Regulation of the extrinsic pathway system in health and disease: The role of factor VIIa and tissue factor pathway inhibitor

Thrombosis

Pergamon

Research. Vol. 79, No. 1. DP. l-47, 1995 Copyright 0 1995 Elsevi% Science Ltd Printed in the USA. All rights reserved 0049-3848/95 $9.50 + .OO

0049-3848(95)00069-O

REGULATION THE ROLE

OF THE EXTRINSIC PATHWAY SYSTEM IN HEALTH AND DISEASE: OF FACTOR VHa AND TISSUE FACTOR PATHWAY INHIBITOR

Lars C. Petersen, Sanne Valentin and Ulla Hedner Novo Nordisk A/S, Health Care Discovery, Vessel Wall Biology, Niels Steensensvej 1, DK-2820 Gentofte, Denmark INTRODUCTION Recent research has provided increasing evidence of the importance of tissue factor (TF) and factor VIIa in initiation of blood coagulation via the so-called extrinsic coagulation pathway [see (l-6) for a review]. Under normal conditions, factor VII circulates in the blood primarily in its inactive zymogen form in itself unable to initiate the formation of a hemostatic plug. Upon vascular injury, however, cell surface TF is exposed and readily forms a one-to-one stoichiometric complex with factor VII. Complex formation with TF has two functions. It augments the activation of factor VII to VIIa, and it enhances the proteolytic activity of factor VIIa towards its substrates. Once complexed to TF, the zymogen factor VII is rapidly converted to factor VIIa by cleavage of a single internal peptide bond located at Arg,,,-IlelS3. The factor VIIa/TF complex, in turn, initiates blood coagulation by proteolytically activating its substrates factors IX and X, eventually leading to thrombin formation and a fibrin clot. TF is a transmembrane cell surface receptor. In this, it differs from other positive modulators of coagulation, like factors V and VIII, which adhere to the surface without anchoring to it. Another distinct characteristics of TF is that it needs no prior proteolytic cleavage to become active. Also, in contrast to factor V and VIII, but consistent with its potent coagulant activity, TF is not exposed to plasma on blood cells and endothelium under normal conditions. The TF antigen is present in cells in the adventitia surrounding blood vessels and epidermal and mucosal lining cells. This is exactly what one would predict if a function of TF is to control hemostasis after tissue injury. By surrounding blood vessels and being present in high concentrations in tissues such as brain, where minor hemorrhages can be catastrophic, the TF distribution provides a hemostatic barrier with the external environment. Keywords: factor VII, tissue factor, tissue factor pathway inhibitor, hemophilia, cardiovascular disease, disseminary intravascular disease. Abbreviations: TF, tissue factor; TFPI, tissue factor pathway inhibitor; KPI, Kunitz-type protease inhibitor; EGF, epidermal growth factor-like; FVII:C, Factor VII coagulant activity; FVII:Ag, Factor VII antigen; FVIIa, activated factor VII (measured with assays based on truncated TF).

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However, the fact that the TF antigen has not been detected by immunohistochemical techniques in cells in contact with blood may not mean that the blood stream is totally devoid of TF in normal individuals. The presence of TF in the blood in minute quantities is indicated by recent sensitive ELISA studies that measured a plasma TF concentration of about 3 pM (7). Also, a small fraction (0.5-10/o) of the total amount of factor VII has been shown to be present as the activated enzyme form, factor VIIa (8-11). The presence of both TF and factor VIIa might account for the very low steady-state level of ongoing activation of coagulation as monitored by split products of coagulation factors (12,13), and a comprehensive description of these basal conditions might be crucial to an understanding of in vivo initiation of the coagulation process. An important aspect of coagulation is its rigorous control and downregulation by proteinase inhibitors, and the clearance from the blood of coagulation factors. Factor VII is unique among the vitamin K-dependent coagulation proteases in that it possesses a halflife of 2-3 h, regardless of whether it is present as the zymogen or as an activated enzyme (14). This is in sharp contrast to other vitamin K-dependent proteases such as factors IX and X, which in their activated forms, factors IXa and Xa, are cleared from the circulation within a few minutes. Unlike these other activated coagulation proteases, factor VIIa in plasma stays free of inactivation by direct inhibitors including antithrombin III. The only physiologically significant inhibitor is tissue factor pathway inhibitor (TFPI), which exerts its inhibitory action only when factor Xa is present, and then only when factor VIIa is bound to TF. TFPI is of particular importance in relation to initiation of the extrinsic pathway. Unlike other coagulation inhibitors and inhibitors of the fibrinolytic system, TFPI is not a serpin but a multivalent inhibitor with three tandemly arranged Kunitz-type proteinase inhibitor (KPI) domains. Apparently, this unique structure serves to provide the protein with complicated functional properties, which makes the inhibitor well suited for a regulatory role. The present review will address the structural properties of the key components involved in initiation of the TF pathway, and covers current knowledge about the molecular interactions between these proteins. Based on this information, pharmacological studies and also measurements of in vivo proteolysis of coagulation proteins, we attempt to develop a conceptual framework for the regulation of the extrinsic coagulation pathway in health and disease.

PROTEIN STRUCTURE Factor VZZ Factor VII is present in human plasma in trace concentrations (400 rig/ml) (15). Initial attempts to characterize the primary structure of factor VII by protein chemical techniques, therefore, resulted in only a partial amino acid sequence (16). It was not until cloning of several factor VII cDNAs that the complete primary amino acid sequence of factor VII was determined (17). In addition to providing the sequence for the mature protein this also provided information about its precursor structure during biosynthesis. The factor VII molecule, like the other vitamin K-dependent proteases, is initially synthesized as a precursor with a 38 amino acid prepro leader segment immediately preceding the mature amino terminus of the protein that circulates in plasma. The first 20 amino acids have a structure resembling a classic signal peptide, which directs the nascent polypeptide to associate with the rough endoplasmic reticulum during translation and subsequent translocation of the protein into

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the lumen of the endoplasmatic reticulum and into the cellular secretory pathway. This first section is, consequently, referred to as the “pre”-peptide or “signal” peptide. The latter 18 amino acids of the leader sequence possesses a strong similarity to the corresponding structure found in the other vitamin K-dependent coagulation factors, and has since been shown to be a recognition signal for an endoplasmic carboxylase enzyme that posttranslationally modifies specific glutamic acid residues present in the N-terminus of the protein (18). This latter section has been called the “pro”-peptide. The remainder of the cDNA encodes for a mature factor VII protein of 406 amino acids with a predicted molecular weight of 45.5 kDa. The sequence shown in Fig. 1 predicts two sites for potential N-linked carbohydrate addition, which together bring the predicted molecular weight up to 50 kDa and thereby in close agreement with that of the plasma protein. The primary sequence of factor VII possesses a great deal of similarity to other vitamin K-dependent coagulation proteases and provides important clues concerning its structure and function. As shown in Fig. 1, the protein sequence can be viewed as follows: the first 152 amino acids comprise a “light” (smaller) chain (Mw -20 kDa), which is connected by a single disulfide bond to the “heavy” chain (Mw -30 kDa) comprised of the last 254 amino acids following cleavage after Arg 152. The light chain contains the “Gla” or y-carboxyglutamic acid domain consisting of approximately the first 38 amino acids and

FIG.

1.

Human factor VII. y: Gamma-carboxyglutamic acid, *: glycosylation site. Active site residues are indicated by circles filled with dark outlines. Arrow indicates activation cleavage site. Adapted from (17).

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probably constitutes the phospholipid binding domain of the protein. The Gla domain is followed by two repeating copies of a 36 amino acid domain with considerable sequence similarity to Epidermal Growth Factor (EGF). These two “EGF”-like domains contain identical cysteine patterns to EGF and, therefore, probably fold into structures resembling the growth factor. An interconnecting sequence (residues 39-44) constitutes the “hydrophobic stack” region highly homologous to similar regions in other vitamin K-dependent coagulation factors. Directly following the two EGF-like domains in factor VII is a sequence containing the activation site (Arg,,,-IlelS3) at which the zymogen, factor VII, is cleaved upon activation, and the two-chain enzyme form, factor VIIa, is obtained. The final 254 amino acids of the protein comprise the domains of the “catalytic” or “serine protease” part because of its striking sequence homology with members of the trypsin family of serine proteases. This unit contains the catalytic triad (consisting of SerjM, Asp,,,, and His& involved in the cleavage by factor VIIa of its protein substrates, factors IX and X. Like other coagulation proteases, factor VII undergoes several posttranslational modifications including N-glycosylation at Asn,,, and AsnJ2*, 0-glycosylation at SerSz and Serm as well as y-carboxylation of several N-terminal glutamic acid residues. This latter modification involves the action of a carboxylase enzyme, which in the presence of reduced vitamin K, fixes carbon dioxide to form a new carboxyl group on the glutamic acid while simultaneously converting vitamin K to vitamin K epoxide (18). Posttranslational processing of glutamic acid residues is limited to 10 sites, all located within the first 38 residues of the factor VII light chain (generally referred to as the Gla-domain). Full y-carboxylation of these residues is essential for calcium binding and expression of factor VIIa coagulant activity. Studies with other vitamin K-dependent proteins such as prothrombin, factor IX, factor X, protein C, protein S, and protein Z have indicated that Ca ‘+ binding is essential for inducing a conformational change, which in turn, is crucial for the interaction of the protein with the phospholipid membrane. Factor VII undergoes posttranslational N-glycosylation of two Asn-X-Thr/Ser sites (19). Both sites (Asn14, and Asn,,,) are normally fully glycosylated, and the structures are composed of N-acetylglucosamine, mannose, galactose, fucose, and sialic acid. Factor VII also contains two O-linked glycosylation sites located within the first EGF domain (20). The first consists of either glucose, glucose-xylose or glucose-(xylose)JO-glycosidically linked to Sersz. The three glycan structures are present in approximately equal amounts. The second glycosylation consists of a single fucose linked to Ser,, (20). Similar glycans have also been found attached to conserved serine residues in factor IX (21), human and bovine protein Z, as well as bovine factor VII (22). Precisely what function these O-linked carbohydrate moieties play is unclear. Recent findings have shown that factor VII mutated at either position 52 and/or 60 (Ser to Ala) possesses essentially normal coagulant activity (20). A number of EGF domains in other proteins contain a ,&hydroxylated Asp or Asn in a position that correspond to AspG3 of factor VII. Based on sequence comparison of homology regions, a consensus sequence required for this modification was proposed (23). However, Asp,, of human factor VII has been shown not to be P-hydroxylated (19), although it is positioned in a putative consensus sequence in EGF domain 1. Tissue Factor

