Cellular Mechanisms for the Activation of Blood Coagulation

Cellular Mechanisms for the Activation of Blood Coagulation Carolyn L. Geczy Heart Research Institute, Camperdown, New South Wales 2050, Australia

1. Introduction

Coagulation is not only an essential defense against injury, but also an integral part of the host’s immune and inflammatory responses. Although normally nonthrombogenic, it is now clear that a number of cell types, principally including blood monocytes (Mo),inflammatory macrophages (Mac), and endothelial cells, can be stimulated to express surface-bound procoagulants. The major activity is attributed to tissue factor (TF), which activates the extrinsic coagulation pathway (Bach, 1988; Nemerson, 1988) although other inducible activators of this pathway are also involved. In addition to the effects of the cascade of coagulation enzymes, amplification also occurs at the level of membrane-substrate complexes (Mann et al., 1990).

This chapter describes the biology of the cellular activators of the extrinsic coagulation pathway. However, these procoagulants do not act in isolation. Upon stimulation of both Mos and endothelial cells, there is a concomitant upregulation of the antifibrinolytic system by decreased or unchanged plasminogen activator synthesis and increased production of inhibitors of plasminogen activator (Vassalli et nl., 1991). Downmodulation of anticoagulation via regulation of thrombomodulin (McCachren et al., 1991), the essential cofactor for the activation of protein C, the inhibitor of factors Va and VIIIa (Stern et al., 1988), also contributes to the outcome of the hemostatic balance. In addition, regulation of the activity of the TF-factor VIIa complex by the extrinsic pathway inhibitor (TFPI) (Rapaport, 1991), and other natural inhibitors of coagulation, including antithrombin, activated protein C and S, and heparin cofactor 11, can all modulate the thrombotic complications of diseases in which cellular procoagulants are involved. lnrernational Review of Cytology. Vol. 152

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CAROLYN L. GECZY

50 II. Tissue Factor A. General

Tissue thromboplastin “substance” was a clot-promoting activity of tissues described by Nolf in 1908. Early attempts to isolate thromboplastin implicated one protein (Chargaff et al., 1944) although attempts to identify a bleeding disorder due to defective thromboplastin cast doubt on the single-protein hypothesis. Purification of a protein with clot-promoting activity which is now known as “tissue factor” was achieved by Bach et al. in 1981. In contrast to other members of the coagulation cascade present in plasma, TF is located in a number of cell types within the blood and tissues. TF is now thought to play a pivotal role in the regulation of coagulation, hemostasis, and thrombogenesis. The pathway of TFmediated coagulation is shown in Fig. 1. TF is a transmembrane cell surface receptor for plasma factor VII. The homogeneous protein was purified from bovine brain and represented a polypeptide of 40-43 kDa (Bach et al., 1981, 1986; Carson er al., 1985). Subsequently, the human protein was purified by immunoaffinity using monoclonal antibodies (Carson et al., 1987; Spicer er al., 1987) and by ligand affinity to human factor VII (Broze et al., 1985; Guha et al., 1986) in quantities sufficient to obtain the N-terminal amino acid sequence. The

JINIRINSICJ Factor IX

Factor IXa factor Vlla

factor Vlla Factor X

Xa-Va

(EXTRINSICI

-

prothrombinase complex Mo, lymphocytes [Gets.

prothrombin

fibrin

thrombin

fibrinogen

FIG. 1 Tissue-factor-mediated pathway of activation of coagulation.

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complete primary structure of the human TF gene, mRNA, and protein is now established. The cDNA was cloned at about the same time by four groups (Fisher et al., 1987; Momssey et al., 1987; Scarpati et al., 1987; Spicer et al., 1987). The TF gene is located on chromosome 1 (Scarpati et al., 1987) at 1~21-22(Kao et al., 1988) and spans 12.4 kbp, excluding the promoter and regulatory enhancer elements (Mackman et al., 1989).The single gene comprises six exons separated by five introns with the 5' region of exon 1 encoding the translational start point and the 3' region encoding the 32residue leader sequence. A number of potential sites for DNA binding and transcriptional regulatory protein binding have been identified and are discussed in detail by Edgington et ul. (1991). The open reading frame encodes a 295-amino-acid polypeptide which undergoes processing to remove a 32-residue leader sequence, yielding a mature protein of 263 residues. The primary sequence indicated a unique transmembrane protein of 30 kDa consisting of an extracellular domain (Serl-Glu2,g),a hydrophobic membrane-spanningregion (residues 220-242), and a cytoplasmic tail (residues 243-263). Two of the three potential Winked glycosylation sites within the extracellulardomain are sites of post-translationalmodification. Glycosylation is apparently not required for functional activity because removal of carbohydrate by endoglycosidase F does not affect the clotting capacity of the native protein (Bach, 1988)and recombinant TF produced in Escherichiu coli is functional (Paborsky et al., 1989). B. Tissue Factor as a Member of the Cytokine, Growth Factor Receptor Superfamily

Tissue factor is a member of a family of receptors for a diverse group of hematopoietic factors, growth hormones, and interferons with apparently unrelated sequences. In contrast, the family of cognate receptors reveals a striking resemblance of binding domains containing a distinctive conservation of four cysteine pairs in the N-terminal half and a WSXWS (one letter amino acid code, X is nonconserved)near the C-terminal end (Miyajima et al., 1992). TF is most closely related to the interferon (aand y) receptors (type 11 cytokine receptors) which contain characteristic cysteine pairs at both N- and C-terminals and which are evolutionarily related to the type I cytokine receptors. They include receptors for growth hormone, prolactin, erythropoietin, interleukin (IL) 2, IL-4, IL6. granulocytemacrophage-colony-stimulating factor (GM-CSF), IL3, G-CSF, IL7, and IL5 (Bazan, 1990a,b). Structural analysis of extracellular segments indicates a common globular protein fold constructed from seven conserved @-strandswith a topol-

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ogy analogous to an immunoglobulin (Ig) constant domain. The binding segment domains from this receptor family are related to a common 90amino-acid sequence known as the fibronectin type I11 domain. Bazan has postulated that this subclass of Ig-like proteins has evolved from primitive adhesion molecules to their present role in specific protein binding. A model of the TF surface domain based on the structure of PapD (a protein with P strand organization (Holmgren and Brandeur, 1989) and aligned according to the analysis of Bazan (1990b)is presented by Edgington et al. (1991). These authors propose a pair of seven-strand Ig-like modules for the TF surface domain, and the model predicts that the residues of T F would align in two sheets of three and four P-strands respectively, in each module. They assume that interactions between the two modules are critical for T F function, based on modeling of the Class 1 receptors, and suggest that this model is consistent with the multiple interactive binding sites on factor VII/VIIa. The four cysteines in the extracellular region of TF are covalently bonded to form two disulfide loops. The tertiary structure of the molecule is required for functional activity (Bach et al., 1981) and the carboxylterminal cysteines 186 and 209 are essential for fully functional ligand binding (Rehemtulla et al., 1991). Furthermore, the amino acid sequences of rabbit and murine TF are respectively 71 and 58% identical to human TF and are consistent with the relative functional activity of each in human plasma. The structural organization of the protein indicates a high degree of conservation of the extracellular domain and the relative positions of the cysteine residues in all three species (Andrews, 1991). The presence of three WKS repeats within human TF (only one of which is found in the mouse and two in the rabbit) (Andrews et al., 1991) and within several proteins involved in coagulation or in proteins that share some functional properties with the coagulation proteins, has been suggested to represent a functional sequence motif on the basis of its high affinity for human but not for murine factor VII (Andrews et al., 1991) although the structural significance of this repeat has been disputed (Bazan, 1991). There are several indications in the literature that TF may exist in dimeric form. Covalent homodimers may occur by self-association of Cys,,, within the cytoplasmic tail as a purification artifact (Bach, 1988) although this cysteine can also be acylated (Bach et al., 1988). In an attempt to draw functional analogies between activation of the large cofactor coagulation proteins V and VIII, which form multidomain complexes on the phospholipid surface, and the activation of factor X by factor VII/VIIa, Roy et al. (1991) used chemical cross-linking experiments to demonstrate that TF exhibits a tendency to self-associate on cell surfaces.

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Mutation experiments indicate that deletion of the cytoplasmic domain does not affect cross-linking and that the transmembrane sequence is necessary for interaction between TF molecules. Furthermore, a study of binding and activity of factor VII on a bladder carcinoma cell line 582, which expresses high levels of TF, indicated at least two sites on TF with positive cooperativity for ligand binding (Fair and MacDonald, 1987). Because the stoichiometry of the TF-factor VII complex is 1:1, Fair and MacDonald postulated that TF was expressed as a dimer and that association of factor VII with the first site may perturb the dimer enough to permit binding of the second ligand. Cross-linking studies have confirmed the presence of a dimer on these cells although high levels of the monomer were also present (Roy et al., 1991). A multisubunit structure is a common feature of a number of receptors and some, like the granulocyte-colony-stimulating factor (G-CSF) receptor, function as homodimers (Fukunaga et al., 1990). It has been suggested that the number of TF molecules correlates with the ability of cetls to initiate the coagulation cascade (Rodgers et af., 1984), but several studies (Fair and MacDonald, 1987; Ploplis et al., 1987; Walsh and Geczy, 1991; Walsh et al., 1992) do not support this view. Variations may occur between cell types and in the composition and integrity of the cell surface phospholipid environment (see later discussion). In addition, and although there has been no correlation with functional activity, TF dimerization, like that observed for other membrane receptors, may represent a magnification of functional activity and therefore a means of modulating coagulation. TF also forms a heterodimer with a 13-kDa polypeptide in some preparations (Carson, 1987; Morrissey et al., 1988a). The resultant 58-kDa form is a functionally active disulfidelinked heterodimer composed of TF and the a-chain of hemoglobin. The interactions between these two molecules appear to be relatively specific and are proposed to occur as a result of lysis of red cells (Momssey et al., 1988a).The physiological significanceof this heterodimer, which might appear following tissue injury or as a consequence of surface shedding of TF (Bona et al., 1987), is unclear.

C. Function Tissue factor is the high-affinity receptor for plasma factor VIIIVIIa. In contrast to factors V and VIII, which also bind their respective enzymes in a 1 :1 stoichiometric complex and which are activated by partial proteolysis, TF requires no further processing. In the presence of Cazc ions, binding of factor VII/VIIa to TF increases the proteolytic activity of factor VIIa for its primary substrate, factor X, to directly initiate the

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extrinsic coagulation cascade (Bach, 1988; Nemerson, 1988). Mapping of TF-binding sequences using monoclonal antibodies to a series of peptides indicates at least two linear sequences within the extracellular domain involved in factor VII binding. It is proposed that interaction within these sites might represent the structural counterparts of the two functional properties of TF, namely to serve as a receptor and as a cofactor for factor VIIa. Observations that two structural sites within factor VIIa-the y-carboxyglutamic acid domain (Sakai et al., 1990) and residues 195-206 in the catalytic domain (Wildgoose et al., 1990)-are involved in highaffinity binding to TF would support this view. TF activity is phospholipid dependent. T F is normally inserted within a membrane lipid bilayer which provides phospholipid cofactors; the pure molecule requires lipid (phosphatidyl choline/phosphatidyl serine) for activity (Bach, 1988; Nemerson, 1988). Recent experiments with a recombinant TF mutant (TFI-219) with membrane-spanning and intracellular domains removed, indicate that free factor VIIa, but not TFl-219or TF,,,,-VIIa complex, forms a stable association with phospholipid (Ruf et al., 1991). These authors suggest that the catalytic function of TFVIIa is independent of its assembly on phospholipid and that the primary protein:protein interactions of factor VIIa with the surface domains of T F are sufficient to markedly enhance the catalytic function of factor VIIa. On the other hand, factor X is recognized as a preferential substrate for the TF-VIIa complex when it is associated with phospholipid surfaces. A situation analogous to the phospholipid association of prothrombin via the Gla domain (Malhotra et al., 1985), which induces a conformational change essential for proper substrate presentation to the prothrombinase complex on cell surfaces, is proposed (Ruf et al., 1991). These studies cannot exclude the possibility that membrane-anchored TF-VIIa preferentially cleaves free factor X (Forman and Nemerson, 1986; Nemerson, 19881, although the decreased apparent Michaelis constant of TF in reconstituted phospholipid vesicles suggests that phospholipid interaction with factor X might facilitate its presentation to the TF-VIIa membrane complex. In an attempt to simulate conditions in uiuo, recent experiments were designed to study phospholipid-dependent coagulation under flow conditions (shear rates 25 sec-I to 1200 slc-I) using a capillary coated with stable phospholipid bilayers containing TF and perfusion with factors VIIa and X (Gemmell et al., 1991). Under these conditions there was apparent increased binding affinity of factor VIIa to TF, and generation of Xa was approximately the same whether factor VII or VIIa were perfused. Furthermore, the functional activity of the immobilized complex was not altered by excess prothrombin fragment I, a split product of prothrombin which displaces factor X from lipid vesicles (Forman and Nemerson,

