity of mutant enzymes for interaction with metal ions Na(+) and Ca(2+) was impaired. These results suggest that although charged residues of the 70-80 loop play an insignificant role in fX recognition by the factor VIIa-tissue factor complex, they are critical for the substrate recognition by factor IXa in the intrinsic Xase complex. The results further suggest that mutant residues do not play a specific role in the catalytic function of fXa in the prothrombinase complex.

Blood coagulation and the risk of atherothrombosis: a complex relationship

The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor – factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin.The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability.

Cloning and expression of rat coagulation factor VII

Smaller and widely available animals such as rats are commonly used to evaluate antithrombotic drug candidates in vivo. However, the isolation and purification of FVII from rats and other species is very challenging because they are present in extremely low levels in plasma (∼10 nM). Furthermore, purification of FVII from other coagulation factors present in the plasma such as prothrombin, factor IX and factor X can often be very challenging and labor-intensive. To facilitate studies on the role of the extrinsic pathway of coagulation in rats, a full-length cDNA-encoding rat factor VII was isolated using polymerase-mediated DNA amplification using a rat liver cDNA library. The cDNA codes for a 41-residue signal/propeptide region, followed by a 405-residue mature protein consisting of the light chain with γ-carboxy glutamic acid (gla) including epidermal growth factor domains (EGF) and the heavy chain with the serine protease catalytic domain. Rat factor VII cDNA was transfected into human embryonic kidney 293 cells and several cell lines that constitutively express rat factor VII were established. The media from the stable lines expressing recombinant rat FVII were rapidly screened for functional activity and were found to normalize clotting time of FVII-depleted human plasma. The supernatants were also functionally active in the presence of tissue factor in chromogenic assays by measuring FVIIa activation using a tripeptide chromogenic substrate and in a two-stage, coupled assay measuring the generation of FXa. Recombinant rat FVII may be an important new tool in the development of novel antithrombotic drugs.

Four loops of the catalytic domain of factor VIIa mediate the effect of the first EGF-like domain substitution on factor VIIa catalytic activity

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII(IXegf1)a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca2+-bound factor VIIa and FVII(IXegf1)a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII(IXegf1)a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII(IXegf1)a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII(IXegf1)a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A + R147A + D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170sdel. The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170sdela (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.

Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: the role of tissue factor and cytochalasin D.

The contribution of platelets and soluble clotting components to clot strength has been the focus of several clinical studies using thromboelastography; it would, therefore, be beneficial to develop an animal model with which to mechanistically approach hemostatic disorders. Thus, we proposed to determine if the contribution of platelet function (G(P), dyne/cm(2)) and soluble components of the coagulation pathway to total clot strength (G(T)) in rabbits were similar to those in humans. Blood was sampled from the ear arteries of conscious rabbits (n = 12); 350 microL of the blood was placed in a thromboelastograph. Ten microliters of normal saline, cytochalasin D (an inhibitor of microtubule function, 10 microM final concentration), or tissue factor (a potent stimulator of platelet function, 0.00625% final concentration) was added to the blood sample, and thromboelastography performed for 1 h. The G(T) (mean +/- SD) was significantly (P < 0.001) different among samples exposed to normal saline, cytochalasin D, or tissue factor, with G(T) values of 7238 +/- 1432, 937 +/- 372, and 16,556 +/- 3314, respectively. G(P) was responsible for 87% and 94% of G(T) in the absence or presence of tissue factor, respectively. G(P) did not significantly correlate with platelet concentration in the absence or presence of tissue factor. The contribution of G(P) to G(T) is similar to that observed in humans. Rabbits may serve as a model of hemostasis that closely approximates human situations to mechanistically determine the etiology of coagulopathy. The contribution of platelet function to total clot strength is similar to that observed in humans.

Factor VII Central: A NOVEL MUTATION IN THE CATALYTIC DOMAIN THAT REDUCES TISSUE FACTOR BINDING, IMPAIRS ACTIVATION BY FACTOR XA, AND ABOLISHES AMIDOLYTIC AND COAGULANT ACTIVITY

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.

Molecular Mechanism of Tissue Factor-mediated Acceleration of Factor VIIa Activity

The mechanism of the acceleration of the catalytic activity of factor VIIa (VIIa) in the presence of tissue factor (TF) was investigated. To explore the VIIa's site(s) that correlates with TF-mediated acceleration, zymogen VII, VIIa, and active site-modified VIIa were prepared, and dissociation constants (Kd) for their bindings to TF or soluble TF in solution were determined. We found that conversion of zymogen VII to VIIa led to an increase in affinity (DeltaDeltaG = 4.3-4.4 kJ/mol) for TFs. Dansyl-Glu-Gly-Arg chloromethyl ketone (DNS-EGRck) treatment of VIIa led to a further increase in the affinity (DeltaDeltaG = 7.3-12 kJ/mol). Neither removal of the Gla domain from VIIa nor truncation of the COOH-terminal membrane and cytoplasmic regions of TF affected the affinity enhanced after DNS-EGRck treatment of VIIa. Treatment of VIIa with (p-amidinophenyl)methanesulfonyl fluoride also enhanced its affinity for soluble TF, whereas treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, phenylmethylsulfonyl fluoride, or diisopropyl fluorophosphate had a slight effect on the affinity. On the other hand, DNS-EGRck and (p-amidinophenyl)methanesulfonyl fluoride treatments, but not diisopropyl fluorophosphate treatment, of VIIa led to protection of its alpha-amino group of Ile-153 from carbamylation. Protection of the alpha-amino group was consistent with formation of a critical salt bridge between Ile-153 and Asp-343 in the protease domain of VIIa. Therefore, TF may preferentially bind to the active conformational state of VIIa. When one assumes that free VIIa exists in equilibrium between minor active and dominant zymogen-like inactive conformational states, preferential binding of TF to the active state leads to a shift in equilibrium. We speculate that TF traps the active conformational state of VIIa and converts its zymogen-like state into an active state, thereby accelerating the VIIa activity.