Purification of the TF apoprotein was hampered by its presence in trace amounts, but also the need to use detergents for extraction of TF from tissues, and the requirement for relipidation prior to functional assays added to the difficulties. Pioneering purification methods

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were reported by Hvatum and Prydz (24), and further progress was obtained with affinity purification chromatographic methods using concanavalin-A (25), antibodies (26), or factor VII (27). This work prepared the way for the isolation and cloning of the human cDNA and the deduction of the complete TF amino acid sequence (28-31). The predicted primary amino sequence is shown in Fig. 2. The primary sequence suggested that TF is a single-pass transmembrane receptor-like protein with a three-domain architecture. The sequence indicated an extracellular domain (residues 1-219) followed by a hydrophobic membrane-spanning domain (residues 220-242) and a cytoplasmatic tail (residues 243-263). Initially, no obvious sequence homology was noted; however, later studies revealed that TF is a distant member of the cytokine/interferon or haematopoietic receptor superfamily [see (32) for a review]. The extracellular domains of these receptors have low but significant homology within the ligand binding region consisting of approximately 200 residues, and this observation led to the suggestion that the regions share a common three-dimensional fold (33). In subclass I, the hemotapoietic recep-

FIG. 2. Human tissue factor. *: Glycosylation site. Predicted residue 220-242. Adapted from (6,28-31).

transmembrane

region

from

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tor family includes receptors for growth hormone, prolactin, interleukins 1 to 7, granulocytemacrophage colony stimulating factors, erythropoitin, and thrombopoitin. Subclass II includes TF and receptors for interferon a! and 0. As an example, a subclass I receptor, the threedimensional structure of the extracellular domain of the human growth hormone receptor was determined by means of x-ray diffraction studies in 1992 (34). Only very recently has this also been possible with the extracellular part of TF (residues 1-219) as the first example of a subclass II receptor (35,36). TF1_219 consists of two domains connected by a single polypeptide linker (Proro2-Asnro7). TF is structurally similar to the growth hormone receptor with respect to the topology and folding of each domain. Distinct differences exists in the domain-domain interface and, as a consequence, the interdomain hinge angle is wider, with TF adopting a more “upright” configuration than the growth hormone receptor. Alaninescanning mutagenesis (37) has identified LysZO, Ilezz, ASPIC, Arg,,,, and Phe14e as the TF residues involved in the interaction with factor VIIa. The structure shows that these residues are all located in the domain-domain interface region, but on the opposite side of the molecule compared to the ligand binding region of the growth hormone receptor. Evidence was provided from site-directed mutagenesis studies (38) that the key residues involved in the interaction with factor VIIa are arranged in three independent contact: a) Ilez2, b) Ly@Asp,,, and c) Arg,,,/Pherd,,. Tissue Factor Path way Inhibitor TFPI is a single chain glycoprotein present in plasma in trace amounts. It was previously known as extrinsic pathway inhibitor, or lipoprotein associated coagulation inhibitor [see (39-43) for a review]. The cDNA coding for TFPI was cloned and characterized in 1988 by Wun et al. (44). The translated amino acid sequence revealed that TFPI has a highly negatively charged N-terminus followed by three tandemly repeated Kunitz-type domains, and a highly positively charged C-terminus (Fig. 3). The mature molecule consists of 276 amino acid residues including 18 cysteines, all involved in disulfide bonds (44). It contains three potential N-linked glycosylation sites at Am I73 Asn167, and Asn,,, (44,45). The molecular weight of the polypeptide backbone is 32 kDa (44). Presumably due to glycosylation, the TFPI present in plasma run, however, with an apparent molecular weight of 42 kDa on SDS-PAGE. Several posttranslational modifications have been reported. TFPI produced by human umbilical vein cells contains a sulphated aspargine-linked oligosaccharide (46). TFPI produced by HepG2 cells has been shown to be partially phosphorylated at Ser, (47). Whether TFPI from plasma is phosphorylated or sulphated remains to be determined. Posttranslational modifications such as glycosylation and phosphorylation are apparently of minor importance for the inhibitory function (48) since recombinant TFPI expressed by E. coli is indistinguishable from recombinant TFPI expressed by mammalian cell lines with respect to factor Xa inhibition. TFPI mRNA was found primarily in megakaryocytes (49), and in endothelial cells (49,50), whereas only a small amount was present in monocytes/macrophages (49). NO TFPI mRNA was found in normal human hepatocytes (49), indicating that the liver is not the primary site of synthesis. Immunohistochemical staining of normal human tissues demonstrated the presence of TFPI on the surface of microvascular endothelium and in megakaryocytes, while no antigen was found on the endothelium of larger vessels or in hepatocytes (5 1). Inflammatory mediators do not significantly affect synthesis of TFPI under conditions of strongly increased tissue factor expression (SO). Furthermore, cultured endothelial cells

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FIG. 3. Human tissue factor pathway inhibitor. *: Glycosylation site, @: phosphorylation site, Pl residues of the three Kunitz-type protease inhibitor domains are indicated by bold circles. Protease cleavage sites are denoted by arrows. HLE: human leukocyte elastase. Adapted from (44,66).

constitutively produce TFPI and are not affected by stimulation (40). Taken together, these data indicate that the endothelium is the principal site of synthesis responsible for maintaining the plasma levels of TFPI in vivo, but more data are needed to confirm this. The average plasma TFPI concentration in normal individuals is approximately 100 rig/ml (range: 54-142 rig/ml) corresponding to a level of about 2.5 nM (45). There appears to be no diurnal variation in the plasma level of TFPI (40,52), and the plasma level is not affected by consumption of a meal (52). Under normal conditions, TFPI circulates in plasma primarily in complex with lipoproteins. Gel filtration analysis of human plasma indicate that TFPI is present as three different molecular weight pools: 50% comigrates with low density lipoprotein (LDL) and very low density lipoprotein (VLDL); 40-45% comigrates with high density lipoprotein (HDL), and 5-10% is free, and not associated with lipoprotein (45,53). Free- and lipoproteinassociated TFPI account, in fact, for only a fraction of the total TFPI available in vivo. Several studies have shown that injection of heparin results in a two- to eightfold increase in plasma TFPI levels (54-56). Heparin releasable TFPI appears not to be associated with lipoprotein (53,57). Also, a small amount of the TFPI activity in plasma is located in platelets, 8 rig/ml (58). TFPI is released upon activation of the platelets by thrombin, and in this way the concentration of TFPI at the site of injury may be elevated and ready to participate in control of the clot formation. TFPI, purified from plasma, is heterogeneous in size and contains mostly 34 and 40 kDa forms, but higher molecular weight forms are also found (45,59,60). When associated with

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LDL, it consists mainly of the 34 kDa molecular weight form, whereas TFPI associated with HDL has a molecular weight of 41 kDa. VLDL particles contain the 34 kDa as well as the 41 kDa form of TFPI (45,59). Western blot analysis suggests that a portion of the plasma TFPI associated with HDL is bound covalently, by mixed disulfide linkage, to apolipoprotein A-II (apoA-II), the major apolipoprotein present in HDL particles (45,59). Native and recombinant TFPI purified from tissue cultures, as well as TFPI released from the vasculature by heparin, has a molecular weight of approximately 43 kDa. These observed differences in molecular weight of TFPI are likely to reflect various degrees of C-terminal truncation (60). In contrast to this, in vitro studies have shown that the C-terminus is essential for TFPI-lipoprotein interaction (61). The reason for this controversy is not known at present. Likewise, it is not known whether TFPI possesses anticoagulant activity when associated to lipoprotein, and also the physiological significance of TFPI in this complex is unknown so far. A nonessential role of lipoprotein associated TFPI could be indicated by the fact that patients with homozygous abeta- and hypo-betalipoprotienemia are not suffering from thrombosis in spite of their very low levels of plasma TFPI (56). TFPI is a heparin binding protein. A predominant binding site is located within the C-terminus, as shown by the much weaker heparin binding of C-termina1 truncated TFPI (62,63). Recent work by Wesselschmidt et al. (64) suggest that heparin binding also involves positively charged epitopes on the third KPI domain of TFPI. The interaction of TFPI with heparin depends on the molecular weight and charge distribution of the heparin ligand (65). Highest affinity was observed with high molecular weight (2 10 kDa) heparin fragments, and/or fragments of high charge density. TFPI also binds to a number of other glycosaminoglycans with the following order of affinity: heparin > dermatan sulphate > heparan sulphate > chondroitin sulphate C (65). Binding of heparin to antithrombin involes a specific pentasaccharide sequence. This heparin site is not involved in the interaction with TFPI (65). Each of the three KPI domains of TFPI may potentially be involved in inhibitory interaction with proteases. Site-directed mutagenesis has been used by Girard et al. (66) to further localize the inhibitory properties to a specified domain. They studied the functional significance of the KPI domains by substitution of their PI residue assumed to be essential for a putative interaction with the active site of a protease. Substitution of the Pl residue in the first KPI domain suggested that it interacts with the active site of factor VIIa. Similarly, the second KPI domain was found to interact with the active site of factor Xa. No inhibitory function could be attributed to the third KPI domain. Expression of separate recombinant KPI domains of TFPI is another approach taken to characterize this multivalent inhibitor. Such studies show that KPI domain 1 inhibits factor VIIa (K, = 250 nM) KPI domain 2 inhibits factor Xa (Ki = 90 nM) and also confirm that KPI domain 3 is apparently without inhibitory function (67,68). In addition to factors VIIa and Xa, TFPI is an inhibitor of trypsin, chymotrypsin, plasmin, and cathepsinG. KPI domain 1 has higher affinity for plasmin and cathepsin G than has KPI domain 2. The reverse is true for trypsin and chymotrypsin (68). An interesting and important property of TFPI was noted independently in 1987 by several investigators (69-71). They demonstrated that factor Xa was required for TFPI to inhibit the activity of the TF/factor VIIa complex, and also showed that factor X or active site-inhibited factor Xa would not suffice. These observations, and also the requirement for Ca*+ ions, noted by Hjort (72), are all accounted for by a model involving a quaternary factor Xa-TFPI-factor VIIa-TF complex (73,74). The TFPI-mediated inhibition of factor VIIa/TF activity is currently thought to occur in two discrete steps. In the first