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1986). This suggests that factor X-phospholipid interactions did not play a significant role under these circumstances although the amount of factor X binding to the lipid bilayer was not measured. TF binds both the active and zymogen forms of factor VII with equal affinity (Zur et al., 1982). The conversion of factor VII to VIIa is most rapidly catalyzed by factor Xa, and at a slower rate by factor IXa, and is more rapid in the presence of TF (Bom and Bertha, 1990; Nemerson and Repke, 1985; Radcliffe and Nemerson, 1976; Rao et al., 1985, 1986). When the active serine of factor VII is inactivated with diisopropylfluorophosphate, the procoagulant activity is lost (Bach et a!., 1981). Based on enzyme kinetic measurements, Nemerson and Gentry (1986) propose a model for TF-mediated activation of coagulation which involves two related ligand-enhanced conformationalactivations. In the absence of TF, factor VIIa is not significantly catalytic and binding to TF is thought to create sites within factor VIIa which allow interaction with its substrate, factor X. The latter in turn may result in a form of factor VIIa which binds more tightly to TF to form a “conformational cage” which precludes the dissociation of factor VIIa from TF while significant concentrations of factor X are present (Nemerson, 1986). The extrinsic clotting pathway can also be activated indirectly by TF via activation of factor IX by TF/VIIa (Osterud and Rapaport, 1977). Comparison of the kinetic parameters for the activation of factor IX and factor X suggests that at plasma concentrations of these substrates, the rate of factor Xa formation would be approximately 2.5 times that of factor IXa formation (Born et al., 1990; Osterud and Rapaport, 1977). The relative importance of the extrinsic factor IX activation to the overall levels of factor Xa generation in uiuo is unclear although when low levels of TF are present (e.g., when limited amounts of factor Xa and factor 1Xa are formed), the contribution of this pathway may be significant (Bom er al., 1990).

D. Localization Procoagulant activity associated with TF was initially described in a number of organs and cell types, including fibroblasts, smooth muscle cells, endothelial cells, Mo/Mac, and a variety of neoplastic cell types by virtue of the dependence of clotting capacity on factors VII and X,and in some cases sensitivity to phospholipase C. Furthermore, many studies were performed using cell lysates, and activity of lysed cells was often much greater than that of intact cells. Levels of intracellular or “cryptic” TF were reported (Leoni and Dean, 1985) and it was suggested that T F activity was mobilized from an intracellular compartment or occurred within the

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cell membrane in a latent form which became available after the cell surface was perturbed (Maynard et al., 1975; Schorer et al., 1985). In the past few years the availability of monoclonal antibodies to TF has made it possible to confirm the location and activity of TF within a variety of cells and tissues (Drake et al., 1989a,b; Faulk et al., 1990; Fleck et al., 1990; Morrissey et al., 1988b). Immunohistochemical studies of normal human tissue show that the majority of TF-reactive cells are not in contact with blood; TF is anatomically distributed in perivascular cells, in capsules surrounding organs, and in cells of epithelial surfaces. In contrast to earlier reports (Zeldis et al., 1972), endothelial cells do not normally express TF. Furthermore, normal peripheral blood cells are also TF-negative, ensuring that the intravascular compartment does not represent a procoagulant environment whereas the normal distribution of extravascular TF suggests that it could activate coagulation following vascular injury. Localization of TF in normal human tissue is described in detail by Drake et al. (1989a,b) and Fleck et al. (1990). It is expressed in high amounts in the gray matter of the brain and spinal cord, and in lesser amounts in the meninges. Recent experiments using fresh brain specimens from the baboon show a distinct pattern of TF antigen expression associated with the microvasculature (cortical gray matter > basal ganglia L cerebellum > cortical white matter) which correlated with functional activity (del Zoppo et al., 1992). To date, there has been no specific neuronal cell associated with the diffuse distribution of TF in gray matter. Del Zoppo and colleagues have made the interesting suggestion that normal migration of Mo/Mac into the cortical parenchyma may shed sufficient T F to account for its diffuse localization. The gray/white matter partition of TF may be accounted for by greater microvascular content and endothelial cell surface area, allowing more Mo/Mac transmigration into cortical gray matter than white matter. The bronchial mucosa, alveolar septae, epithelial cells, and macrophages of the lung are TF positive. Fibroblast-like cells and Mac, but not trophoblasts, in connective tissue of the villi of the placenta and in amnion encasing the placenta and umbilical cord are generally strongly positive (Faulk et al., 1990; Fleck et al., 1990). Epithelial cells delimiting body/ environment boundaries (e.g., squamous epithelium of the skin and cervix, gut mucosa, cuboidal epithelium of the bladder) strongly express TF. TF in the kidney is limited to the glomeruli in which epithelial and mesangial cells of the glomerular tuft react strongly, and epithelium of Bowman’s capsule is also positive whereas glomerular capillary endothelium is negative. Skeletal muscle cells do not express T F whereas cardiac myocytes possess a cytoplasmic distribution and only smooth muscle cells of the muscularis mucosa of the esophagus showed positive reactivity. In addi-

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tion, cells composing the fibrous caps of liver, spleen, kidney, and adrenals express varying levels of TF. Expression of TF by connective tissue fibroblasts occurs most prominently in the adventitia of blood vessels whereas its location within these cells at other sites is apparently variable (Drake et al., 1989a; Fleck et al., 1990; Wilcox et af., 1989). As expected from the widespread distribution of human TF described by immunohistochemistry, constitutive expression of TF mRNA has been found in many mouse tissues and is especially abundant in lung, brain, midterm placenta, testis, and kidney, with lower levels apparent in heart, spleen, and intestine (Hartzell et al., 1989). In addition, mRNA in human tissue is found in adipose tissue, placenta, adrenal gland, small intestine, kidney (Fisher et al., 1987), and brain (Scarpati et af., 1987), whereas it was not detected in pancreas, liver, and spleen (Fisher et al., 1987). In situ hybridization studies (Wilcox et a[., 1989) located strong TF mRNA expression in adventitial fibroblasts, which correlated well with antigen expression in samples of normal human saphenous vein and internal mammary arteries. Scattered smooth muscle-like cells within the tunica media had levels of mRNA similar to those of adventitial fibroblasts, but had lower levels of protein.

1. Monocyte-MacrophageTissue Factor Mo/Mac procoagulant activity (MPCA)is induced by a number of intrinsic and extrinsic stimulants and is the subject of a number of reviews (Dean er al., 1984; Edwards and Rickles, 1980a; Geczy, 1984; Ryan and Geczy, 1987). It is induced by a variety of infections, including both gram-negative and gram-positive bacteria, bacterial toxins, viruses, parasites [e.g., Plasmodulinfalciparum-infected erythrocytes (Pernod et af., 1992)],activated complement components (Osterud et al., 1984), immune complexes (Schwartz et al., 1982a), modified lipoproteins (Levy et al., 1981), free cholesterol (Lesnik et al., 1992), and cytokines which regulate cell-mediated immune reactions and inflammation (see later discussion). One of the best-studied inducers of MolMac, TF is bacterial lipopolysaccharide (LPS) (endotoxin), which in a clinical situation plays a pivotal role in the development of gram-negative septicemia. The hematological manifestations of this condition include activation of the coagulation, fibrinolytic, and complement systems (van Deventer ef al., 1990). Fibrin deposition and complement activation can cause extensive damage of vessel walls and may be associated with multiple organ failure. A role for TF in the disseminated intravascular coagulation (DIC) associated with administration of LPS is substantiated by the reduction of fibrin formation and DIC by pretreatment of rabbits with anti-TF antiserum and the promotion of DIC and pulmonary artery thrombosis by infusion with TF (Warr

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et al., 1990). Furthermore, a monoclonal antibody to TF administered to baboons prior to injection of E. coli attenuated the coagulopathy and protected against its lethal effects by reducing the degree of cardiovascular collapse and cell injury (Taylor et al., 1991). Although part of the procoagulant effect has been attributed to increased levels of IL1 and tumor necrosis factor-a (TNF-a) (Bauer et al., 1989; Conkling et al., 1988a; van der Poll et al., 1990) which have variable effects on Mo T F and which modulate TF expression by endothelial cells, markedly more TF is expressed by Mo/Mac in direct response to LPS. Furthermore, delayed treatment with anti-TNF antibodies did not reverse the consumption of fibrinogen in plasma of baboons given E. coli (Hinshaw et al., 1990), supporting the proposal that an additional pathway of procoagulant induction contributes to lethality. In addition, peritoneal Macs (Robinson et al., 1978), blood Mos, and spleenic cells (Rothberger et al., 1983) from animals injected with LPS express high levels of procoagulant activity. Actinomycin D, which sensitizes mice to the lethal effects of LPS, renders murine Macs sensitive to levels of LPS some 100,000-fold less than normal, whereas TNF production is only doubled (Wheeler et al., 1991). Human Mos are exquisitely sensitive to LPS, responding to levels as low as 0.1-1 pg/ml in our experiments. Blood Mos are not heterogeneous with respect to their responsiveness; our immunohistochemical studies show that >98% of these cells convert from surface TF-negative to TFpositive following incubation for 16 hr with I ng/ml LPS (Walsh and Geczy, 1991). Our studies have also consistently found that blood Mos from female donors are more sensitive to LPS than those from male donors ( J . D. Walsh and C. L. Geczy, unpublished). Although the reason for this observation is uncertain, the recent evidence that estrogen can regulate TF gene expression in the immature rat uterus (Jazin et al., 1990) suggests that TF levels in Mos may also be under hormonal control. TF transcripts are detectable in human Mos 0.5 hr postinduction with LPS and reach maximal levels within 4 hr, coinciding with expression of TF activity on viable cells (Gregory et al., 1989). Interestingly, LPS coordinately initiates the transcription of the IL- l p and TNF-a genes over the same time course as that of TF although the selective reduction of TF mRNA expression in LPS-stimulated Mos from patients with advanced AIDS indicates that these genes can segregate under pathological conditions (Lathey et al., 1990). Furthermore, reduction of TF in this condition may contribute to the diminished resistance to infection observed in AIDS patients. Experiments with the monocytoid cell line THP-1 indicate that after stimulation, TF gene transcription increases threefold and at 1 hr T F mRNA is stable over 60 min with a half-life of >120 min, whereas at 2 hr the half-life declines to 25 min, suggesting that both transcriptional and post-transcriptional mechanisms control its synthesis (Brand et al.,

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1991). Possible mechanisms of transcriptional control of the TF gene are described by Mackman et al. (1989) and reviewed by Edgington et al. ( 1991) .