High-affinity calcium-binding site in the gama-carboxyglutamic acid domain of bovine factor VII.

The calcium-mediated interaction of factor VIIa with tissue factor is considered to be the primary trigger of blood coagulation. To determine the role of calcium ions in the action of factor VII, we prepared monoclonal antibodies whose binding to factor VII was calcium-dependent. A monoclonal antibody designated C6 strongly inhibited factor VII-induced clotting at a molar ratio of factor VII to antibody of 1:1. The half-maximal binding of factor VII to the C6 antibody was observed at a concentration of calcium ions of 80 microM. Proteolytic fragments of factor VII were assayed for their ability to inhibit competitively the binding of 125I-factor VII to immobilized C6 antibody. The binding was inhibited by increasing amounts of factor VII, by a fragment that contained the gamma-carboxyglutamic acid (Gla) domain linked to first epidermal growth factor-like domain, and by a Gla domain peptide (residues 1-41), over a range of concentration of 10(-9) to 10(-7) M. The antigenic site recognized by the monoclonal antibody C6, which was generated upon the high-affinity binding of calcium ions, was located in the Gla domain. The C6 antibody inhibited the activation of factor X and the amidolytic activity of factor VIIa in the presence of tissue factor. These results demonstrate that a high-affinity calcium-binding site(s) is located in the Gla domain of factor VII, which is concerned with the initiation of tissue factor-mediated blood coagulation by factor VIIa.

Tissue Factor Expression during Coculture of Endothelial Cells and Monocytes

The role of monocytes as initiators of coagulation through the expression of tissue factor has been well documented in vitro, and the relationship of monocyte tissue factor to the thrombotic complications of atherosclerosis has been suggested. Tissue factor antigen has been identified in the plasma membranes of monocytes adherent to the vascular endothelium overlying atherosclerotic plaques and the presence of tissue factor in adherent mononuclear cells correlates with the polymerization of fibrin at these same sites. To further understand the relationship of cellular adhesion to tissue factor expression, human monocytes were cocultured for periods ranging from 30 min to 24 hr with endothelial cells isolated from human umbilical veins (HUVEC). Tissue factor antigen, as assayed by both ELISA and immunogold electron microscopy, was minimal on either monocytes or HUVEC maintained in homogeneous cultures or on the cells when cocultured for 1 hr or less. This was true whether the HUVEC were in a native state or if they had been stimulated with interleukin-1 (IL-1β) or lipopolysaccharide (LPS) prior to monocyte adhesion. Typically, less than 13% of the cells in short-term cocultures were positively labeled through anti-tissue factor immunogold microscopy. The level of tissue factor, however, was increased 3-fold above baseline when monocytes were cocultured with unstimulated HUVEC for 4 hr, and it was more than double this if the HUVEC had been exposed to IL-1β or LPS (7-fold increase). By 24 hr, the expression of tissue factor antigen was nearly 50-fold higher in cocultures involving stimulated HUVEC, and at the later times greater than 70% of the cells were labeled with immunogold. Through the use of quantitative immunogold electron microscopy, the increase in tissue factor was most pronounced on monocytes which had three times greater increase in tissue factor than HUVEC in the same cultures. These studies document the stimulation of tissue factor expression by monocytes upon coculture with endothelial cells, and the data document an enhancement of this coculture effect upon HUVEC stimulation with cytokines. These observations have relevance to atherosclerotic disease by suggesting that interaction of monocytes with dysfunctional endothelial cells overlying atherosclerotic plaques would be sufficient to induce tissue factor and by so doing predispose to localized thrombotic events.

Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.

Complex-dependent inhibition of factor VIIa by antithrombin III and heparin.

The regulation of the factor VIIa-tissue factor complex is essential for control of the hemostatic response. However, the role of the inhibitor antithrombin III in the regulation of factor VIIa has remained in question. The inhibition of factor VIIa activity by antithrombin III and heparin in the presence and absence of tissue factor was evaluated using the fluorescent substrate m-LGR-nds. Our data show that the activity of recombinant human factor VIIa is inhibited by antithrombin III in the presence of heparin at a rate of 1.7 x 10(2) M-1 s-1. In the presence of tissue factor, the rate constant for this reaction increases to 5.6 x 10(3) M-1 s-1. A 1:1 stoichiometric complex between factor VIIa and antithrombin III, with an apparent molecular weight of 110,000, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A heterogeneous mixture of factor VIIa products with molecular weights between 50,000 and 80,000, most likely representing proteolytically degraded factor VIIa-antithrombin III complexes, was also observed.