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step, TFPI binds reversibly to factor Xa with a 1: 1 stoichiometry, causing loss of factor Xa catalytic activity; and in the second step, the quaternary factor Xa/TFPI/factor VIIa/TF complex is formed with the resultant loss of factor VIIa/TF catalytic activity. Direct inhibition by rTFP1 of factor VIIa/TF amidolytic activity as well as proteolytic activity has been reported (62,75,76). This inhibition of the factor VIIa/TF complex requires an unphysiologically high concentration of TFPI, and seems to be independent of the presence of factor Xa. Furthermore, TFPI is capable of inhibiting the cleavage of factor IX by factor VIIa-TF in the absence of factor Xa (39,76), but a higher concentration of TFPI is required than when factor Xa is present (6). Very little is known about the function of protein structures originating from the N-terminal sequence in front of KPI domain 1 and the interlink sequences between the KPI domains in TFPI. Slightly more is known about the positively charged C-terminal tail of TFPI. An abundancy of lysine residues may account for its binding to negatively charged glycosaminoglycans and may also be the reason why the tail is very susceptible to proteolytic cleavage. Without special precaution to avoid degradation, both plasma and recombinantderived TFPI are cleaved at several positions in the C-terminal tail during purification (77,78). Compared to the intact protein, this results in a significant loss of anticoagulant activity (77,78), but apparently this does not affect the TFPI activity as assessed by a chromogenic assay for factor VIIa-TF inhibition (77). The C-terminal part of TFPI is involved in the interaction with factor Xa and strongly affect the transient inhibition of this protease (79). An inhibitor homologous to TFPI, also containing three tandemly repeated KPI domains, has recently been cloned and partially characterized (80). It was provisionally refered to as tissue factor pathway inhibitor-2 (TFPI-2) because of its ability to inhibit factor VIIa/TF (K: = 10 nM in the presence of heparin). In contrast to TFPI, the homolog did not inhibit factor Xa. A retrospective examination of the literature (81) revealed that TFPI-2 is identical to placental protein 5 (PP5), that was originally isolated as a glycoprotein (30-36 kDa) from human placenta on heparin-Sepharose (82,83). PP5 is present in sera from both men and women at a level of about 13 and 16 pM, respectively, whereas in pregnancy plasma samples it rizes dramatically to a level of about 1 nM (84). PPS has been reported to inhibit thrombin-induced clot formation, although it did not inhibit the amidolytic activity of thrombin (85). The physiological role of this inhibitor remains to be seen. ACTIVATION

OF ZYMOGENS

Initiation of the Tissue Factor Pathway by Reciprocal Zymogen Activation The TF pathway of coagulation constitutes a series of consecutive zymogen activation reactions, a cascade. It is initiated when factor VIIa/TF activates factor X by specific cleavage, and is further substantiated when factor Xa activates prothrombin. Although the classical cascade model adequately accounts for the amplification effect, it does not explain how an initial activity is induced as a result of an external stimulus, e.g., exposure of TF. Neither does it explain why this initial activation does not lead to full and total activation of all zymogens involved in a given cascade. To provide a more complete picture of the regulation of the TF pathway, the original cascade model has to be further developed. For this purpose it is important to consider the implications of “reciprocal zymogen activation” (see below), and also the fact that activation takes place in the presence of potentially very efficient physiological inhibitors. Of further importance is the fact that kinetically the cascade ought to be treated as an open system rather than a closed system, as has traditionally been the case.

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Thus, instead of a “dormant,” closed system, which upon exposure to external stimuli can be brought to action by some sort of “triggering” mechanism, the cascade might be considered an open system, which under “resting” conditions operates at a very low steady-state rate of zymogen activation. Under these conditions, de nova synthesis of zymogens is counterbalanced by enzyme clearence, presumably by way of receptor-mediated catabolism involving inhibitor complexes. Transition from a “resting” to an “activated” state may be induced by several regulatory mechanisms. Exposure of surface-bound cofactors to obtain localized assembly of multicomponent complexes resulting in rate enhancement of zymogen activation is one such mechanism that has been frequently emphasized [see, e.g., (86) for a review]. Another event of importance for the transition from a resting to an activated state is the feed back regulation caused by proteolytic cleavage of the cofactors (factors V and VIII) by thrombin. It is suggested in the present review that neutralization or destruction of inhibitors combined with reciprocal zymogen activation may provide an alternative and perhaps equally important mechanism by which such transition can be induced. Studying the initiation of the intrinsic system, the concept of “reciprocal zymogen activation” was first introduced by Cochrane et al. in 1973 (87), and subsequent investigators have found that the very start of any known cascade system apparently consisted of a zymogen couple capable of reciprocal activation. With reference to the TF pathway, factor VII and X represents such a couple capable of reciprocal zymogen activation in the sense that the activated form of factor X can activate factor VII and the activated form of factor VII can activate factor X. But also the factor VII/factor IX zymogen couple could be relevant in this context. Other such examples of reciprocal zymogen activation comprises the factor XII/prekallikrein and the pro-uPA/plasminogen zymogen couples. When two zymogens undergoing reciprocal activation are mixed in a closed system (e.g., a test tube) without inhibitors, it is a common experience that the mixture is unstable. It will eventually end up as a mixture of the two activated enzymes. No matter how carefully you prepare your proteins, the zymogen mixture will always possess a finite, although small, activity capable of initiating an autoactivation circle. The question whether or not this activity occurs as a result of genuine “zymogen activity” or it occurs as a result of trace amounts of “enzyme contamination” is merely of academic interest without importance for the practical outcome. To get an impression of the conditions that determines the initial phase of reciprocal zymogen activation, it is perhaps useful to analyze this problem in terms of the simplest possible system shown below:

Factor VII

------+ A

Factor VIIa

Factor Xa f---------

Factor X

The model describes a system consisting of a zymogen X, which is activated to the enzyme, Xa, by an enzyme, Ya, which, again, is obtained as a result of activation by Xa of the zymogen, Y. It is assumed that the reactions take place without the analytical complications induced when localized assembly of multicomponent complexes are considered. An analysis of the kinetics of this model suggests that the initial activation of the mixture proceeds

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with a progress curve described by a sum of two exponential functions (Petersen, in preparation). The lag phase of the progress curve appears to be strongly dependent on the initial concentration of active enzymes, Xa and Ya, whereas the later phase is described by an exponential increase determined by an apparent exponential constant containing the square root of the product of the concentrations of zymogens. The time it takes before a significant activation is observed depends strongly on the amount of initial activity, whereas the rate of the subsequent transition is influenced by the concentration of the zymogens. In vitro activation studies reveal that factor VII is most rapidly activated by factor Xa (88-90), although several other proteases including factor IXa (91), factor XIIa (92,93), and thrombin (89), are also capable of activating factor VII. Like all vitamin K-dependent zymogen activation reactions, this also requires the presence of calcium ions and phospholipid. The activation of factor VII differs from the activation of other vitamin K-dependent proteases, however, in that it is greatly enhanced by binding to its cofactor, TF. Formation of a zymogen factor VII:TF complex renders the scissile bond at Arg,,,-IlelSj extremely susceptible to cleavage by trace amounts of factor Xa or IXa. Activation by factor Xa was shown to be profoundly enhanced by relipidated TF but not by soluble TF1_219 (94). Furthermore, the enhancement with acidic PS vesicles was much more efficient than that obtained with PC vesicles (95). This suggest that factor Xa-mediated activation, like many membrane-dependent coagulation reactions, requires binding of the Gla-domain to phospholipid head groups. The factor VII:TF complex is also susceptible to specific cleavage by factor VIIa in a reaction termed autoactivation (96). The progress curve was well described by the kinetic model applied to autoactivation of factor XII (97) and prekallikrein (98); however, it is of interest to note that whether these reactions are enhanced by surfaces of high negative charge density, this was not the case with factor VII autoactivation, which was enhanced by polyp-lysine (99). Importantly, resent studies have shown that autoactivation is dramatically enhanced by the presence of TF (IOO), and further kinetic studies revealed that the reaction takes place by lateral diffusion when a factor VII/TF complex in the membrane encounters a factor VIIa/TF complex (94). Increased density of factor VII/TF in the membrane strongly enhanced autoactivation, and it was also shown by these investigators that autoactivation occurred preferentially on neutral phospholipid membranes and did not require exposure of PS. Because intact cells normally sequester acidic phospholipids away from the outer leaflet of the plasma membrane, it was suggested that this reaction should permit factor VII autoactivation to predominate on unactivated/undamaged cell surfaces when other clotting reactions are dormant (94). It was of interest to note that a large excess, relative to TF, of soluble, unbound factor VII was activated at a much decreased rate, indicating that dissociation of factor VIIa from the complex is a slow, rate-limiting reaction. It is also notable that factor VII autoactivation on cells with constitutively expressed TF takes place undisturbed in the presence of physiological concentrations of antithrombin III (100). Precisely which of the proteases, factor Xa, IXa, or VIIa, is responsible for the activation of factor VII under in vivo conditions is less clear. Recent studies have attempted to mimic what happens in vivo by assessing the ability of normal pooled plasma and various congenitally factor-deficient plasmas to support the activation of an inactive factor VII mutant (S344A factor VII) on the surface of a TF expressing cell line (582 bladder carcinoma celis) (101). It was found that incubation of the S344A FVII:TF complex with a lo-fold dilution of normal pooled plasma in the presence of hirudin resulted in a time-dependent conversion of S344A FVII to its cleaved counterpart. Substitution of the normal plasma with plasma