2. Cytokine-Mediated TF Induction on Monocytes and Macrophages: Implications in Cell-Mediated Immune Reactions Fibrin deposition is a common feature of inflammation and of numerous diseases in which cell-mediated immunity (CMI) plays a role (Dunn and Willoughby, 1981; Edwards and Rickles, 1980a; Geczy, 1983; Geczy et al., 1984). The swelling typical of classical delayed-type hypersensitivity (DTH)reactions is caused by water trapped within fibrin (Colvin and Dvorak, 1975; Colvin et al., 1973). A role for extravascular fibrin in these reactions is confirmed by observations that afibrinogenemic patients fail to respond to skin test antigens (Colvin et al., 1979) and anticoagulants inhibit induration induced by skin test antigens (Edwards and Rickles, 1978; Nelson, 1965). Mo/Mac procoagulants, induced in response to cytokines released as a consequence of immune activation, are now considered important initiators of coagulation. In addition to the exogenous inflammatory stimuli which influence MCPA (see earlier discussion), our earlier studies (Geczy and Hopper, 1981) and those of Edwards and Rickles (1980b) and van Ginkel and colleagues (1981) implied a role for T lymphocytes and Tlymphocyte-derived products in this response. In addition, a number of other products of an active immune response, particularly autoantibodies (Tannenbaum et al., 1986), antigen-antibody complexes (Lyberg et al., 1982; Rothberger et al., 1977; Schwartz et al., 1982a),and activated complement components C5a and C3b (Muhlfelder et al., 1979; Prydz et al., 1977) all influence Mo/Mac procoagulant expression. Our earlier experiments showed that generation of MPCA following culture of mononuclear cells with microbial antigens is a close in v i m correlate of DTH skin test reactions in man (Geczy and Meyer, 1982) and there is a parallel between the capacity of mouse strains to develop DTH and the intensity of MPCA generation in uitro (Geczy et al., 1983). Although there may be a requirement for direct T-lymphocyte-Mac contact under some circumstances of TF activation (Edgington et a)., 1981;Helin et al., 1983; Levy and Edgington, 1982;Tsao et al., 1984; Fan and Edgington, 1988), a clear involvement of lymphocyte-derived cytokines is now well accepted although the identity of the cytokines involved is still somewhat unclear. This pathway of Mo/Mac procoagulant induction may contribute to the pathology of many immunologically mediated diseases such as multiple sclerosis [evidence from an animal model system-experimental autoim-

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mune encephalomyelitis (Geczy et al., 1984)l the Guillain-Barre syndrome (Geczy et al., 1985), influenza (Schiltknecht et al., 1984), coeliac disease (Devery et al., 1990), allograft rejection (Halloran et al., 1985; Zappala et al., 1989),glomerulonephritis (Cole et al., 1985),and malignancy (Lyberg, 1984b; Rickles and Edwards, 1983)and other diseases, such as atherosclerosis (Wilcox et al., 1989)in which a role for lymphocyte-derived cytokines has only recently been recognized (Serneri et al., 1992). Our studies indicate that murine Lyt 1'2- T, spleen cells in the presence of a major histocompatibility complex (MHC) class 11, antigenpositive, adherent accessory cell produce a factor which we called macrophage procoagulant-inducing factor (MPIF) (Geczy et al., 1983; Ryan and Geczy, 1986). Antigen presentation by monocytes in the context of MHC class I1 antigen was necessary for MPCA induction by protein antigens, confirming a classical immune response to antigen (Schwartz, 1985). Furthermore, in contrast to CD 8 + cells, alloantigen-activated murine CD4+T cells produce MPIF (Fan and Edgington, 1988)and the human alloantigen response is mediated by T cell clones of the CD3+,CD4+, CD8- phenotype (Gregory and Edgington, 1985). Moreover, cyclosporin A, an immunosuppressive drug which alters T cell function, inhibits MPIF induction in uitro (Chung et al., 1991; Thomson et al.. 1983a) and in vivo (Thomson et al., 1983b). Because much of the background work in this area has been the subject of a number of reviews (Geczy, 1983, 1984; Ryan and Geczy, 19871, only the more recent work will be presented here. Cytokines modulating MPCA are given in Table I. Several studies clearly implicate cytokines, in addition to MPIF, in MPCA induction although it is interesting that procoagulant responses of various cell types appear to be somewhat cytokine-specific. In contrast to their effects on cultured endothelial cells, ILl-a and -p induce weak activity on human blood Mos (Carlsen et al., 1988; Carlsen and Prydz, 1988). We find that these mediators have no effect on murine Macs. Although high levels of TF induction on U937 monocytoid cells and blood Mos by TNF has been described (Conkling et al., 1988b), we and others have been unable to demonstrate this effect (Carlsen et al., 1988; Gregory et al., 1986; Ryan and Geczy, 1986). Carlsen and Prydz (1988) demonstrated a 15-fold enhancement of TF activity on human Mos by IL2 whereas others (Gregory et al., 1986) found that 100-1000 U/ml IL2 induced low levels of TF compared with MPIF, and we failed to induce procoagulant on murine exudate Macs with IL2. Interferon-? (IFN-y), a product of both CD4+ and CD8+ lymphocytes, apparently plays a regulatory role in the procoagulant response. IFN-y is an important activator of many cell types, particularly Macs, which are produced as a consequence of infection, activated CMI, and inflammation (Adams and Hamilton, 1984), and is thought to play a significant role in

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TABLE I Modulation of MolMac Procoagulants by cytokines ~

Cytokine T-lyrnphocyte-derived IFN-y MPIF IL2 IL4 MolMac-derived TNF-CY ILI-a. p GM-CSF IL6

Tumor-derived VPF

Procoagulant effect Primes inflammatory Macs Induces TF expression on both Mos and Macs Induces TF on Mos Inhibits MPCA induction by some stimulants Some reports of TF induction on Mos Induces moderate levels of TF on Mos Primes inflammatory Macs to respond to LPS Weak potentiation of LPS response on Macs Induces weak TF activity on Mos

the pathology of septic shock (Heinzel, 1990). Our studies show that viable, thioglycollate-elicited murine peritoneal exudate (TG-PEC) Macs primed with IFN-y expressed 10-40-fold more MPCA in response to suboptimal levels of LPS (Moon and Geczy, 1988). The major procoagulant activity had characteristics of TF which were maximal after 24-hr culture and kinetics similar to those described for induction of tumoricidal or microbicidal activities exhibited by these cells in response to IFN-yI LPS (Adams and Hamilton, 1984). In addition to the synergy observed between IFN-y and LPS, we have investigated the possibility that, although some cytokines may not act alone, they may synergize with IFN-y to initiate procoagulant expression or TG-PEC. Of a number of cytokines tested, we found that IL6 synergizes weakly with IFN-y to induce TF levels approximately four times greater than basal levels. MPIF apparently also synergizes with IFN-y although further studies with pure MPIF are required to confirm this (A. Jones and C. L. Geczy, unpublished data). We also found that IFN-y synergized with PMA, but not LPS, to induce TF in a myelomonocytoid human cell line, RC2a (Geczy and Jones, 1988), although its potential interaction with other cytokines has not been tested in this system. There is one report that GM-CSF primes cultured (1-14 days) adherent TGelicited Macs to respond to LPS (Zuckerman and Surprenant, 1989) al-

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though we failed to demonstrate this activity using TG-PEC cultured with GM-CSF and LPS in nonadherent conditions. Although IFN-y alone failed to induce significant levels of procoagulant in viable TG-PEC, lysed cells express an activity similar to factor VIIa. There are a number of reports of factor VII/VIIa induction on Mo/Macs (Chapman et al., 1985; Tsao et al., 1984), and murine exudate Macs may constitutively express factor VII (Shands, 1984; Shands et al., 1988), which could combine with TF within disrupted cell membranes to provide active factor VIIa. Our recent experiments show that rIFN-y upregulates T F mRNA expression in murine TG-PEC to levels somewhat greater than those induced by LPS (Fig. 2), although we cannot detect increased amounts of TF antigen in lysates of cells cultured over 2-24 hr with IFNy in Western blotting experiments (Jovanovich and Geczy, 1994). These experiments confirm that IFN-y can modulate TF gene transcription although factors regulating expression of functional activity are still unclear. In contrast to exudate Macs, neither resident Macs nor blood Mos respond to IFN-y, either to express intracellular activity or extracellular T F when cultured with LPS (Moon and Geczy, 1988). Furthermore, IFNy does not induce TF on human blood Mos, and suppression of T F induction by allogenic lymphocytes or LPS occurs when these cells are cocultured with IFN-y (Carlsen et al., 1987; Carlsen and Prydz, 1988; Conkling et al., 1988b). By contrast, human Mo-derived Macs (blood Mos cultured in Teflon bags for 6-8 days) grown in suspension culture express procoagulant activity in response to IFN-y (Miserez and Jungi, 1992), and a murine monocytoid leukemic cell line (WEHI 265) is also directly responsive

FIG. 2 Northern blot analysis of mRNA extracted from murine TG-PEC incubated for 4 hr with (i) control media, (ii) LPS (Ipg/ml), (iii) IFN-y(100 U/ml) and probed with (A) a cDNA fragment probe to murine TF (kindly provided by Dr. D. Nathans) and (B) an oligonucleotide probe to rat 18s ribosomal RNA for comparative quantitation of total RNA.

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(Wheeler et al., 1991). These experiments add weight to the proposal that the maturation and differentiation states of Mo/Mac may determine their reactivity to different stimulants of TF expression (Geczy, 1984; Lyberg et al., 1983b). In contrast to the narrow range of activity induced by IFN-y, human and murine MPIF directly stimulate TF expression on human Mos (Gregory and Edgington, 1985) and murine TG-PEC respectively, and on some monocytoid cell lines (Farram et al., 1983). In contrast to the slow response elicited by IFN-y/LPS on TG-PEC, MPIF induces high levels of activity after 6-8 hr of culture which are maintained over 24 hr. MPIF has been purified to homogeneity in our laboratory but its complete amino acid sequence is still not determined (M. Lackmann and C. L. Geczy, unpublished). Our earlier studies (Ryan and Geczy, 1986) and those of Gregory et al. ( 1986) indicated that MPIF was a heparin-binding protein which displays heterogeneity with respect to size and charge. We isolated two active murine components with PIS of 8.5 (a)and 8.8-9.0 (p) and a third component (PI 5.5) which we now believe to be IFN-y (M. Lackmann and C. L. Geczy, unpublished data). The human and murine MPIFs are apparently novel proteins. A large panel of cytokines tested for MPIF activity was inactive and purified fractions are devoid of many of the cytokines, including CSFs, IL1, IL2, TNF-a, and $3, and IFN-y, and antibodies to a number of cytokines fail to alter activity. Furthermore, human MPIF enhances TF on RC2a cells whereas LPS fails to alter TF expression on those cells, confirming our suggestion that the activity is not merely due to contaminating LPS or to a synergistic response with LPS (Geczy and Jones, 1988). The relationship between MPIF, factors present in supernatants of a number of murine tumor cell lines which induce MPCA on TG-PEC (Inoue et al., 1983), and vascular permeability factor (VPF) produced by murine meth A fibrosarcoma cells (Clauss et al., 1990a,b) (which induces TF on endothelial cells and somewhat weaker activity on human Mos) is unknown, although the species specificity of murine MPIF is different than that of VPF. There have been relatively few studies to determine whether cytokines affecting MPCA induce fibrin deposition in uiuo. The presence of fibrin in DTH lesions is well established and recent enzyme and immunohistochemical studies confirm the presence of thrombin (Imamura and Kambara, 1992) and of TF-positive Macs at these sites (Imamura et al., 1992). Our studies showed that a highly enriched fraction containing MPIF induced an indurated lesion containing extravascular fibrin, an early influx of neutrophils, and a sustained influx of mononuclear cells over 24-48 hr (Ryan and Geczy, 1988) when injected intradermally into rat skin.

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We subsequently separated MPIF from a unique protein member of the family of SlOO Ca*+-bindingproteins (called CP-10; chemotactic protein, 10 kDa) which has potent chemotactic activity for neutrophils and Mos (Lackmann et al., 1993). This protein induces a strong neutrophil infiltrate 4-6 hr postinjection, with increased numbers of Mo/Mac evident after 24 hr (Lackmann et al., 1993; Devery et al., 1994). The time course and cellular characteristics of the inflammatory response are similar to a DTH response and in marked contrast to the more transient infiltration of neutrophils elicited by other proinflammatory cytokines such as ILl (Cybulsky et al., 1986), macrophage inflammatory protein (MIP) (Wolpe et al., 1987) and IL 8 (Foster ef al., 1989), and by C5a (Yancey et al., 1985). Interestingly, the classical chemotactic stimulants for phagocytic cells, C5a and bacterial cell wall peptide fMet Leu Phe (FMLP), induce T F on human Mos (Janco and Morris, 1985; Muhlfelder et al., 1979) and platelet activating factor (PAF), a chemotactic factor and potent inflammatory mediator produced by a variety of cells, including activated neutrophils, Mo/Mac, platelets, and endothelial cells, primes TG-elicited murine Macs to express 2- to 5-fold higher procoagulant levels (Kucey et al., 1991). In addition, PAF is a potent neutrophil stimulant which enhances LPSinduced blood Mo TF expression when these cells are cultured together with neutrophils and platelets. Osterud (1992) has suggested that this reaction is mediated by neutrophil-derived cathepsin G, which is a more potent platelet activator than PAF (Selak et al., 1988). On the other hand, we have been unable to influence procoagulant with CP-10 or a number of other chemotactic proteins, including MIPl and 2, macrophage chemotactic proteins (MCP) 1 and 2 (Yoshimura et al., 1989), platelet factor 4, and transforming growth factor-p (TGF-p). Chemotactic peptides may contribute to the activation of cogulation in uiuo by virtue of their ability to release enzymes which may indirectly modulate the response and/ or alter expression of adhesion receptors on infiltrating cells (see later discussion). These agents induce cellular migration into tissues, a process which causes changes in the vasculature. This event alone may be sufficient to promote fibrin deposition as proposed by Dvorak and colleagues (1985). 3. Regulation of Endothelial Cell TF

The endothelium was originally considered to be a passive barrier between blood plasma and cells and the interstitial matrix, referred to by Florey (1966) as the “cellophane wrapper” of the vascular tree. However, studies over the past 10 years suggest that the normally anticoagulant surface of endothelial cells may promote coagulation when inflammatory and cellmediated immune responses are activated. TF is not normally expressed

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by endothelial cells in tissues or by these cells from a variety of sources in vitro. Many studies indicate however, that TF can be induced in culture by intrinsic stimuli such as the proinflammatory cytokines IL1 and TNFa,by thrombin and platelets, by antibodies and immune complexes, by some components of normal plasma and serum, and by extrinsic agents such as LPS, phorbol esters, and allogenic lymphocytes (see Table 11). Despite extensive studies to characterize TF induction on endothelial cells in culture, there is little evidence of its expression by these cells in pathological situations in uiuo. Although only a small number of studies have been performed, sensitive in situ hybridization techniques failed to demonstrate TF in endothelial cells lining normal mammary artery and saphenous vein. Furthermore, no TF mRNA or protein is detected in either the endothelium lining the vascular surface or small vessels within human atherosclerotic plaque specimens, whereas high levels of TF are associated with Macs (Wilcox et al., 1989). Histologicalevidence of TF associated with fetal stem vessel endothelial cells in areas of placental chorionic villi associated with chronic inflammation has been presented by Faulk et af. (1990), although these lesions contain activated Macs which represent a potential source of TF. Endothelial cells in culture were first shown to produce TF by Maynard et a!. (1979, who concluded that it was functionally dormant in undisturbed TABLE II Inducersof Endothelial Cell TF

Agent Extrinsic LPS Phorbol esters Intrinsic Platelets Thrombin Interleukin I Tumor necrosis factor-a Allogenic lymphocytes Vascular permeability factor Diacylglycerol Normal plasmalserum "Procoagulant" albumin Antibody, immune complexes Antiphospholipid antibodies Minimally oxidized LDL Histamine EpineDhnne

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cells but that scraping, freeze-thawing, or tryptic digestion-procedures that perturb the cell surface-made TF available to initiate extrinsic coagulation. These findings confirmed earlier histological observations by Ashford and Freiman (1968) that the endothelium performed a protective function and had no coagulation activity in uiuo unless it was traumatized. The first clear demonstration that TF was an inducible protein in endothelial cells (reviewed by Galdal, 1984; Prydz and Pettersen, 1988) has led to many studies to define the mechanisms of TF induction by these cell types. The TF of unstimulated endothelial cells is intrinsically low but is enhanced to varying degrees by the agents listed in Table 11. Functional activity has usually been measured using lysates of human umbilical vein endothelial cells (HUVEC). It occurs optimally after 4-8 hr in culture and returns to basal levels after 24 hr. Decay of TF activity is accompanied by a reduction in TF antigen, suggesting that it is degraded in culture rather than inhibited (Andoh et al., 1990). Cell surface expression of T F induced by LPS comprises approximately 30% of the total TF activity (Bevilacqua et al., 1984). In one study using a monoclonal antibody to assess surface TF, maximum antigen expression in response to LPS preceded maximal functional activity and capacity to bind radiolabeled factor VIIa by 2-4 hr (Noguchi et al., 19891, and followed kinetics similar to those described for induction of mRNA synthesis (Crossman et al., 1990). Noguchi et al. (1989) propose that the TF apoprotein may require a post-translational conformational change at the cell surface as described for the insulin proreceptor (Olson and Lane, 19871, reorganization of phospholipid cofactors important for factor VII binding, and/or proteolytic processing to achieve optimal functional capacity. The functional properties of surface T F on HUVECs stimulated with thrombin has been compared with purified TF apoprotein in reconstituted mixed phospholipid vesicles. With both types of TF, the rate of factor VIIa/TF activation of factor X is severalfold faster than that of factor IX, indicating that the microenvironment of T F within the membrane does not alter the kinetic parameters of factor VIIalTFdependent activation in favor of factor IX as the substrate (Almus et al., 1989). The estimated affinity constant for TF-mediated binding of factor VII/ VIIa on LPS-stimulated HUVECs is 17.2 ?Z 5.2 nmol and approximates levels of FVII in plasma (10-20 nmol) with an estimated 342,000 +- lo00 binding sites/cell (Clozel et al., 1989). Effective triggering of the extrinsic coagulation pathway on the cell surface may depend on the number of molecules of FVIWIIa bound rather than on the affinity of FVII/VIIa for TF, as long as the affinity is sufficient to achieve receptor occupancy under physiological conditions. Furthermore, the recent demonstration

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of at least two independent binding sites for factor VIUVIIa on stimulated endothelial cells, of which about 10% are TF-specific and the rest are shared with equal affinity by other vitamin K-dependent proteases via the Gla-domain common to these proteins, suggests that the nonspecific sites may facilitate the assembly of components of the extrinsic coagulation pathway on the cell surface by lateral recruitment of these factors (Reuning et al., 1993). Treatment of cells with cycloheximide or actinomycin D during T F induction confirmed the response to be dependent on de nouo protein and RNA synthesis (Galdal et al., 1985; Lyberg et al., 1983a; Nawroth and Stem, 1986; Schorer et al., 1986). Northern blot analysis indicates that T F mRNA is rapidly induced in HUVECs by LPS, phorbol myristate acetate (PMA), TNF-a and ILI-@,with maximal peak activity variously reported to be between 1 and 3 hr, after which it rapidly declines (Busso et al., 1991; Crossman er al., 1990; Scarpati and Sadler, 1989). Recent studies confirm that PMA stimulates transcription of the TF gene (Scarpati and Sadler, 1989), causing a 10-fold increase compared with the twofold increase in transcription levels following LPS stimulation and that these values parallel levels of functional acti-.,i:y (Crossman et al., 1990). Crossman er al. (1990) suggest that LPS stimulation of T F mRNA in HUVEC is substantially controlled by message stability whereas PMA stimulates transcription. Functional studies by Andoh et al. (1990) indicated that cycloheximide increased the half-life of TF induced by PMA from about 8 to 30 hr, and other studies suggest that a labile protein may mediate T F transcription (Busso et al., 1991; Scarpati and Sadler, 1989). In addition to direct induction by a single agent, T F expression can be augmented either by coculture with a second reagent or by some other cell types. LPS has been almost universally used as a standard stimulant for T F induction in cultured endothelial cells. In contrast to the exquisite sensitivity of human Mos to LPS (in the picogram range; see earlier discussion), endothelial cells require approximately 103-106higher doses. These levels are higher than those frequently measured in plasma from humans suffering with sepsis or in patients with sustained organ failure (Wharram et al., 1991), suggesting that the potential contribution of endothelial cell T F to the pathological consequences of septicemia may be due to a combination of LPS and cytokines. Furthermore, Drake and Pang (1989) suggested that the fibrin formed in the vegetations of infective endocarditis was apparently not a consequence of endothelial cell T F produced as a result of infection. Using endothelial cells isolated from human cardiac valves, these authors demonstrated high levels of T F in response to LPS but not following exposure to viable enterococci, viridans streptococci, or an enterococcal cell wall preparation. In this situation, indirect activation by other mediators produced locally as a result of

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infection (e.g., thrombin and ILI and TNF-a produced by activated Macs) may contribute to TF-initiated thrombosis in endocarditis. Early studies demonstrated that TF induction on HUVECs cultured with phytohemagglutinin (PHA), PMA (Johnsen et al., 1983)or LPS (Brox et al., 1984) or heat-aggregated IgG and sera from two patients with lupus (Galdal et al., 1985) was augmented by platelets and platelet aggregates whereas platelet-derived mediators played only a minor role. Although the procoagulant activity had plasma coagulation factor dependencies consistent with TF, it is possible that platelets provide an increased phospholipid surface that serves to assemble the prothrombinase complex in close proximity to TF induced on cultured cells, rather than enhancing TF gene transcription/antigen per se. Studies by Stern and colleagues (1985a) support this view. Scanning electron microscopy (EM) showed that LPS-activated rabbit aorta endothelial cells generate fibrin in the presence of factors VIIa, IX, VIIJ, X, prothrombin, and fibrinogen. Platelets increased thrombin formation about S f o l d , and platelet-derived factor V was thought to contribute to the response. Platelet-associated ILI expressed following activation by thrombin, ADP, and collagen (Hawrylowicz, 1993) may contribute to this response. Cooperation between endothelial cells and leukocytes and mononuclear cell lines has also been described (Lyberg et al., 1983a). Lymphocytes and Mos directly stimulated TF synthesis, which was augmented by PMA or PHA. Both CD4+ and CD8+ allogenic lymphocytes induce approximately a 6-fold increase in HUVEC TF. In contrast to other agents, this response is biphasic, with peak activity evident following 16 and 72 hr of coculture and may be mediated, at least in part, by cytokines released into the medium during allogenic stimulation although these were not identified (Lyberg et af., 1983a). Normal neutrophil/Mo suspensions, either directly in contact with cultured endothelial cells or separated by a semipermeable membrane, also enhance procoagulant levels in the absence of an exogenous stimulus (Schaub et af., 1990). Approximately 45% of the activity was attributed to ILl-/3 and was possibly produced by any or all of the three cell types in the mixture. The contribution of ILI to endothelial cell TF induction has been confirmed in coculture experiments by Wharram e? al. (1991), who showed that human Mos slightly increased basal procoagulant levels whereas they act in a synergistic manner to increase the sensitivity of endothelial cells to LPS by a factor of lo4. The response was amplified some 40-fold by heat-aggregated IgC, which has been shown by others to provoke cytokine secretion by LPS-sensitized monocytes and/or endothelial cells (Arend et al., 1985). Stern and colleagues (1985a) suggested earlier that an integral part of the endothelial cell response to injurious stimuli was the regulatory effect

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of endothelial cell-derived ILI on both the inflammatory process and the vessel wall, and showed that thrombin and LPS-stimulated endothelial cell supernatants contained procoagulant-inducing activity attributed to IL1. Supernatants from the stimulated cocultures described above (Wharram et al., 1991) induced TF, 98% of which was inhibited by anti rILI-p but not by anti-r ILI-a or anti-rTNF-a, which suggests that IL1-p is the principal cytokine involved. Although human ILl-a and -p stimulate endothelial cell procoagulant with approximately equal efficiencies (Dejana er al., 1987), ILl-p is the major soluble product of activated Mo. On the other hand, TNF-a is also a major product of activated Mo and was detected in these supernatants at levels approximating 20 ng/ml. In comparison with ILI-p, TNF-a induces weak T F activity in endothelial cells (Yong et al., 1991); the activity of rILI-0 is about 200 times that of rTNFa and these two cytokines act in synergy over a narrow dose range to induce TF. Additional factors are apparently also involved because anti-r-lL 1-p and -aand anti-TNF inhibit stimulated cell coculture by only 58%. This proposal is supported by the fact that T F induced by rIL1-P alone peaks at 6 hr and rapidly declines whereas the procoagulant response is prolonged through at least 24 hr by Mos. Although the majority of studies describe T F induction 4-8 hr after stimulation with recombinant TNF-a or IL1, Schorer et al. (1986) describe persistent T F activity induced by native ILI on HUVECs over 24 hr. The differences may reflect variations in assay or culture conditions or ILI preparations. The contribution of ILI-p derived from other cell types is also worth considering. The rapid release of neutrophil-derived ILI may play a role in early inflammatory response (Goto et al., 1984), particularly in conditions such as vasculitis, in which these cells play a key role (Bacon, 1991). Furthermore, neutrophil ILl-/3 is upregulated by a number of stimuli, including LPS, GM-CSF, TNF, and IL1 (Goh et al., 1989; Lindemann et al., 1988; Marucha et al., 1990). The combination of IL1-p and TNF cooperatively induce on neutrophil IL1-p gene expression, which is rapid and which may augment inffammationbefore mononuclear cells are prominent within the exudate. IFN-y is an important T-lymphocyte-derived mediator involved in activation of a number of cell types and which can also influence endothelial cell function (Pober, 1988). As described earlier, IFN-y can profoundly augment the procoagulant responses of inflammatory Macs. rIFN-y weakly enhances T F induction by LPS, PHA (Almus et al., 19891, and IL1-p (Yong et al., 1991) on HUVECs although it may not act through priming since it increased T F when it was added after LPS or IL1-p, and prolonged responses to these agents over 16 hr. In contrast to its effect on TG-elicited Macs, the reported enhancement of the LPS response