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congenitally deficient in either factor IX, factor VIII, factor XI, factor V, or prothrombin resulted in qualitatively similar activation rates. Incubation with factor X deficient plasma also resulted in activation of S344A FVII, however, at a reduced rate of that observed with normal pooled plasma. This seems to indicate that reciprocal factor VII activation via factor X is more efficient than activation via factor IX. Incubation of the “‘1 S344A FVII with factor VII deficient plasma produced no detectable activation. From this experiment, the authors suggest that the autoactivation of factor VII by VIIa plays a role in the initiation of extrinsic blood coagulation. However, lack of factor VIIa-mediated activation of factors X and IX can, in fact, also account for the lack of subsequent factor VII activation. Activation of Factors IX and X As already discussed, it is not until formation of a complex with TF that factor VIIa possesses proteolytic activity to activate its substrates, factor IX or factor X, at a significant rate. Because factor VIIa:TF can directly activate factor X, and the function of factor IX is also to activate factor X, there was early skepticism as to the physiological importance of the factor VIIa:TF-dependent mechanism of factor IX activation. Precisely which of these proteases is the preferred substrate has been a matter of considerable debate. Detailed kinetic parameters for the activation of factors IX and X by factor VIIa:TF have been reported by a number of groups, utilizing a variety of TF sources. With TF producing cell lines as a source of TF, factor VIIa activates factors IX and X with a K, corresponding to 200 nM and 300 nM, respectively (101-103). Significantly lower K, values (20-250 nM) are obtained when the intact TF-expressing cells are substituted with purified reconstituted TF apoprotein or lysed and sonicated cell extracts. As expected, the reported k,,, values also vary widely with the source of TF used. Reported k,,, values vary between 0.25 s-i and 2.0 s-l for factor IX activation with values between 0.5 SC’ and 3.0 SC’ reported for the activation of factor X by FVIIa:TF. In practice, a number of theoretical and practical complications can account for the observed variability in kinetic parameters mentioned above. Probably the most serious complications are caused by the effector function of TF and the phase heterogeneity of the system under investigation. The fact that TF is an essential activator of factor VIIa implies that the apparent K, is not a single constant but rather a function of the relative concentrations of TF and factor VIIa (104). Other circumstances that add to the complexity of the apparent constants derived from kinetic studies arise as a consequence of binding of substrate (factor IX or X) to phospholipid membranes. The reaction rates on biological membranes may be limited by diffusion (105), in which case interactions of factors IX and X with the factor VIIa:TF complex may depend on the size, charge, and geometric shape of the lipid structure surrounding the TF apoprotein. Thus direct extrapolation of kinetic data, obtained under in vitro conditions to the physiological situation, must be undertaken with extreme caution. MODULATION

OF FACTOR

VIIa ACTIVITY

TF Stimulation As mentioned previously, factor VIIa possesses little coagulant activity in itself, and it is only upon binding to its cell surface cofactor, TF, that factor VIIa possesses significant proteolytic activity to activate its substrates factors IX and X. Although factor VIIa possesses full activity towards simple peptide substrates solely in the presence of TF apoprotein and calcium

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(106), this is not the case for physiological substrates such as factor IX or factor X. These latter reactions also require negatively charged phospholipids for full expression of factor VIIa proteolytic activity (107). In this regard, it has been shown that the procoagulant activity of a TF-producing cell line can be modulated by calcium-mediated changes in the asymmetric distribution of negatively charged phospholipids such as phosphatidyl serine (108). Because phospholipid is not directly required for the formation of a factor VIIa:TF complex, the functional increase in factor VIIa activity probably reflects increased substrate binding to the phospholipid:factor VIIa:TF complex. Numerous studies have now been conducted to assess which structural regions of the factor Vlla molecule are involved in the formation of a factor VIIa:TF complex. Although it has long been known that y-carboxylation of the factor VII Gla-domain is crucial for manifestation of full biologic activity, it is only recently that the Gla-domain has been directly implicated in TF binding. Experiments conducted with Gla-domainless factor Vlla indicate that binding to TF is markedly decreased or absent (109,110). Proteolytic degradation with cathepsin G results in des (l-38) factor Vlla, whereas autodigestion in the absence of Ca’+ gives rize to the des (l-44) factor VIIa (111). Recent studies with these des Gla forms have confirmed that removal of the Gla domain strongly affected the binding to TF but also suggested that truncation, which leaves the hydrophobic stack region (residues 39-43) intact, provides a structural determinant that partly preserves the interaction between TF and factor VII (112). Although these experiments clearly indicate the involvement of the FVII Gladomain, it is not clear whether this is due to direct protein-protein interactions or a more indirect effect due to allosteric interactions mediated by the binding of calcium ions to the factor VII Gla-domain. Competition binding experiments indicate that the purified FVII Gladomain (residues l-38) is incapable of inhibiting the specific binding of factor VIIa to cell surface TF (113), thus suggesting that the factor VII Gla-domain does not, in itself, contain the structural elements necessary for the formation of a stable binary factor VIIa:TF complex. Other studies (114,115) using synthetic peptides and specific antibodies to factor VII have implicated the hydrophobic stack region and regions in the protease domain as important in TF binding (112). An interaction between this region and the factor VII Gla-domain is suggested by the observation that antibodies raised against residues 195-206 are calcium dependent, and bind tighter to full length factor Vlla than to Gla-domainless factor Vlla (115). This suggests that the factor VII Gla-domain is necessary in inducing a calcium dependent conformational change in the factor VIIa molecule that, in turn, expresses one or more neoepitopes directly involved in TF binding. Other calcium binding sites such as the one identified in the factor VII protease domain (residues 210-220) also appear to contribute to factor VIla:TF binding, presumably by a similar mechanism (116,177). Binding of TF to structures outside the Gla-region is also indicated by studies that show that the amidolytic activity of Gla-domainless factor Vlla is stimulated by TF and calcium ions (I 10). An interesting observation (118) suggests that a dansyl-Gly-Gly-Arg chloromethyl ketone treated bovine factor Vlla des Gla form bound to TF, whereas bovine des Gla factor VII did not. The EGF domains of factor Vlla have also been implicated as possible TF binding sites. It has been demonstrated that a monoclonal antibody that specifically reacts with the first EGF domain (residues 51-88) directly inhibits the binding of factor VIIa to TF (119). Others have shown that factor VII/factor IX chimeras, consisting of various portions of the factor VII light chain connected to the factor IX heavy chain, bind to TF apoprotein with an affinity essentially equal to that of wild-type recombinant factor VIIa (120). This has been used as evidence to support the notion that the high-affinity TF binding site is located in

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the EGF domains. More recent studies have attempted to address this problem by studying the TF binding properties of factor VII fragments obtained by tryptic digestion of fully carboxylated wildtype factor VIIa (121). Fragments corresponding to residues l-109 have been purified and found to inhibit the binding of factor VIIa to cell surface TF. Interestingly cleavage of the factor VII Gla-domain by treatment with cathepsin G resulted in complete loss of inhibitory activity, suggesting that the calcium ion-dependent conformation of the Gla-domain is providing some degree of structural integrity necessary for the interaction of the EGF domain with TF. Taken collectively, these studies indicate that the physical interaction of factor VIIa with TF involves multiple regions of the factor VIIa molecule, including the N-terminal Gla-domain, the EGF domains, as well as residues located in the factor VII protease domain. Studies with synthetic peptides (117) have suggested that residues 285-305 and residues 376-396 of factor VII are involved in factor X recognition, and also that these epitopes on factor VII interact with the heavy chain of factor X. The involvement of Arg,, in factor X recognition was confirmed in a study with a factor VIIa (Arg 290Ala) substitution variant. This replacement did not affect TF binding (122). TFPI as an Inhibitor of Factor VIIa Initiation of the TF pathway is under strict control by TFPI apparently exerted via a complex reaction mechanism. The direct interaction between TFPI and factor VIIa/TF as indicated by the inhibition of the amidolytic activity (Ki = 200 nM) (62,75,76) does not in itself explain the very strong inhibition of coagulation assays initiated by TF where inhibition at subnanomolar levels is often observed. TFPI inhibition of the factor VIIa/TF-mediated factor X activation is currently thought to occur in two discrete steps. A model has been proposed (48,70,73,74) in which TFPI binds initially to factor Xa in a reversible reaction, and in the next step factor Xa/TFPI interacts with factor VIIa/TF and a quaternary complex is formed. To explain that an efficient inhibition of factor VIIa/TF was only observed in the presence of factor Xa, the model postulates a quaternary complex (48) involving a strong binding of the product, factor Xa, to the enzyme. Three groups have recently studied the inhibition kinetics (48,79,123). In essence, this type of inhibition might be described by the simplified model shown below:

E+S

=,

ES-

EP =>

E+P

TFPI

3 TFPI

l

EP

where E is factor VIIa/TF, S is factor X, P is factor Xa, and I is TFPI. Binding of the product, P, to the enzyme, E, is strongly stabilized by specific interaction with the inhibitor. Formation of this dead-end inhibitor complex prevents interaction with the substrate, S. KPI domain 2 of TFPI obviously contribute to the specificity of the inhibitor and allows it to distinguish between the substrate, factor X, and the product, factor Xa. Generally KPIs are characterized by a rigid protein structure serving as a scaffold that presents a matching