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on HUVECS is comparatively weak. The increase in total cell activity (lysed cells) induced was only about 2-fold and much higher doses (102-104U/ml) were required for endothelial cells than for Macs (0.1lo2 U/ml). The significance of IFN-y-enhanced endothelial cell TF is unclear although its principal function may be to maintain the response induced by other stimulants. A recently described 44-kDa protein isolated from supernatants of murine fibrosarcoma cells and related to vascular permeability factor (Keck et al., 1989) amplified the TF mRNA expression induced in HUVECS by low concentrations of TNF-a (Clauss et al., 1990a,b).The factor had little effect alone but enhanced TF antigen expression approximately threefold over 4-6 hr when it was added in the picomolar dose range. Clauss and colleagues suggest that it may increase the affinity and number of endothelial binding sites for TNF-a to cause enhanced TF activity. This may result in the formation of occlusive thrombi within the vasculature and alterations in vascular function within the tumor microenvironment. In addition to the cytokines discussed above, endothelial cell procoagulants are induced by some modified proteins, including glycosylated albumin, which are produced to resemble advanced glycosylation end products (AGE) of proteins (Esposito et al., 1989), and a “procoagulant albumin” constituent of normal plasma, the precise modification of which is unknown (Faucette et al., 1992). Such modified proteins may accumulate in the vasculature of older subjects and patients with diabetes, and may be associated with vascular damage. The effect of AGE-bovine serum albumin (BSA) on endothelial procoagulant was slower in onset than that observed for “procoagulant albumin” and other cytokines and required at least 2-3 days for induction of maximal activity. Both forms of albumin induced relatively weak activity approximately equivalent to that produced by TNF-a whereas AGE-BSA-activated cells (2 days) expressed about eightfold more activity when subsequently stimulated for 6 hr with TNF-a. This suggests that such proteins may sensitize the vasculature to exhibit amplified procoagulant activity in response to other inflammatory stimulants. Because of the potential importance of cellular procoagulants in mediating tissue injury and clot formation in atherosclerosis, modified lipoproteins have been implicated in TF induction in endothelial cells. Native low-density lipoprotein (LDL) is ineffective whereas minimally oxidized LDL (mm-LDL), a form of LDL which alters several functions of endothelid cells that modulate interactions with blood cells, enhances TF mRNA levels approximately 30-fold within 2 hr, with peak activity in lysed cells evident after 4-6 hr (Drake et al., 1991). These studies differ from the

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majority of those described above in that cultured aortic and cardiac valve endothelial cells were used in the experimental system. The effective form of mm-LDL was recognized by the LDL receptor, indicating minimal modification of the apoprotein whereas both highly oxidized LDL and malondialydehyde-modified LDL, forms of LDL recognized by the scavenger receptor, failed to induce TF. In contrast to the levels of TF produced by many agonists, optimal doses of mm-LDL preparations (40 pg/ml) enhanced TF activity from 8- to 50-fold, which is equivalent to levels achieved for maximal stimulation with LPS. Low concentrations had an additive rather than synergistic effect when tested in combination with IL-I , whereas low levels of LPS caused no enhancement. In spite of the large number of studies of endothelial cell TF induction in uitro, the paucity of reports demonstrating TF antigen or mRNA in pathological specimens raises questions about its physiological relevance. Furthermore, discrepancies exist within the literature concerning the optimal time of induction of TF mRNA by a variety of stimulants and the duration of TF expression. These differences may be explained, in part, by the source of endothelial cells used (generally human umbilical vein, which may be inappropriate for studies aimed at questions concerning thrombosis or atherosclerosis), the growth conditions and factors used, the number of passages cells have undergone before testing, and measurement of total cell (lysed) versus viable cell TF activity. For example, secondary cultures (4-5 doublings)apparently respond better than primary cultures, but at higher doublings the response declines (Andoh et af., 1990; Busso et al., 1991; Prydz and Pettersen, 1988). Endothelial cells develop hyporesponsiveness to a stimulant followingprolonged incubation so that serum factors or LPS may alter the procoagulant potential of these cells even though they may respond well to a second agent (Busso et al., 1991; Moore et al., 1987). Sensitization is not limited to TF induction and may represent another level of modulation of endothelial cell function by inflammatory agents in uiuo. Growth factors may also influence the experimental outcome (see later discussion). A variety of model systems have been used in attempts to simulate conditions in uivo. Effects of human rIL-IP (1 pg/kg) on coagulation in rabbits 0.5 or 4 hr after systemic injection, after which stasis was induced in isolated segments of each jugular vein, were studied microscopically. Perturbed endothelium was indicated by increased surface microvilli, blebs, and gaps at cell junctions. Fibrin was seen as single strands in close association with the surface rather than as a clot, and single disk-shaped platelets (nonactivated) sometimes overlay the fibrin strands (Merton et al., 1991). Fibrin formation was consistent with studies by Nawroth et af. (1986) who infused ILl into rabbits and demonstrated fibrin deposition

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on intact arterial endothelium. Although activated platelets, leukocytes, and red cells were found within the fibrin network, Merton and colleagues suggest that the amplification of procoagulant activity by IL1 alone is insufficient for the formation of an acute occlusive venous thrombosis. Studies using perfusion chambers to simulate conditions of blood flow showed that HUVECs cultured with LPS or TNF-a induced fibrin deposition at relatively low shear rates (100-300 sec-I) (Clozel et al., 1989; Tijburg et al., 1991b). Under these conditions LPS exposed some of the extracellular matrix (ECM) between cells and this also supported platelet and fibrin deposition. Weiss et al. (1989) found that the critical pathway of fibrin deposition on subendothelium of segments of rabbit aorta after exposure to flowing blood was TF dependent. Procoagulant activity was located immediately beneath the endothelium in both human umbilical artery and rabbit aorta, confirming the immunohistochemical location of TF in the deep layers of the vessel wall rather than at the surface. Although TF may be derived from fibroblasts or smooth muscle cells (Maynard et al., 1975; Zacharski and McIntyre, 1973a), endothelial cell-derived TF has been shown within the ECM. Using small diameter capillaries lined with HUVECS, Lindhout et al. (1992) demonstrated a 20-fold greater activity in the ECM than at the surface following perfusion with TNF for 4-8 hr. Scanning electron microscopy indicated reduced integrity of the monolayer under these conditions, exposing the ECM. The cellular origin of TF protein trapped within the ECM of the necrotic core of atherosclerotic plaque (Wilcox et al., 1989) is, however, unknown. Initiation of coagulation by TF-factor VIIa on perturbed endothelium is some 10-fold greater in the presence of factors IX and VIII than when only factor X is present (Stern e f al., 1985b; Tijburg et al., 1991b). Highaffinity factor IXa binding sites are proposed to mediate this response (Dorian et al., 1989) although the relationship between this receptor and the vitamin K-dependent Gla-binding domain of TF described by Reuning et al. (1993) is unclear. Active site-blocked IXa (IXaJ blocked the TFfactor VIIa-initiated responses of TNF-activated endothelial cells in plasma whereas activity of the ECM (some 10- to 20-fold greater than on cells) was inhibited by IXq only when low concentrations of TNF were used to induce TF. By contrast, Xa(i) inhibited reactivity induced by all concentrations of TNF. These studies emphasize the link between TF-factor VIIa-mediated activation of factors IX and X, and again indicate the importance of the IX/IXa pathway in circumstances where low levels of TF are expressed. TNF-a-induced TF that is associated with membrane vesicles at the subendothelial matrix but not on the apical surface has recently been

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described (Ryan et al.. 1992).TF that is associated with membrane vesicles shed from a variety of TF-bearing cells has been reported by others (Bastida et al., 1984;Bona et al., 1987)and this may represent amechanism of sequestering TF in the subendothelium to promote the formation of thrombin at sites of vascular injury. The importance of the subendothelium in the rapid initiation of the hemostatic process after its exposure is well accepted. In addition to sequestering TF, thrombin interacts with cell surface heparan sulfate receptors (Shimada and Ozawa, 1985) and with dermatan sulfate in the ECM (Bar-Shavit et a!., 1989). Thrombin bound to the ECM can convert fibrinogen to fibrin and activate platelets. Sequestered thrombin may contribute to the enhanced platelet adhesion to the ECM observed followingperturbation of endothelial cells (de Groot et al., 1987). Furthermore, ECM-immobilized thrombin is protected from inactivation by antithrombin and heparin, which suggests an altered conformation of the bound enzyme (Bar-Shavit et al., 1989). The sequestration of procoagulants on the ECM may not only play an important role in acute thrombosis, but may have functional significance during inflammation. 4. Tissue Factor on Other Cell Types

Zacharski et al. originally described a coagulant expressed by fibroblasts in tissue culture which had properties associated with TF (Zacharski et al., 1973; Gordon and Lewis, 1978; Green et al., 1971). The procoagulant increased several hundredfold over 12-24 hr of incubation, was correlated with cell adhesion (Zacharski and McIntyre, 1971), and required new protein and RNA synthesis (Zacharski and McIntyre, 1973a,b). Subsequent studies indicated that TF associated with the surface of cultured smooth muscle cells and fibroblasts was in a dormant state until perturbed (Maynard et af., 1975). This lead to the proposal that TF expression by selected extravascular cells is fundamental to hemostasis and initiates clot formation as a consequence of injury. In spite of the fact that TF expression by these cells has been known for many years, and murine (Hartzell et al., 1989; Ranganathan et al., 1991)and human (Morrissey et af.,1987)fibroblasts were used as a source of cDNA for its cloning, relatively few studies have been performed using these cells. The sequential expression of specific genes designated immediate-early genes, which mediate the growth response of fibroblasts to growth factors, include those encoding TF (Hartzell et al., 1989). Platelets enhanced TF expression (Smariga and Maynard, 1982a), possibly via a soluble platelet protein (Smariga and Maynard, 1982b). TF mRNA is now known to be upregulated in mouse 3T3 fibroblasts within 20 min by

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platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (Hartzell et al., 1989). The gene is transcriptionally activated by growth factor and superinduced by cycloheximide. In comparison with PDGF, epidermal growth factor, and insulin, which induce relatively low levels of T F activity on murine AKR-2B fibroblasts, TGF-/31 induces high amounts and this effect is apparently not coupled to mitogenesis (Ranganathan et d., 1991). These studies have resulted in speculation that T F may have functions in addition to those involved in hemostasis and that they may include embryonic development, cell growth, and wound healing. Calcium ionophore A23 187 rapidly and reversibly enhances the surface expression of TF on bovine fibroblasts to levels achieved by disruption. Bach and Rifkin (1990) suggest that surface expression of TF is mediated by Caf2-dependent changes in the asymmetric distribution of phosphatidylsenne in the plasma membrane. Redistribution of phosphatidylserine may be important in the association of TF with factor VIIa. Human fetal lung fibroblasts bind factor VIIa in a Ca2+-dependent manner to about 100,000 high-affinity binding sites per cell (Ploplis et al., 1987). Unlike Mos, specific high-affinity binding is abolished by high levels of Ca2+ whereas dissociation constants for factors VII or VIIa are similar on both cell types. In addition, fibroblasts bind factor X, with the estimated number of binding sites approximately 7% of those estimated on endothelial cells. Association of factor VIIa and X at levels less than normally present in plasma (10 and 100 nM, respectively) with the fibroblast surface may be important in inflammatory lesions, or in tissue injury, where plasma extravasation would provide sufficient protein to render these cells functionally active. In addition to the procoagulant activity associated with alveolar Macs in inflammatory lung diseases, and in support of the immunohistological data of Drake et al. (1989a), alveolar epithelial cells express some 10- to 20-fold greater TF activity than Macs. The activity is on the surface of unstimulated cells, and mRNA and activity levels increase about twofold following stimulation with PMA (Gross et al., 1992). The failure of LPS to enhance TF expression may be due to the desensitization of these cells in a manner similar to that described for endothelial cells and Macs, due to the likelihood of their constant exposure to inhaled LPS. Mesangial cells and epithelial cells within the glomerulus also normally express TF, which is increased in glomerulonephritis (Drake et al., 1989a; Neale et al., 1988)although the majority of the procoagulant activity may be derived from infiltrating Macs. Little more is known about the regulation of epithelial cell procoagulant activity, particularly its modulation by cytokines, and its physiological importance may be restricted to specific tissues.