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contact area to interaction with a substrate binding pocket of a serine protease. In itself, such interactions can be very strong as exemplified by the binding of aprotinin to trypsin, Ki = 6 x lo-l4 M (124). Thus, it might be expected that TFPI inhibition is described primarily by the interaction between the active site of the protease and the respective KPI domain. Experiments with truncated TFPI derivatives and separately expressed KPI domains (Fig. 4A) clearly indicate that this is not the case. The figure shows that inhibition of factor Xa by KPI domain 2 from TFPI, Ki = 4 x lop8 M, is several orders of magnitude weaker than that of full length TFPI,-276, Ki = 2 x 10-i’ M. The results, therefore, suggest that secondary site interactions with structures outside the KPI domain play a significant role for the interaction with factor Xa (Fig. 4). As reported by many investigators [see, e.g., (6,40)], and also shown in Fig. 4B, the inhibition by full length TFPI of factor VIIa/TF-mediated factor X activation is still an order of magnitude stronger (apparent Ki about 10-l’ M); and it can, therefore, not be accounted for by factor Xa inhibition alone. These results, and the very low apparent Ki value, suggest that the quaternary IEP complex is extremely stable, and that this stability is obtained as a result of multiple strong interactions. Complex formation with truncated TFPI derivatives

1,

VP1 derivative](M)

-1

[TFPIderivative](nM)

FIG. 4. Inhibition by TFPI derivatives.(A) Inhibition of factor Xa amidolyticactivity. Factor Xa (10 PM) was incubated with various concentrationsof TFPI derivative for 15 min. Substrate(0.3 mM MeOCO-CHA-GlyArgpHA) was then addedand residualactivity measured.(B) Inhibition of factor VIIa/TF/factor Xa activity (modified Sandsetassay). Factor VIIa (2 PM), lipidatedTF, and 1 nM factor X wasincubatedwith variousconcentrations of TFPI derivativesfor 15min. Factor X (99 nM) and 0.3 mM MeOCO-CHAGlyArgpNA was then added and residualactivity measured.The experimentswere performed in 50 mM TrisCI, 100mM NaCI, 5 mM CaCl,, 0.01% Tween 80, pH 7.4. q : TFPI,_,,6, 0: TFPl,_,,, A: TFPI,_,6,, n : TFPI,j_,,o (domain 2).

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(TFPII-i6r or TFPIimZ4,) leads to less efficient dead-end inhibition of the factor VIIa/TF activity and the inhibition with KPI domain 2 appears to affect only the factor Xa activity. When heparin is present together with full length TFPI this further stabilizes the quarternary complex, as indicated by an apparent Ki = 1 pM (results not shown). This effect of heparin is observed only with TFPI derivatives comprising the KPI domain 3. A synergistic anticoagulant effect between TFPI and heparin is observed in in vitro coagulation assays (125-127). The higher affinity of TFPI in the presence of heparin may partly explain this effect, but it is also of importance in this context that heparin accelerates the inactivation of factor Xa by TFPI approx 40-fold (43). A relatively rapid inactivation of factor VIIa/TF is observed in the presence of heparin and plasma concentrations of antithrombin III (128,129). Whether or not this reflects an anticoagulant effect of physiological relevance has been a matter of discussion (130). Regulation on Cofactor and Inhibitor Levels

Of the numerous coagulation factors assigned to the intrinsic and extrinsic pathways of coagulation, TF is the only factor that is fully active in its native state and not activated by proteolysis. The expression of procoagulant activity in the blood requires only cell membrane damage or perturbation with subsequent exposure of TF. Thus, the very low TF level measured in the blood (11) may reflect primarily normal cell death and destruction. Possible ways in which blood cells and vasculature can be altered from a normal quiescent state to express TF in vivo has recently been reviewed by Esmond (131). Studies with cells in culture have resulted in detailed information about the expression of TF and its regulation; however, it is often difficult to extrapolate to in vivo conditions. In vitro studies have shown that most cells express detectable TF albeit predominantly at a low concentration [see, e.g, (132)]. Furthermore, addition of various growth factors are able to produce a rapid, but transient, appearance of TF mRNA. As a result of these observations, the TF gene might be classified as a growth-related immediate early gene (133), whose transcription is initiated coordinately with the induction of cell proliferation. This may indicate a role for TF beyond hemostasis. Recent experiments with sarcoma cells suggest that TF may control angiogenic properties of tumor cells in mice (134). In relation to the hemostatic response, the TF expression by endothelial cells and monocytes/macrophages is of particular importance. When quiescent, these cells normally lack TF activity but in culture they can express TF in response to inflammatory agents, and it is generally believed that in vivo inflammation constitutes a major mechanism by which an upregulation of coagulation takes place on a cellular level. In endothelial cell cultures TF synthesis can be stimulated by cytokines, while monocytes are known to respond to endotoxin with a transient increase in TF expression with a maximum of 4-6 h after stimulation. TF gradually decreases to baseline levels 12-24 h later, even in the presence of a continuous high endotoxin level (132). Whether the endothelial cells in vivo actually expose TF as a response to an inflammatory stimuli remains uncertain. Monocytes, on the other hand, is generally assumed to produce TF in response to endotoxins and cytokines (135). In addition to stimulating TF synthesis, endotoxins can suppress the expression of thrombomodulin (136), thereby changing the cell surface also in this way to become more thrombogenic. TF is expressed on monocytes from patients with inflammatory diseases such as septic shock and organ transplant rejection (137,138), and TF expression on monocytes/macrophages has also been observed in atherosclerotic lesions (139).

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Coagulation might also be initiated by cell-mediated, TF-independent mechanisms. Recent studies have suggested that monocytes, after activation by ADP, can bind factor X to the Mac-l receptor and somehow stimulate its activation without the participation of TF (140). In contrast, polymorph neutrophil leukocytes might initiate coagulation via the intrinsic pathway by assembly of the prekallikrein/kininogen/factor XI complex (141). It is important that the activator function of TF and subsequent propagation of coagulation is counterbalanced by the action of inhibitors. In general, the blood is swamped with inhibitors with the capacity to neutralize active proteases as fast as they are generated. Antithrombin III, which is present in high plasma concentrations (2-5 PM), is important in that respect as the primary inhibitor of coagulation proteases. A number of activated coagulation factors, including thrombin, factor Xa, and factor IXa, are rapidly inhibited by antithrombin, especially in the presence of heparin or other glycosaminoglycans. In spite of this, the blood is not devoid of activated coagulation factors; they are only present at a very low steady-state level. This level might be determined by the ratio v,//c; [I], where v, is the rate of activation and kj is the on-constant for inactivation of the protease with a practically irreversible inhibitor, (I). Thus the steady-state level might be increased with an enhanced activation rate (e.g., via cofactor stimulation), or an elevated level could occur as the result of an impeded reaction with the inhibitor. With this interpretation, one would, for example, expect a transition to a more activated state, simply as a consequence of the decrease in the antithrombin reaction, when the blood passes a segment in which the glycosaminoglycans has been denuded from the vessel wall. It may be discussed whether inactivation of factor VIIa/TF by antithrombin is of physiological significance (130), however, TFPI is generally thought to be more important for the initial events of TF-dependent reactions, and, indeed, it possesses inhibitory properties with interesting implications for the regulation of coagulation. These properties are perhaps best illustrated in the flow model introduced by Gemmell et al. (142,143) in which a reaction mixture containing factors VIIa and X is perfused through a flow reactor consisting of a procoagulant, TF-containing surface. Figure 5 shows our own results from a flow study with the extracellular matrix of fibroblast cells as the procoagulant surface (Valentin, Nordfang, and Lindhout, submitted). Factor VIIa and factor X were perfused through the flow reactor and the generated factor Xa was measured at the outlet. Under these conditions, a factor VIIa- and TF-dependent activation on the surface produces a constant level of factor Xa (curve a). In accordance with previous results (143), discontinuation of factor VIIa from the reaction mixture does not result in a significant decrease in the factor Xa level at the outlet (results not shown). This is a demonstration of an impressive stability of the factor VIIa/TF complex reflecting the strong interaction of factor VII with TF (144,145). In Fig. 5, curve b shows the effect of including 0.25 nM TFPI. The results illustrates what might be inferred from the proposed reaction mechanism, namely that factor Xa has to be accumulated to a certain level before an effective inhibition of the factor VIIa/TF activity takes place. Generation of the product, factor Xa, results in a quaternary complex with TFPI and factor VIIa/TF thereby preventing further activation of factor X. It has been pointed out (3,4) that this property of TFPI may imply that factor VIIa/TF-induced coagulation is shut down after a certain boost of factor Xa. This transient generation of active factor X might, however, be sufficient to activate prothrombin; and coagulation is then continued mainly as a result of an upregulation of the intrinsic pathway involving thrombin-catalyzed back activation of factors XI, VIII, and V followed by subsequent activation of factors IX and X.

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0.5 0 20

10

30

Time, mln FIG. 5. Comparison of the inhibition profile of TFPI and inactive FVlIa towards factor Xa generation at the surface of fibroblast extracellular matrix. The matrix was perfused at a wall shear rate of 30 s-l with (a) 50 mM factor X and 1 nM factor VIIa; (b) 50 nM factor X, 5 nM factor VIIa, 0.25 nM TFPI; and (c) 50 nM factor X, 0.1 nM factor VIIa, 0.9 nM S344A factor VIIa. Samples were collected at the outlet of the flow reactor and assayed for FXa.

Figure 5 (curve c) also shows that inhibition of factor VIIa/TF-mediated factor X activation by an inactivated factor VII derivative is strikingly different, This form (factor VIIai), which has lost its catalytic activity due to reaction of its catalytic site with a chloromethylketone, still binds TF with a high affinity, When factor VIIai is added to the factor VIIa containing reaction mixture and perfused through the flow reactor, it competes for TF binding and effectively blocks complex formation between TF and factor VIIa. However, whereas TFPI is capable of inhibiting factor Xa generation after the factor VIIa/TF complex has been formed, this is not the case with factor VIIai. As a consequence of the high stability and slow off-rate of the factor VIIa/TF complex, factor VIIai did not significantly replace factor VIIa within the time limit of the experiment. Thus, the the two types of inhibition might be significantly different also in a physiological setting. Preincubation with TFPI compared to a strong direct competitor of factor VIIa binding to TF is likely to be less efficient in preventing initiation of coagulation. On the other hand, displacement of factor VIIa from TF, when the complex is first formed, seems less attractive than a factor Xa-dependent neutralization of the factor VIIa/TF complex. As indicated above, the structure of TFPI as a multivalent inhibitor with three tandemly repeated KPI domains gives rise to a complex reaction mechanism, and also permits multiple ways of regulatory interactions. Each KPI domain may provide a putative contact region for the active site of a protease; however, the fact that several segments of the protein are susceptible to proteolytic cleavage also reveals possible ways to control its inhibitory action. It has been shown recently (146,147) that specific cleavage at Thrs,-Thrs8 by leukocyte elastase has a dramatic effect on the inhibitory properties of TFPI. The cleavage segregates KPI

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domain 1 from the rest of the molecule, and this event not only interferes with the inhibition of factor VIIa/TF, it also abrogates the TFPI inhibition of factor Xa. As a consequence of the weak interaction with the separate KPI domain, factor Xa is released from the complex with an intact procoagulant activity (147). Although the physiological importance of this reaction remains to be elucidated, it is of interest to note that an inflammatory response leading to stimulation of neutrophils to release their content of active proteases (elastase, cathepsin G, and protease 3) might result in upregulation of the procoagulant activity by this mechanism. One could even imagine that the release of factor Xa resulted in factor VII activation and initiation of coagulation. In addition to TFPI, antithrombin III has also been shown to be very susceptible to cleavage by human leukocyte elastase (148), and this might as well lead to an increased procoagulant activity. Antithrombin III is cleaved efficiently only in the presence heparin in contrast to TFPI, which is protected from cleavage in its presence (Petersen, unpublished results). Additional possible regulatory interactions include the binding of TFPI to the vascular wall, to heparin, and to phospholipids. Sandset et al. (54) demonstrated that injection of heparin, subcutaneous as well as intravenous, increased the plasma concentration of TFPI two- to eightfold, and that the effect was dose dependent. A finding later confirmed by others (55,56). No difference in TFPI release was observed between low molecular weight heparin or unfractionated heparin (54,55,149). Released TFPI activity peaks 3-5 min after intravenous heparin administration, whereas subcutaneous administration induced a less immediate and more prolonged release. The heparin releasable pool is believed to originate from TFPI bound to glycosaminoglycans present on the luminal side of the endothelium. TFPI might be displaced to the circulation from these sites by heparin. Eight repeated heparin injections with time intervals of 9-19 h to the same volunteer(s) was shown not to change the level of released TFPI, demonstrating that depletion of this TFPI pool did not occur (150). This observation suggest that TFPI most likely reattaches to the endothelium. As mentioned earlier, heparin released TFPI is not associated with lipoproteins (53,57). Using an in vitro assay, it was demonstrated that heparin, in a dose-dependent manner, can prevent complex formation between TFPI and lipoproteins (61). It was furthermore shown that heparin was capable of releasing TFPI from a preformed TFPI-VLDL complex, although this required an unphysiological high amount of heparin (61). In vivo administration of heparin does not displace the TFPI complexed to lipoproteins in plasma because the elution profile from the gel filtration column remains unchanged when control and postheparin plasma are compared (57). In humans, TFPI released by heparin stays in circulation as long as heparin is present, as demonstrated by the administration of protamine sulfate or protamine chloride following heparin treatment, which cause the TFPI activity to decrease immediately to pretreatment values (149,151). In accordance with this, it has been observed that heparin released TFPI has a half-life similar to the elimination of heparin (54). Intravenous injection of radiolabelled TFPI into rabbits showed that 30 min following injection the radioisotope was found primarily in liver and kidneys (152), indicating a clearance of TFPI by this route. Also, the low density lipoprotein receptor-related protein (LRP) most likely is involved in the clearance of TFPI (153). LRP mediates endocytosis of several circulating plasma proteins, predominantly in t.he liver. If the LRP activity on hepatoma cells were blocked by antibodies, the cellular degradation of ‘25I-labeled TFPI was reduced by more than 80% (153). The initial TFPI binding site on hepatocytes is not LRP, but more likely heparan sulfate proteoglycans, or other Cell surface proteins, that after binding of TFPI presents it to LRP for uptake and degradation (153).

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Various affects of heparin and calcium on the transient inhibition of factor Xa has been observed, depending on the nature of the TFPI derivative. Heparin enhances the inhibition of factor Xa by both full length and C-terminal truncated TFPI when calcium is present, whereas in its absence, only the inhibition of factor Xa by full length TFPI is moderately affected by heparin (78). Heparin has no effect on the factor Xa inhibition either in the presence or absence of calcium, with a TFPI variant comprising only KPI domain 1 and 2 (68). ACTIVATION OF THE TISSUE FACTOR PATHWAY UNDER BASAL CONDITIONS

The human coagulation system continuously generates very small quantities of factors IXa, Xa, and thrombin under basal conditions. This has been shown by measurement of plasma levels of activation peptides released from factors IX, X, and prothrombin (12,13,154). A substantial amount of this activation originates as a result of the presence of both factor VIIa and TF. Evidence for this notion comes from measurements of circulating factor VIIa levels in normal plasma using a factor VIIa specific coagulation assay (7,155) and from measurements of the TF antigen level (11). Taken together, these measurements suggest that small, but significant amounts of factor VIIa/TF are present under basal conditions. In addition, experiments with chimpanzees have shown that neutralization of TF by an anti-TF monoclonal antibody resulted in a significant suppression of basal activation of factors IX and X (156). The question about in vivo activation of factor VII has also been addressed by studying the degree of activation under basal conditions. Using the factor VIIa specific coagulation assay, it has been possible to measure and directly compare the circulating factor VIIa levels in plasma from severe factor VIII or factor IX deficient patients (10). The data from this study indicate that normal individuals possess an average functional factor VIIa level corresponding to 1% of their total FVII antigen (assuming an average factor VII antigen level of 400 rig/ml). Factor VIII deficiency has a small but statistically significant effect on circulating factor VIIa, with patients on average possessing a level corresponding to 60% of the factor VIIa level observed in normals. Factor IX deficiency, on the other hand, has a far greater effect with factor IX-deficient patients possessing a basal factor VIIa Ievel corresponding to < 10% of that observed in normal individuals. When taken in conjunction with previous observations that factor VIII and factor IX-deficient individuals possess essentially normal levels of factor Xa (12), these data would support the notion that factor IXa is responsible for factor VII activation under in vivo conditions. It is important to stress, however, that this factor IX-dependent activation may only be important under nonstimulatory and nonthrombotic conditions, and it is quite possible that other activation mechanisms such as autoactivation and/or factor Xa-mediated activation takes over immediately following vascular injury. THE ROLE OF THE TISSUE FACTOR DEPENDENT IN DISEASED STATES

PATHWAY

Cardiovascular Disease A number of studies reporting on increased plasma factor VII antigen levels (FVII:Ag) as well as factor VII coagulant activity (FVII:C) in patients at risk for cardiovascular disease

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have been published during the last decade. In 1986 it was claimed that FVII:C levels were as powerful as total cholesterol in predicting risk for ischemic heart disease (157). The increase in FVII:C was at least partly attributed to a fraction of plasma FVII:C that was sensitive to phospholipase C, and showed a significant positive correlation with plasma triglycerides (158,159). The same authors also found the presence of increased levels of the phospholipase C-sensitive FVII:C in male survivors of acute myocardial infarction (159). The increase of the factor VII-phospholipid complex in risk patients such as those with an acute myocardial infarction was confirmed by Sandset et al. (160), who also found a highly significant correlation to the triglyceride level. A number of reports have then appeared showing a correlation between the increased FVII:C levels in plasma and plasma triglycerides (161), total cholesterol (162), low density lipoprotein (LDL), and the very low density lipoproteins (VLDL) (163-166). The reduction of FVII:C by phospholipase C reported (158,167) was proposed to involve an effect of this enzyme on triglyceride-rich lipoproteins (VLDL, chylomicrons), which is consistent with the observed correlation between the phospholipase C-sensitive fraction of factor VII:C and the plasma triglyceride concentration. Adding chylomicrons to plasma with a constant factor VII concentration in vitro did not, however, affect FVII:C activity (168). This is in conflict with the original theory by Dalaker et al. (159), who suggested that phospholipase C reduces FVII:C activity by releasing normal zymogen factor VII from its complex with phospholipids, implying that binding of factor VII to the phospholipase C-sensitive plasma lipids causes an increase in FVII activity that can be reversed by phospholipase C. The alternative theory offered by Hubbard and Parr (167,168) suggests that phospholipase C reduces FVII:C activity by modifying triglyceride-rich lipoproteins to a form that binds to factor VII. When it comes to the prediction of cardiovascular disease, the phospholipase C-sensitive FVII:C activity may, therefore, have no greater significance than the measurement of plasma triglyceride levels. The Northwick Park Heart Study published in 1980 and 1986 including middle-aged men (mean age 52 years) (157,169) as well as later studies, all find increased level of factor VII in plasma to be predictive for ischemic heart disease (158,161,170). In 1989, Hoffman et al. (171) published a study showing significantly increased FVII:Ag as well as FVII:C levels in young first-degree relatives of patients with ischemic heart disease (mean age 34.9 years). The study included also one group of patients with ischemic heart disease (14 men and 1 woman between 45 and 66 years old) and one group consisting of 15 individuals (10 men and 5 women, aged between 23 and 42 years) at low risk for ischemic heart disease. The finding of increased FVII:C levels in patients with ischemic heart disease as compared to low-risk individuals was confirmed. As compared with the low risk individuals, elevated serum levels of total cholesterol and triglycerides were found both in the first-degree relatives and in the patients with ischemic heart disease. It has been discussed extensively whether the increased FVIIC-level in plasma reflects an increased

level of activated

factor

VII or mainly the zymogen level. To elucidate this,

assays claimed to be more sensitive for the activated form of factor VII have been introduced. Clotting assay using bovine thromboplastin and the amidolytic assay described by Seligsohn et al. (172) were used and the results compared to determinations of the total amount of factor VII analyzed by clotting assay using rabbit thromboplastin or determination of FVII:Ag by ELISA. These studies indicated that the increased factor VII level primarily was related to zymogen factor VII (171,173,174). In the study that used bovine thromboplastin to measure the activated form of factor VII, no correlation was found between the factor VII level and triglycerides (174,175). The group of Negri et al. (175) also