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111. Procoagulants A. Regulation of Procoagulant Activity by Adhesion Receptors

It has been known for many years that adherence of Mos to plastic is sufficient to induce TF expression (van Ginkel et af., 1977). Many earlier studies of Mo/Mac TF induction used cells (blood Mos, splenic Macs) adhering to plastic or fibronectin-coated plates and activity of cell lysates was usually measured. By contrast, all the studies carried out in our laboratory have used nonadherent conditions to study procoagulant expression by viable cells. It is possible that adhesion may itself function as a priming step to amplify responses to other agents, but few careful comparative studies have been made. Miserez and Jungi (1992) recently showed that although IFN-y is a strong inducer of TF on human Moderived Macs grown in suspension, it fails to activate adherent Macs whereas LPS induces activity under both circumstances. The leukocyte-restricted differentiation antigen, CDll/CD18, plays a major role in inflammation. CDl lb/CD18 (Mac-l) is important in adhesion reactions of neutrophils and Mos. Furthermore, CDl lb/CD18 on Mos binds factor X with reasonable affinity (2 x M ) in a Ca’+-dependent manner (Altieri and Edgington, 1988a; Altieri et af., 1988b). Altieri and colleagues have shown that a number of agonists, including ADP, ionomycin, and the chemotactic peptide FMLP, induce functional epitopes within CD1 lb/CD18, absent under resting conditions, to an “activated” state capable of binding factor X (Altieri and Edgington, 1988b).Activation is rapid, with optimal binding after 20 min; factors 11, VII, IX, IXa, or Xa fail to compete for binding. A sustained increase in cytosolic free [Ca2+Ii,coupled with variations in transmembrane potential, is produced by ADP. An intracellular signaling pathway, possibly involving protein kinase C in a manner similar to that of other classical mobilizers of cytosolic Ca2+,such as FMLP, C5a, and IL8, is proposed to modulate the transient and functional upregulation of this receptor (Altieri et al., 1990). In the absence of demonstrable TF or TF-VIINIIa complex, ADPstimulated Mos and myeloid cells expressing Mac- 1 directly convert surface-bound factor X to the active form, Xa. Neutralizing antibodies to TF fail to block this response and an unknown enzyme of cellular origin is thought to process the inactive zymogen (Altieri et af.,1988a). Furthermore, engagement of the CDl lbKD18 complex either with antibodies or by binding to immobilized fibrinogen enhances the Mo TF response to LPS or MPIF some two- to eightfold (Fan and Edgington, 1991).

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These findings suggest that augmentation of the procoagulant response after ligation of CD1 lblCD18 may represent an important amplification mechanism in inflammatory lesions. It may be particularly important in the enhanced TF production which may occur under some circumstances of T cell-Mo cooperation (Farram et al., 1983) and in the interactions between endothelial cells and Mos or neutrophils described later. In addition, CD1 lb/CD18 binding of opsonized (C3bi) microorganisms may have a similar effect, thereby potentiating fibrin formation in infection. The levels of CDllb/CD18 on neutrophils are also modulated by a number of proinflammatory agents. Both blood neutrophils and a myeloid cell line HL60 (differentiated with dimethylsulfoxide to a cell with granulocyte properties) express functionally competent Mac- 1 receptor after ADP stimulation, with characteristics of factor X binding similar to those of circulating Mos or related cell lines (Altieri and Edgington, 1988a; Altieri et al., 1988b).This pathway of procoagulant induction has many physiological implications. Adenine nucleotides are continuously generated during normal hemostasis at sites of vessel injury and inflammation (Born and Kratzer, 1984),and platelets represent a major source. Inflammatory sites characteristically involve localized activation of neturophils and/or Mos, and migration of these cells from the blood in response to chemotactic stimulation upregulates CD11b/CDl8. Because neutrophils possess no

activates endothelial

Factor Xa on Mo/Mac endothelium

thrombin

FIG. 3 Neutrophils can mediate procoagulant responses (see text).

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other known activators of the extrinsic coagulation cascade, the CDl Ib/ CD 18-mediated pathway may induce rapid localized fibrin formation following transmigration of these cells. Furthermore, CDl 1b/CD18 rapidly binds fibrinogen although under comparable conditions factor X competes for fibrinogen binding (Altieri et al., 1988a). In addition, CDl lc/CD18 on TNF-a-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the Aa chain of fibrinogen (Loike et al., 1991). Neutrophil binding to fibrin occurs via the 95-kDa P-chain common to the three members of the leukocyte integrins (Cooper et al., 1984). The properties of neutrophils which indicate their potential role in activation of coagulation are illustrated in Fig. 3. Neutrophils rapidly accumulate within fibrin thrombi (Cooper et al., 1984), and Macs at sites of antigen challenge are enmeshed in a fibrin network (Hopper et al., 1981). A number of years ago we suggested that fibrin formation may serve to localize phagocytic cells at inflammatory sites and that this process was the basis of migration inhibition by such cytokines as macrophage migration inhibition factor (MIF) (Geczy and Hopper, 1981; Hopper et al., 1981). The data reported here indicate the feasibility of this hypothesis although the ability of the recently cloned MIF (Weiser et al., 1989) or MIF-related proteins (Odink et al., 1987) to influence CDl 1/CD 18 expression and/or procoagulant induction has not been reported. B. Other Cellular Procoagulants

Activated Mo or Mac have been reported to assemble or express all of the proteins of the extrinsic coagulation pathway. Differences in experimental design and assay procedures, source of cells (differentiation and maturation state of the cells, for example, can affect the procoagulant response), and a variety of stimulants may contribute to the differences in results obtained in different laboratories. In addition, many studies using animal cells, particularly those from mice, have used human or bovine plasmas and/or coagulation factors to define a particular procoagulant response and the species specificities of these factors makes identification difficult (Bach, 1988; Janson et al., 1984). In the absence of specific reagents to identify the murine proteins, MoMac procoagulant characterization was mainly derived from functional studies. Shands (1984) summarized the early work describing procoagulants, in addition to TF, detected on murine peritoneal Macs (factors IX, X, VII, V, 11),murine TG-PEC (factor VII, factor X activator), and murine and human blood Mos (prothrombinase and factor VII). It is surprising

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that although cDNA probes and antibodies to human factors VII and X have been available for some time, little is known about the regulation of these factors in human Mo/Mac or other cell types. Only activities characterized recently are described here (see Table 111). A unique prothrombinase produced in response to specific lipoproteins and first reported by Schwartz, Levy and colleagues (Levy et al., 1981; Schwartz er al., 1981, 1982b) was not neutralized by antifactor X serum or inhibited by a panel of factor Xa inhibitors, indicating the activity was not factor Xa (Ottaway er al., 1984). In response to murine hepatitis virus type 3 (MHV-3) infection, Mo/Mac prothombinase increased to maximal levels within 4-6 hr and its expression correlated strongly with susceptibility or resistance to the disease (Dindzans et al., 1986).The Ca2+-dependent activity within the cell membrane is a serine esterase. Fung et al. (1991) recently produced a panel of monoclonal antibodies which failed to react with TF but some of which influenced the prothrombiTABLE 111 Some Cellular Activators of Coagulation

Activity

Cell type

Factor VII

Mo/Mac

Factor V/Va

Factor Va light chain-like protein (Effector cell protease receptor-], EPR-1) Cysteine protease

Platelets, Mo/Mac, lymphocytes, neutrophils, endothelial cells Mo, monocytoid/myeloid cell lines, some neutrophils, NK cells, and some lymphocytes Endothelial cells

Glycoprotein gC

Endothelial cells

CDIIb/CD18 (Mac- 1)

Mo/Mac, neutrophils

Unique prothrombinase (two-chain protein, 74 and 70 kDa)

Mo/Mac

Properties May be inadequately ycarboxylated. Antigensensitized lymphocytedependent induction Induced by thrombin, LPS, PMA on endothelial cells Binds factor Xa, lymphocyte EPR-I enhanced by PHA, conA, allogenic cells Activates factor X induced by hypoxia Facilitates binding and activation of factor X; HSV infection; gC encoded by HSV genome Binds factor X-processed to factor Xa, binds fibrinogen; activated by ADP, FMLP, IL8 Activates prothrombin induced by viral infection (murine hepatitis virus)

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nase activity on MHV-Istimulated murine Macs. The protein is not detected on normal cells and requires new protein and RNA synthesis for expression. Numbers of positive Macs and staining intensity correlated with multiplicity of infection. Antibodies reacted with proteins of 140, 74, and 70 kDa (nonreduced) and 74 and 70 kDa (reduced). Interestingly, LPS-stimulated Macs (TF-positive)failed to bind the antibodies and the molecular weight of the prothrombinase is substantially greater than the reported mass of TF. Although the amino acid sequence of the prothrombinase has not been reported, this is apparently a novel procoagulant associated with viral infection. Sinclair et al. (1990) suggest that Mo/Mac procoagulants induced by bacteria and viruses may represent disparate routes of procoagulant induction. Our finding of an as-yet uncharacterized prothrombinase-like activity together with TF induced by LPS following IFN-y-priming (Moon and Geczy, 1988; Wheeler et al., 1991) suggests a unifying pathway. In addition to its antiviral role, IFN-y is thought to be a key mediator in the Schwartzman reaction (Heremans ef al., 1990) and in endotoxemia (Heinzel, 1990), and may therefore contribute to the coagulopathies associated with bacterial sepsis. Furthermore, the synergistic response of two procoagulants induced on the cell surface by different combinations of stimulants would represent a potent amplification of the procoagulant potential at inflammatory sites. The role of platelets in the assembly of the prothrombinase complex is well documented and occurs via Ca2+-dependent I :1 stoichiometric complex formation of factor Xa with the nonenzymatic cofactor Va. The cofactor contributes to the amplification of prothrombin activation by altering the kinetics of its cleavage and by stabilizingthe enzyme-substrate complex (Mann er al., 1990). A number of agents which activate platelets induce the release of microparticles which are enriched for binding sites (possibly phospholipid in composition) for factor Va, and their release parallels the expression of the catalytic surface for the prothrombinase complex. Factor Xa activates the factor V associated with microparticles 50-100 times more effectively than thrombin (Monkovicand Tracy, 1990). Thus factor Xa generated by cellular TF-factor VIIa may activate factor V bound as platelet microparticles or aggregates. In addition, membrane surfaces provided by Mo, and lymphocytes (Tracy ef af.,1983), neutrophils (Tracy et af., 1985), and intact (Rodgers and Shuman, 1983) and perturbed (Annamalai et af., 1986; Rodgers and Kane, 1986; Rodgers and Shuman, 1983) endothelial cells, but not nonvascular cells such as fibroblasts and visceral lung cells, express factor V. Rodgers and Shuman suggest heterogeneity within the endothelial cell population with aortic cell 2 umbilical cell > microvascular cell activity. Most of the factor V is present within the membrane of endothelial cells although a

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secreted, unactivated form is produced by cultured cells (Cerveny et al., 1984). Thrombin, LPS, and phorbol esters upregulate factor V production on endothelial cells but to date there is no evidence of cytokine involvement (Rodgers, 1988).In contrast to earlier studies suggesting that platelets were required for the formation of prothrombinase on the endothelial cell surface, Stem et al. (1985b) and Tijburg et al. (1991a) showed no requirement for exogenous factor V and propose that endogenous factor V(a) is involved in prothrombin activation on the cell surface and that activation of the zymogen by thrombin may regulate the rate of reaction. Relatively few studies have been performed to define the contribution of factor V to the procoagulant response of mononuclear cells. Tracy et al. (1983) showed that Mo prothrombinase activity was mediated by highaffinity factor Va binding sites, whereas factor V was intracellular rather than membrane-associated, More recent studies by this group (Worfolk et al., 1992) indicate that factor Xa interacts with the Mo through two sites distinguished by their requirements for factor Va and their expression of different functional activities. Thrombin is generated from prothrombin solely by a membrane complex of factors Va and Xa, whereas in the absence of added factor Va, Mo-bound factor Xa cleaves factor IX to the nonenzymatic intermediate IXa. This may be processed to the active form by TF-factor VIIa (Lawson and Mann, 1991) following Mo stimulation. Factor Xa binds to monocytoid/myeloid-cell lines THP, U937, and HL60 via a membrane protein of approximately 74 kDa which cross-reacts with a monoclonal antibody to factor Va light chain (Altieri and Edgington, 1989). The term “effector cell protease receptor 1” (EPR-1) was given to this protein, which it is suggested represents a cell-surface analog of plasma factor V/Va (Altieri and Edgington, 1990). The claim that EPR-1 provides a cofactor-like effect in factor Xa-catalyzed prothrombin activation is disputed by Worfolk et al. (1992) who found that added factor Va is essential for this activity on Mo and that factor IX is the preferred substrate in its absence. These studies are in keeping with the current knowledge of how factor Va functions in the prothrombinase complex. The light chain of factor Va forms part of the factor Xa binding site at the cell surface (Higgins and Mann, 1983;Tracy and Mann, 1983)whereas the heavy chain binds prothrombin (Guinto and Esmon, 1984), indicating a requirement for both subunits to assemble the enzyme and substrate of the prothrombinase complex. Apart from the reported ability of lymphocytes to assemble a functional prothrombinase complex (Tracy et al., 1983), normal lymphocytes were considered to be devoid of procoagulant activity. However, Altieri and Edgington (1990) recently reported EPR-1 expression on a subset of CD3+ cells coexpressing CD2, CD4 or CD8, CD57, CDl lb, and apT-cell receptor. Furthermore, peripheral blood lymphocytes stimulated with PHA,