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determined the complex between factor VII and phospholipids as reflected by the difference in activity found before and after treatment with phospholipase C, and found this complex to correlate with triglycerides, confirming the results of Sandset et al. (160). The effect of dietary fat on the various factor VII fractions in plasma has been extensively studied during the last years. The diurnal variation in plasma triglyceride concentration and FVII:C was studied in healthy individuals put on different diets, high on either polyunsaturated or saturated fatty acids. The plasma FVII:C followed the triglyceride concentration, with a delay of 160 min showing the highest levels in the late afternoon and evening. No corresponding influence on the FVII:Ag was noted. The increased FVIIC was suggested to be the result of a conversion of single-chain factor VII into the activated two-chain form. The negatively charged surfaces provided by the large lipoproteins (VLDL) and chylomicrons were inferred to facilitate the activation of coagulation factors involved in the intrinsic coagulation pathway such as factors XII, XI, and IX and the further conversion of factor VII to VIIa by these factors (176,177). In 1994, two articles appeared describing the effect of dietary fat on factor VII levels. One described the effect in healthy individuals (178), whereas the other included both a control group of healthy individuals and a group of 33 patients with a previous acute myocardial infarction divided into one group with hypertriglyceridemia and one being normotriglyceridemic (179). In the study of Mitropoulos et al. (178), the normal individuals were given 4 weeks of a saturated fat diet, after a washout period of 12 weeks, an unsaturated fat diet, and after another washout period, a carbohydrate diet. Blood samples were taken on the last day of each diet period. FVII:Ag did not differ between diets of unsaturated fat and carbohydrate, but was significantly higher following the saturated fat diet. The FVII:C also was highest following the period of the saturated fat diet, lower after the unsaturated fat diet, and even lower after the carbohydrate diet, the differences being significant for both variables. Factor VIIa directly determined with the specific method of Morrissey (180) showed the same pattern as FVII:C, although all values still were within the normal range for FVIIa (3.6 f 1.44 rig/ml). The study of Silveira et al. (179) describes the changes in the factor VII parameters following the intake of a standardized oral fat load. Basic levels before the meal showed the highest FVII:Ag levels in the hypertriglyceridemic patients and the lowest in the normohyperglyceridemic patients, with the controls in between. FVII:C was highest in the controls and so was the FVIIa, measured with bovine thromboplastin. Interestingly, the highest fraction of FVIIa was found in the control group before the fat meal. After the fat load, no changes were seen in FVII:Ag or FVII:C, while the FVIIa levels increased, peaking at 3-6 h after the meal. The controls showed the highest levels in all samples throughout the study period, The activation of factor VII seemed to be related to the free fatty acid production during lipolysis of triglycerideric lipoproteins. The lower FVIIa activity found in the hyperglyceridemic patients was explained by the less efficient formation of free fatty acids as a result of a lower lipoproteinlipase activity in such patients. In this study, an increased level of TFPI was found in the hypertriglyceridemic patients, which was taken as an indication of a more efficient defense against an activated extrinsic coagulation pathway initiated by the increased FVIIa concentrations. The apparent discrepancy between various studies, especially with regard to the activated factor VII levels, may be due to the different methods used. Not until recently a specific method determining only the activated form of the factor VII molecule was described (180). Using this method, or slight modifications hereof, a significantly lower level of FVIIa was found in a Japanese population (mean 2.5 rig/ml) as compared to the value previously reported for the Western populations (3.6-4.3 rig/ml). In Japanese, the mean FVIIa concen-

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tration was 39% higher in patients with ischemic heart disease than in controls (181). A later publication by the same authors (182) failed to show any direct association of FVIIa with lipids, and suggested that the FVIIa-lipid dissociation constant is relatively high. An agerelated increase of FVIIa and FVII:Ag, the ratio FVIIa-to-FVII:Ag being higher in men than in women, was described. Increased FVIIa levels were found in hypertensive and diabetic patients and arterial cardiovascular disease was more common in the high-FVIIa group than in the low-FVIIa group. During the latest decade, the importance of thrombus formation as the immediate cause of most major clinical episodes of ischemic heart disease has increasingly been focused. In addition, a potentially important role of the hemostatic mechanism in the development of atheroma is being discussed. The epidemiological Northwick Park Heart Study demonstrated a strong relationship between the level of factor VII activity and the later incidence of ischemit heart disease (157). Several studies, including the Northwick Park Heart Study, reported an association between high levels of plasma fibrinogen and ischemic heart disease. The high fibrinogen levels were linked to smoking (183-185). Provided various lipid fractions may facilitate the activation of the zymogen factor VII into the active two-chain molecule, the formation of thrombin may be initiated through the exposure of tissue factor by rupture of an atheromatous plaque. The fragment l-2 (Fl-2), formed as a result of the conversion of prothrombin into thrombin, has been claimed to stimulate the synthesis of the vitamin K-dependent coagulation factors including factor VII (176). In accordance with a tentative importance of the factor VII-dependent coagulation pathway in the development of atherosclerotic disease, or at least for the major events such as acute myocardial infarction and stroke, inhibition of this coagulation pathway should be of major importance in patients with extensive atherosclerotic disease. The infusion of an inactivated factor VII molecule in baboons subjected to carotid endarterectomy in fact has shown a significant reduction of the deposition of platelets and fibrinogen at the site of vessel wall injury (186). In this context it is of interest to mention the results reported by Sosef et al. (187), who used ultrasonography to measure carotid intima-media thickness in healthy volunteers aged 18-56 years. An increased artery wall thickness is seen in advanced atherosclerosis. In this material of healthy subjects having signs of early atherosclerosis, no association of factor VII, or its polymorphism, with intima-media thickness was seen. No attempts were made to assay FVIIa specifically. In another material (Wallmark et al., personal communication), consisting of 503 individuals aged between 48 and 67 years, factor VIIa was determined using a modification of the method of Morrissey et al. (10). In these individuals, the plaque size, intima-media thickness, and maximal media thickness were determined using ultrasonography without any signs of acute ischemic heart disease and were found to decrease with increasing FVIIa levels confirming the results of the study by Sosef et al. (187). Recently, in a study on seasonal variations, it was reported (188) that plasma levels of fibrinogen and FVII:C were significantly higher during the winter season. The authors suggest an association between this variation and the marked seasonal variation in death from ischemit heart disease and stroke in the elderly, which also culminates during winter seasons. Recently, a genetic polymorphism of the factor VII gene that changes arginine at residue 353 to a glutamine (R353Q) was described (189). This polymorphism was associated with lower plasma levels of FVII:C. The frequency of the allele coding for this substitution was 0.1, and carriers of this allele had levels of FVII:C 22% lower than the sample mean. Individuals homozygous for the R353Q allele had both low FVII:C and low FVII:Ag levels. Similar findings have been reported in healthy women (190). It was speculated (189) whether the

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amino acid substitution may alter the conformation of the protein leading to reduced secretion from the liver or increased catabolism. Others have suggested that the substitution influences interactions of factor VII with phospholipid surfaces or tissue factor, thereby having an indirect effect on factor VII activation and plasma levels hereof. The sequence may also be involved in the interaction between triglyceride-rich lipoproteins and the factor VII molecule. The determination of the factor VII genotype may be useful in finding individuals at risk for development of ischemic heart disease (191).

Hemophilia The importance of the extrinsic pathway system in normal hemostasis has been increasingly notified. Accordingly the limited bleeding problems in patients with deficiencies in the contact activation system (factor XI and factor XII-deficiencies) (192), as compared with the severe bleedings seen in patients deficient in factor VII (193), has been emphasized. In fact, factor XII does not seem to be involved with hemostasis because patients with factor XIIdeficiencies do not bleed (192). As mentioned before, the development of sensitive assays for the determination of a number of cleavage peptides (12,13,194) generated by the activation of coagulation proteins has made it possible to show that coagulation is a permanently ongoing process. Thus, a certain basic level of factor X-activation peptide as well as the prothrombin fragment (F1+2) was found in normal individuals, at a level that was significantly lowered in patients with factor VII deficiency. In contrast, patients with factor VIII deficiency (hemophilia A) or factor IX deficiency (hemophilia B) had peptide levels within the normal range, indicating the importance of the factor VII-dependent coagulation system for the activation of factor X and prothrombin (194). Because not only patients with severe factor VII deficiency but also hemophilia A and B patients suffer from severe bleedings, these findings also indicate that a basal activation of factor X and prothrombin is not enough to ensure an optimal hemostasis. Interestingly, hemophilia A and B patients were shown to have a lower than normal plasma level of factor VIIa, as measured with the specific assay using a truncated tissue factor (10). This may indicate an impaired capacity of activating factor VII in hemophilia, especially hemophilia B, having the lowest levels of FVIIa in plasma. Thus, the FVIIa level in plasma might be a sensitive monitor of coagulation abnormality stressing the importance of the extrinsic coagulation pathway in normal hemostasis. The adequate treatment of patients deficient in factor VIII or factor IX is considered to be the administration of the lacking coagulation protein. However, in a substantial fraction (lo-25%) of hemophilia patients complicated by the development of antibodies against factor VIII/factor IX, such therapy is met with limited success. Much effort has, thus, been focused on the search for so-called “factor VIII bypassing” agents for use in these patients. A “factor VIII bypassing” agent should be capable of initiating hemostasis independent of the presence of factor VIII/factor IX. Most commonly used are so-called prothrombin complex concentrates containing variable amounts of the vitamin K-dependent coagulation factors (factor IX/factor IXa, factorX/factor Xa, factor VII/factor VIIa, prothrombin, protein C, protein S). However, such concentrates are limited in their effectiveness, and a general supplement of activated coagulation factors is associated with thrombotic side effects (195). Because factor VIIa is proteolytically active only when complexed with TF, the administration of factor VIIa alone should minimize the risk of inducing a generalized systemic activation of the coagulation system.