CELLULAR MECHANISMS FOR ACTIVATING COAGULATION

81 concanavalin A, or allogenic cells expressed 8- to 10-fold more EPR-1. A population of natural killer (NK) cells and a heterogeneous population of neutrophils also express EPR-1 although nothing is known concerning its regulation in these cells. These findings suggest a role for EPR-1 in immune effector function and possibly in fibrin formation in the early stages of DTH reactions. Together with the capacity of CDIIb/CD18 on infiltrating leukocytes to bind and activate factor Xa, these localized cellular reactions may result in sufficient thrombin formation to initiate an early response. In addition to its ability to alter the procoagulant potential of endothelial cells and platelets, thrombin is chemotactic for phagocytic cells (Bar-Shavit et al., 1983), processes IL8 (Herbert et al., 1990) and ILl (Jones and Geczy, 1990), and alters endothelial cell permeability, all of which would contribute to enhanced and/or sustained inflammation. Infection of endothelial cells with herpes simplex virus 1 (HSV-1) (Visser et al., 1988) or cytomegalovirus (CMV) (van Dam-Mieras et al., 1992) leads to membrane perturbations resulting in enhanced factor X binding and prothrombinase activity. Visser et al. (1988) suggest that infection causes a rearrangement of negatively charged phospholipids on the outer leaflet which facilitate factor Va and Xa assembly. The recent demonstration by Etingin et al. (1990), of an HSV-encoded endothelial cell surface glycoprotein (gC) which binds and promotes activation of factor X on infected, but not uninfected cells, indicates a novel pathway for procoagulant induction. Although gC encoded by the HSV genome can function as a complement (C3b) receptor, it has no primary structural homology to Mac- 1 but may bind factor X in a manner similar to that described above for this ligand. These findings have been implicated in the pathogenesis of atherosclerosis since both HSV (Hajjar et al., 1987) and CMV (Adam et al., 1987) have been located in human atherosclerotic plaque, and in the coagulopathies associated with HSV infection (Visser et al., 1988). There are several reports suggesting that factor VII is produced by activated Mo/Mac although further definition of this activity is required. Factor VII-like activity secreted by cultured murine LPS-stimulated Macs (Shands, 1983) may contribute to the factor X activator activity of these cells by binding to TF. In contrast, human Mos can be induced by LPSactivated CD4' T lymphocytes to express a factor VII/VIIa activity which is predominantly of intracellular origin since viable cells express only 5-20% of the total functional activity (Tsao et al., 1984). Similarly, factor VII expression on sensitized blood Mos stimulated with antigen (tuberculin; Godfrey et al., 1986) or allogeneic cells (Carlsen et al., 1989) indicates a requirement for T cells and suggests involvement of factor VII in fibrin formation in cell-mediated responses. In addition, factor VII synthesized and secreted by alveolar Mac may contribute to inflammatory diseases of the lung, including idiopathic pulmonary fibrosis and adult respiratory

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distress syndrome (Chapman et al., 1985). Interestingly, these authors found increased alveolar Mac-associated factor VII activity in sarcoid patients, and propose that the disproportionate increase in alveolar T cells which occurs in these individuals may contribute to its expression. Alveolar Macs synthesize factor VII in the 48-kDa single-chain zymogen form but not the two-chain activated derivative, factor VIIa. Chapman el al. (1985) and Carlsen et al. (1989) suggest that much of the synthesis may occur in the undercarboxylated precursor form. Although factor VII mRNA has been demonstrated in extracts of freshly isolated alveolar Macs, the lack of vitamin K-dependent y-carboxylase, which is required for processing the factor into the functional form, implies that activity may be limited (McGee et al., 1989). The effect of lymphocytes on ycarboxylase activity is worthy of study. Interestingly, we found that IFNy, the T-lymphocyte product, induced a factor VIIa-like activity in murine inflammatory Macs (Moon and Geczy, 1988). The regulation of functional factor VIIa, by virtue of cytokine induction of a y-carboxylase, may represent another level of control by these mediators. The relationship of a tumor-derived cysteine protease and the factor X activator recently described by Ogawa et al. (1990) is unclear. The latter enzyme is expressed maximally 72 hr after endothelial cell cultures are exposed to hypoxic conditions but is not observed with intact or disrupted endothelium grown in ambient conditions. The Ca2+-dependentenzyme directly activated factor X. It was not inhibited by neutralizing anti-TF antibody, but HgCI, blocked activity, suggesting a cysteine protease. The physiological relevance of this enzyme is still to be determined, particularly since the V,, is lower than that of the factor IXa-VIIIa complex although alterations in the endothelial milieu (reduced pH and blood flow) may favor the factor X activator during hypoxic episodes. These data suggest that this enzyme may be expressed in response to stress, and experiments investigating this possibility may be informative. C. Procoagulants and Malignancy

The association between recurrent thrombotic episodes and malignancy was described over a century ago by Trousseau (Rickles and Edwards, 1983). The prevalence of subclinical coagulation abnormalities in patients with cancer has been reported to be as high as 98% (Sun et al., 1979) although the differences in study design among various groups make comparisons difficult. Nanninga et al. (1990) recently concluded that approximately 22% of untreated primary cancer patients exhibited subclinical coagulopathy although the percentage may be higher in patients with more advanced disease (Nand and Messmore, 1990).

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Fibrin has been demonstrated histologically within the mass of many tumors, and anticoagulant drugs have antimetastatic effects. The role of coagulation in metastasis has been discussed for many years and is thought to contribute to tumor cell invasion and localization in normal tissue (Goldfarb and Liotta, 1986), and to development of blood supply (Laki, 1974). The formation of a fibrin barrier may prevent immune recognition and lytic interaction of tumor cells with cytotoxic lymphocytes (Donati et a f . , 1986) although activated macrophages associated with tumor cells within a fibrin meshwork have been associated with tumor rejection (Dvorak et al., 1978). Alternatively, fibrin may inhibit tumor cell detachment, thereby protecting the host from tumor cell invasion (Dvorak et af.,1979). It is now clear that fibrin formation may be a consequence of direct activation of the extrinsic coagulation cascade by tumor procoagulants or their shed membrane vesicles, or indirectly by stromal cells (e.g., Macs) stimulated to express procoagulant activity by soluble tumor-derived products or by cytokines produced as a consequence of an activated immune response to the tumor. A thrombosis-inducing activity in the plasma of lung cancer patients has been described (Maruyama et al., 1989). A summary of the characteristics of human tumor procoagulants has recently been published (Edwards et al., 1993). Immunohistochemical techniques indicate a heterogeneous pattern of procoagulant expression in which cells from some tumors are poorly reactive whereas in others coagulation factors are localized either to the tumor cells or to tumor-associated Macs (Zacharski et al., 1990). T F activity is found in cells from a number of solid tumors, including adenocarcinomas of the colon, ovary, gastrointestinal tract and pancreas; squamous cell carcinomas; melanoma; neuroblastoma; and bladder; lung, and teratanocarcinomas (Edwards et al., 1993). TF is the major procoagulant identified on cells and cell lines of the myeloid lineage, many of which have been used in the studies discussed earlier to investigate T F regulation. A "thromboplastic activity of leukemic white cells" was reported by Eiseman and Stefanini in 1954 although definitive proof of the involvement of TF has been relatively recent, Increased TF activity by leukemic cells from patients with acute nonlymphoid leukemia closely correlates with the occurrence of DIC (Andoh et al., 1987;Guarini et al., 1985). Inducible T F activity has been reported on monocytoid, early myeloid, monoblastic, and myelomonocytic cell lines (Edwards et al., 1993). In addition, and unlike normal or activated blood lymphocytes, TF has also been reported on a variety of T-lymphoblastoid lymphoma cell lines (Barrowcliffe et al., 1 989). Acute promyelocytic leukemia is associated with a high incidence of hemorrhage attributed to DIC and secondary fibrinolysis (Bauer and Rosenberg, 1984). HL60 cells are derived from human leukemic promyelo-

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cytes and have high basal levels of TF activity (Wijermans et af., 1989). Differentiation into Mac-like cells with phorbol esters (Rovera et af., 1979) induces high levels of TF expression before these cells acquire other Mo/ Mac characteristics. LPS and PHA, which stimulate TF expression on normal Mos, have no effect on these cells (Kornberg et af., 1982). If HL60 cells are differentiated into granulocytes by culturing with retinoic acid or dimethylsulfoxide, there is a concomitant decrease in procoagulant activity (Wijermans et af., 1989). Cells differentiated to either Mos or granulocytes also have decreased proteolytic activity (elastase, cathepsin G), which may account for the fibrinolytic activity of normal neutrophils. The combination of high TF and granulocyte protease levels may be unique to the promyelocyte and may explain the common bleeding complications in patients with acute promyelocytic leukemia (Wijermans et af., 1989). The recent finding that neutrophil elastase and cathepsin G both inactivate tissue factor pathway inhibitor by a cleavage which restores factor Xa activity after its initial inhibition by TFPI (Petersen et af., 1992) would strengthen this hypothesis. In addition to TF, hepatoma cells secrete single chain factor X and prothrombin (Fair and Bahnak, 1984),and some carcinoma cells and their shed membrane vesicles can support assembly of a functional prothrombinase complex. Factor Va bound to some tumor cell surfaces serves as a receptor for factor Xa, with the number of factor Va-Xa binding sites comparable to values reported for platelets (Van de Water et al., 1985). In addition to the expression of normal extrinsic pathway activators, a malignancy-associated procoagulant has been described. A factor Xactivating activity first isolated from extracts of colon and kidney carcinomas and rabbit V, carcinomas (Bauer and Rosenberg, 1984) acts independently of TF/VIIa. It is a unique 68-kDa acidic protein with cysteine protease activity (Falanga and Gordon, 1985; Gordon and Cross, 1981) which has only been described in neoplastic and fetal cells (Gordon et af., 1985). Varying levels of the enzyme were isolated from primary and metastatic human melanomas, but not benign nevi, with the highest activity in metastatic samples (Donati et al., 1986). Its involvement in metastasis has been strengthened by a close correlation between its expression and the metastatic potential of B16 melanoma cell variants (Gilbert and Gordon, 1983). This procoagulant has also been located in extracts of blast cells from patients with acute nonlymphoid leukemia, and Falanga et af. (1988) suggest varying proportions of factor X activator and TF in different cytological subtypes although the relative contributions of these two procoagulants in coagulopathies is still unclear. Furthermore, the recent demonstration in extracts of rat sarcomas of another factor X activator, a serine protease with properties distinct from TF-factor VIIa, which acti-

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vates factor X perfused within the tumor bed in uivo (Pangasnan et al., 19921, indicates that additional factors may also be involved. Increased incidences of thrombotic problems in nonlymphoid malignancies have been associated with the use of chemotherapeutic drugs (Doll et al., 1986; Goodnough et al., 1984; Levine et al., 1988; Sills et al., 1978; Weiss et al., 1981) and Fibach et al. (1985) suggested that these agents may contribute to the thrombotic complications of patients undergoing treatment for acute promyelocytic leukemia. Our earlier experiments indicated that doses of cycloheximide suboptimal for the inhibition of protein synthesis (0.5-1 pg/ml) superinduced procoagulant activity on murine TG-PEC in response to IFN-y or LPS (Moon and Geczy, 1988). Similar effects were noted with human blood Mos although we demonstrated a discordant relationship between the levels of TF antigen, which fell by about 70%, and functional activity, which rose by approximately 100% (Walsh and Geczy, 1991). The binding capacity of TF on LPS/cycloheximide-stimulatedMos is about the same as that of LPS-stimulated cells, suggesting that factor VII binding need not directly correlate with TF cofactor activity as suggested by Rodgers et al. (1984). We propose that the reduction in protein synthesis caused by cycloheximide may affect membrane fluidity and/or stabilization by altering protein and phospholipid turnover. Whereas exogenous phospholipid enhanced the TF activity of LPS-activated human Mos, it had no effect on LPS/cycloheximide-stimulatedactivity, which suggests that optimal levels of acidic phospholipids were available within the membrane to facilitate factor VII binding and the allosteric change in factor VIIa necessary for enhanced catalytic function (Walsh and Geczy, 1991). Bach and Rifkin (1990) suggest that modulation of the distribution of phosphatidyl serine in the plasma membrane of cells expressing TF may regulate its function. These studies prompted us to investigate whether other cytotoxic agents which intercalate with DNA or RNA, may modulate Mo/Mac procoagulant activity in a similar manner. In contrast to the antimetabolites methotrexate and 5-fluorouracil, we found that pharmacological levels of both cisplatin (which blocks DNA polymerase) and the anthracycline drugs doxorubicin and donarubicin (which inhibit DNA strand separation during replication) modulate TF expression on both murine TG-PEC and WEHI 265 monocytoid cells (Wheeler and Geczy, 1990) and on human Mos and RC2a cells (Walsh et al., 1992; Yen et al., 1993). Cisplatin and Adriamycin induce weak TF activity and synergize with low levels of LPS (10100 pg/ml) to enhance TF expression on human Mos. At concentrations which do not cause cell death, cisplatin (20 pg/ml) enhances TF induced by LPS some 6- to 20-fold whereas the quantity of TF antigen on viable cells indicates that the synergy observed with these agents is not due to

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CAROLYN L. GECZY

enhanced TF antigen expression even though all the procoagulant activity is neutralized by an anti-TF monoclonal antibody. We propose that a mechanism similar to that observed with cycloheximide-treated cells is involved. D . Suppression of Procoagulant Expression

In contrast to the large number of reports describing the induction of TF by the cells described, there is relatively little information on factors regulating this response. The contribution of TFPI, which is produced by activated platelets, endothelial cells, and possibly by Mo/Mac (Werling et al., 1993), and which inhibits the TF-factor VIIa complex in a factor Xadependent reaction, is the subject of a number of recent reviews (Rapaport, 1991; Sandset and Abildgaard, 1991). Although it is an important regulator of TF-mediated reactions, it will not be described here. Apart from the inhibitory effects of TFPI, there are few reports of effective suppression of cellular TF once the thrombotic reaction is under way. Since the activity of TFPI is dependent on factor Xa and because the only requirement for activation of coagulation is the binding of factor VII/VIIa to TF, other regulators of this complex may be expected. Conkling et al. (1989) recently showed that sphingosine, the basic building block of the sphingolipids, profoundly inhibited TF on activated human Mos. Although it may function by inhibiting protein kinase C, the inability of a number of sphingosine analogs to alter activity suggests that the amino group of the polar head of sphingosine inhibits formation of the TF-factor VII complex. Alternatively, sphingosine may disturb the phospholipid cofactor activity within the cell membrane by becoming inserted into the bilayer. Although sphingosine and other glycosphingolipids may represent a new class of inhibitors of coagulation, their pharmacological use has not been explored. Several investigators have tested a number of agents known to affect the inflammatory response in an attempt to find clinically useful inhibitors of the development of TF (see Table IV). Dexamethasone inhibits TF generation in Mos stimulated with LPS in uitro (Lyberg, 1984a) although PEC from rabbits injected with LPS have higher procoagulant levels when injected simultaneously with cortisone acetate (Robinson et al., 1978). This observation is in accord with the finding that cortisone can substitute for the first of the two LPS injections which provoke the Schwartzman reaction (Thomas and Good, 1952). Our studies support this observation and indicate that in contrast to blood Mos, glucocorticoids enhance the procoagulant potential of inflammatory Macs, particularly in response to IFN-y ( J . D. Walsh, H. Wheeler, and C. L. Geczy, unpublished observa-

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TABLE IV

Some Modulators of TF ~

Modulating agents CD8 lymphocytes +

Interferon-y Interleukin 4

Heparin-binding growth factor1 plus heparin Agents elevating [cAMPIi (including pentoxyfylline, HSW 138) Prostacyclin, iloprost Sphingosine Retinoids

Effect Inhibit TF induction on endothelial cells Suppresses LPS-induced TF on Mos Variably suppresses LPS, ILl, and TNF-induced TF on endothelial cells and Mos Inhibit TF induction on endothelial cells Inhibit TF induction

Inhibit TF induction Inhibits preformed TF Inhibit TF induction

tions), a key mediator of the Schwartzman reaction. On the other hand, suppression of production and secretion of several cytokines, including ILl, TNF-a, IL2, and GM-CSF by steroids (Kelley, 1990)could indirectly downregulate procoagulant induction in a number of clinical situations. Retinoids modulate several functions of mononuclear phagocytes. Human blood Mos pulsed with therapeutic doses of retinoids for 10 min exhibit markedly depressed TF expression in response to LPS whereas there is little reduction of preformed TF (Conese et al., 1991). Inhibitor studies suggest that the effect may be mediated by a product of the lipoxygenase pathway although this is undefined. The ability of retinoids to downregulate procoagulant generation may contribute to the antiinflammatory effects of these compounds. Pharmacological mediators of intracellular metabolic events can also regulate TF expression. Elevation of intracellular levels of CAMP by dibutyryl CAMP inhibits induction of TF on Mos (Lyberg, 1984a),and phosphodiesterase inhibitors suppress TF expression on HUVECS (Galdal et al., 1984).Although the mechanisms of TF induction are still largely unknown, protein kinase C is thought to be involved in the processes leading to TF synthesis (Brozna, 1990; Pettersen et al., 1992) and inhibitors of protein kinase C, H7, H8, and sphingosine suppress the induction of TF on endothelial cells by allogenic lymphocytes and ILI-j3 (Pettersen e l al., 1992).

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The regulatory role of arachidonic acid metabolites on inflammatory reactions is generally well accepted. Although cyclooxygenase inhibitors have little effect on TF induction on Mos (Crutchley, 1984; Edgington and Pizzolato, 1983; Prydz and Lyberg, 1980), suggesting that the prostaglandins (PG) are not involved, accumulated PGE, and PGE, suppress Mo TF activity, possibly by increasing intracellular levels of CAMP. A recent study by Crutchley and Hirsh (1991) confirms the earlier observations and shows that the stable prostacyclin analog, iloprost, and to a somewhat lesser extent, prostacyclin, inhibit development of TF on stimulated blood Mos and THP-1 cells although the mechanism remains speculative. These observations add to the clinical potential of prostacyclin analogs which may inhibit both platelet aggregation and Mo procoagulant activity, in antithrombotic therapy. Furthermore, a recent report describes the protective effect of the xanthine derivative HSW 138 on the life-threatening coagulopathy induced in rats by LPS. Like pentoxifylline and theophylline, it acts by inhibiting phosphodiesterase and increasing intracellular levels of CAMP. Pentoxifylline suppresses TF induction on Mo and endothelial cells (Archipoff et al., 1989; de Prost et al., 1989) and TNF production (Bahrami et al.,1992), supporting a role for CAMPin the procoagulant response. PGE, suppresses the development of fulminant hepatitis in MHV-3-infected mice, and mononuclear cells recovered from infected mice treated with PGE, have no prothrombinase activity. Furthermore, PGE, prevents prothrombinase induction by MHV-3 in uitro, suggesting that the immunosuppressive effect of this agent may be linked with the procoagulant potential of the cells involved (Levy and Abecassis, 1989). However, an indirect effect of the prostaglandin on induction of a T-cell-dependent cytokine thought to mediate this response cannot be excluded. Induction of TF on endothelial cells induced by allogenic stimulation with CD4+-Tlymphocytes is suppressed by CD8+ lymphocytes (Pettersen et al., 1992). This may be mediated by IL4, a CD8+-Tlymphocyte product recently shown to downregulate a number of proinflammatory functions of blood Mos (Hart et al., 1989). IL4 suppresses the expression of TF on cultured bovine aortic endothelial cells by ILl-j3 and TNF-a (Herbert et al., 1992). Although IL4 suppressed induction of TF on HUVECs by LPS, activity expressed in response to ILl-p and TNF-a was not affected by IL4, confirming the results of Kapiotis et al. (1991). In addition, TF induction on adherent Mos by ILl-j3 was reduced by IL4 whereas the LPS response was not. Herbert et al. (1992) found no suppressive effect of IL4 on murine TG-elicited Macs stimulated with LPS. The apparent discrepancies in responsiveness of different cell types may reside in the differences in magnitude of procoagulant responses induced by different

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stimulants (e.g., ILl-/3 is a weaker stimulant of Mo T F than LPS) and in the origin of the cell used. Recent experiments by Hart and colleagues suggest that whereas blood Mos are sensitive to the suppressive effects of IL4, human Macs isolated from inflammatory sites are refractory (P. Hart, personal communication), again indicating the importance of the maturation and differentiation state of these cells in the outcome of the inflammatory response. Furthermore, the dichotomy of responsiveness of Mo/Mac to IFN-y (it suppresses LPSinduced blood Mo T F and inhances Mac T F expression) suggests additional levels of control. Suppression by varying levels of TFPI, as may occur on Macs in some tumors (Werling et al., 1993), and by other anticoagulants such as antithrombin (upregulated by IFN-y in inflammatory Macs; Kakakios and Geczy, 1994) or other cytokines which may affect T F gene transcription, offer a number of possibilities. Investigations with other cytokines, particularly ILlO and IL13, recently shown to downmodulate a number of Mac functions, should be pursued. The functional properties of TF-factor VIIa can be modified by heparin. Heparin increases the substrate specificity of TF-factor VIIa to enhance activation of factors X and IX by HUVEC in a pure system (Almus et al., 1989). It does not affect the cofactor activity of factor Xa for TFPI, indicating that it does not regulate the anticoagulant potential of TFPI in plasma. We also found that heparin weakly enhances the T F response of blood Mos (Kakakios and Geczy, 1994) measured in a plasma recalcification assay, suggesting a possible procoagulant role for heparin in clinical situations, such as DIC, in which anticoagulant proteins are depleted by excessive coagulation. Primary cultures of HUVEC grown with heparinbinding growth factor 1 (HBGF-1) and heparin have decreased levels of both surface and total T F in response to thrombin than when grown without heparin. Decreased T F mRNA coincided with decreased TF antigen (Almus et al., 1991) in a manner similar to suppression of PAI-1 expression (Konkle and Ginsburg, 1988), fibronectin synthesis (LyonsGiordano et al., 1990), and diminished prostacyclin production (Hasegawa et al., 1988),which suggests that heparin modulates a generalized response by endothelial cells to HBGF-1, which could alter the procoagulant potential of these cells. IV. Conclusion

The importance of cellular procoagulants in the pathology of sepsis, infection, thrombosis, and inflammation has become increasingly obvious over the past decade. The role of T F in these conditions is now relatively

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well defined although there are many questions still unanswered. It is interesting that the expression of TF in several cell types is induced in what seems to be a “cytokine-specific” manner. For example, IL1-p is a potent inducer of endothelial cell TF whereas its effect on MolMac is rather weak, and FGF is effective on fibroblasts but not Mo/Mac or endothelial cells. Because T F is apparently an early-response gene product of some cell types, studies investigating its role in cell growth and embryonic development and its transcriptional control in various cell types may yield interesting results. Furthermore, the relative contributions of TF, sequestered to the ECM of stimulated endothelial cells, and of Mo/Mac TF, which is expressed at much higher levels, to initiation of coagulation, should be objectively assessed. It is possible that the sequestration of coagulation factors to the ECM may have additional functions in inflammation. Under these circumstances, their effects on cell migration and activation may be of major significance. Early experiments using anticoagulants suggested that fibrin formation was an important event in inflammatory reactions. However, with the exception of thrombin, the possible contributions of products of activated coagulation to other components of the inflammatory response (e.g., cytokine processing, cell activation) warrant further investigation. An understanding of the control of procoagulants, in addition to TF, by cytokines and growth factors is essential for assessing their contribution to fibrin formation, particularly within the extravasculature. Downregulation of procoagulant expression may occur via inhibitors, cytokines, and other agents which regulate gene transcription and translation, by availability of phospholipids and other cofactors, and by the physiochemical presentation of these factors within the cell membrane. Further investigation in this complex area are likely to have important clinical relevance, particularly in the development of more specific and potent inhibitors, and should be extended to include the possible interactions of TF/factor VIIa/Mac-l/factor X on adhesive substrates and on the ECM. Studies of the biology of cellular procoagulants will continue to be important in linking our understanding of the basic mechanisms of the activation of blood coagulation and inflammation, and human diseases as diverse as atherosclerosis, thrombosis, multiple sclerosis, and cancer. Acknowledgments The author acknowledges support from the National Health and Medical Research Council of Australia and the members of her laboratory who have contributed to the work. The excellent secretarial assistance of Tina Lum is gratefully acknowledged.

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