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A few hemophilia A patients with antibodies were successfully treated with factor VIIa derived from human plasma, thereby indicating that the administration of exogenous factor VIIa was capable of inducing hemostasis in patients with an impaired factor VIII/factor IXdependent coagulation system (196). Later, this concept was further substantiated when recombinant factor VIIa was shown to shorten the cuticle bleeding time in hemophilia A as well as in hemophilia B dogs (197). With the availability of recombinant human factor VIIa (19) it has now been possible to apply this treatment to a number of patients with a variety of serious bleeding complications (198,199). In total, 279 patients have been given recombinant factor VIIa, and 1270 bleeding episodes have been treated. Most patients suffered from hemophilia A with inhibitors (n = 254), 13 had hemophilia B with inhibitors, and 12 were nonhemophiliacs who had developed inhibitors against factor VIII. One patient with coagulopathiea due to liver impairment was treated. In approximately 90% of these instances the clinical response was judged as being effective (198). Also, major surgery was carried out without any bleeding problems under the cover of factor VIIa as the sole hemostatic agent. This indicates that supplementary factor VIIa is capable of temporarily restoring the coagulation process in hemophilia patients. A factor VII plasma level of approximately 5-7 U/ml seems adequate to sustain hemostasis, although a higher level (20-30 U/ml; 8-12 @g/ml) seems necessary to initiate hemostasis in moderate to severe bleeds (198). It should be emphasized that a level of 5-7 U/ml is five times higher than the normal endogenous factor VII antigen level. A total of 10 patients with congenital factor VII deficiency (five females and five males aged between 2 months and 83 years, median 16.8 years) have been treated with factor VIIa in association with 21 bleeding episodes. The doses given to these patients varied between 15 and 30 pg/kg every 4-6 h, and were extrapolated from the amount of factor VIIa necessary to normalize the prothrombin time of the patient’s plasma in vitro. It was obvious that substantially lower doses are needed to obtain an adequate hemostasis in patients with an intact intrinsic (factor VIII/factor IX-dependent) coagulation system. The efficacy rate of factor VIIa in the factor VII-deficient patients was 95%. No immediate side effects were seen. One patient received by mistake an overdose of factor VIIa (about 20-40 times the recommended dose) without any signs of a systemic activation of the coagulation. However, this patient developed IgG antibodies against factor VIIa. She is the only patient among the total number of individuals [279] having received factor VIIa who has developed antibodies, and it should be emphasized that she belonged to the group of patients with no factor VIIantigen (factor VII-deficient patients). Hemophilia patients, on the contrary, have their own factor VII antigen and are helped to compensate for their impaired intrinsic coagulation by the addition of extra factor VIIa in addition to the factor VII of their own. Therefore, any risk of developing antibodies against factor VII in these patients should be minimal. The mechanism behind the hemostatic effect of high doses of factor VIIa (about five times the normal concentration) in hemophilia patients is still unclear. Tissue factor-bound factor VIIa is inhibited by TFPI as well as by antithrombin III (200). The TFPI inhibition requires factor Xa before it acts as a factor VIIa/TF/inhibitor forming the quatenary factor Xa/TFPI/factor VIIa/TF complex. This complex is only minimally influenced by the factor VIIa concentration as shown in vitro by Rao et al. (200). On the contrary, the inhibition of factor VIIa bound to cell surface TF by antithrombin III does not require the presence of factor Xa. Furthermore, binding of antithrombin III to factor VII accelerates its dissociation from cell surface TF. High concentrations of factor VIIa restored the catalytic activity of cell surface factor VIIa/TF complexes inhibited by antithrombin III/heparin. One

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explanation that hemostatic efficacy is only obtained at a relatively high dose of factor VIIa in hemophilia may, therefore, be this interaction with antithrombin III in the presence of cell surface glycosaminoglycans. The active factor VIIa bound to TF might be neutralized by antithrombin III and dissociated from the TF sites, thereby creating free TF ready to complex with fresh factor VIIa molecules. Another possibility would be that factor VIIa forms hemostatically active complexes also with other structures than TF. It was shown that factor VIIa was capable of activating factor X in the presence of phospholipid vesicles, although much slower than in the presence of TF (201). Recently, monocytes were demonstrated to support TF-independent factor Xa generation mediated by factor VlIa, although the rate was less than 10% of that achieved after induction of TF expression on the monocytes (202). The authors suggested that tissue macrophages or stromal cells at sites of injury may support factor Xa production in the presence of high doses of factor VIIa. The fact that this TF-independent factor Xa generation would not be inhibited by TFPI may argue in favor of the importance of this putative mechanism in achieving hemostasis in hemophiliacs by the administration of exogenous factor VIIa. A normalizing effect on bleedings in hemophilia basically caused by deficiencies in the intrinsic coagulation pathway obtained by manipulating the extrinsic system is illustrated by hemostasis achieved by addition of extra factor VIIa as described above. Another possibility to enhance the activity of the extrinsic system would be to neutralize the natural inhibitor of the system, TFPI. Following the shut down of the factor VIIa/TF-dependent system by TFPI complexed with factor Xa, there will be no thrombin-generating system in hemophilia patients lacking the enforcement loop that includes factor VIII and factor IX (the intrinsic coagulation system). Therefore, TFPI has been attributed an important role in hemophilia (43). Addition of full-length TFPI was also recently shown to prolong the clotting time of normal plasma as measured in a dilute TF coagulation system (77,203). In contrast. the inhibition of the TFPI shortened the coagulation time of both normal and hemophilic plasma (204,205). Injection of antibodies against TFPI was later found to significantly shorten the cuticle bleeding time in a rabbit model in which the rabbits were made transiently hemophilic by administrating antibodies against factor VIII (206). In this model, the mean basic rabbit cuticle bleeding time was 5.4 min (range 3-l 1 min; n = 13) increasing to 26.1 min (range 15-30 min; n = 14) after given antifactor VIII. Following the blocking of TFPI, the mean cuticle bleeding time again shortened to 11 min (range 6-15 min; n = 4). In addition, both the APTT and diluted tissue factor (dTF) clotting time were prolonged following the injection of antifactor VIII; the APTT up to more than 120 s and the dTF from 200 to 300 s. Also, these parameters were shortened following the administration of anti-TFPI (APTT: 80 to 57 s; dTF: 250 to 152 s). Thus, pushing the extrinsic coagulation system either by adding extra factor VIIa or by blocking the inhibitor TFPI seems to induce hemostasis independent on the intrinsic coagulation pathway. Disseminated Intravascular Coagulation (DIG) DIC usually occurs as a complication of conditions in which the circulating blood may be exposed to TF (207). Monocytes (135) and vascular endothelial cells (208) have been shown to express TF following incubation with gram-negative endotoxin in vitro. TF activity has also been demonstrated on peritoneal macrophages (209) as well as on peripheral blood mono-

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nuclear cells (210) from rabbits given endotoxin. In addition, mononuclear cells from the blood of patients with meningococcemia (211) were shown to possess TF activity. The availability of TF on cells in the circulating blood may facilitate the formation of factor VIIa/TF complexes, thereby inducing an activation of factor X that may result in the development of DIC. In such a situation, the inhibition of factor Xa by TFPI followed by the formation of the quaternary complex factor Xa/TFPI/factor VIIa/TF may be of major importance in the defense against the development of a full-blown DIC. Also, it was shown by Sanders et al. (212) that the TFPI activity of plasma could be exhausted in vitro by increasing the TF concentration of the reaction mixtures. No unanimity has, however, been achieved with regard to plasma levels of TFPI in patients diagnosed as having sepsis (213-215). A series of experimental work have been performed in order to elucidate the role of TFPI in endotoxin-induced DIC. Normal levels of TFPI did not prevent the development of DIC in rabbits with TF- or endotoxin-induced DIC (207). However, rabbits immunodepleted of TFPI were shown to be sensitized to DIC induced by TF (216) as well as by endotoxin (217). This was taken as supporting evidence for the role of TFPI as a natural anticoagulant regulating factor VIIa/TF activity expressed on cell surfaces in vivo. These findings also speak in favor of TFPI as a modulator of factor VIIa/TF activity up to a certain level, although the physiological concentration of TFPI seems to be insufficient in preventing the effect of extensive TF exposure that may occur under certain conditions such as a fulminant endotoxin influx. It should be stressed though that the plasma level of TFPI did not decrease substantially during the experiments described above. This suggests that DIC may progress not because TFPI is exhausted but because factor VIIa/TF complexes are continuously formed at a rate that normal plasma TFPI inhibition cannot match (39). The trigger function of TF in the development of DIC was, however, stressed by the fact that pretreatment with anti-TF IgG prevented the fibrin precipitation in the kidneys in endotoxinas well as TF-induced DIC in rabbits (207). Similar results were also obtained in a baboon model in which administration of monoclonal antibodies against TF prevented lethal E. coli septic shock. However, in the rabbit model (207), the animals were found to develop shock in spite of pretreatment with anti-TF IgG and attenuated DIC. By giving TFPI-depleted rabbits factor Xa and phospholipids in the absence of TF, Warn-Cramer and Rapaport (218) studied to what extent the rabbit DIC model depended on inhibition of factor Xa by TFPI. In contrast to the results obtained on infusion of TF or endotoxin, the TFPI-depleted rabbits were not sensitized to coagulation initiated by factor Xa and phospholipid. This led to the conclusion that the physiological function of TFPI in regulating TF-dependent coagulation stems primarily from its ability to inhibit factor VIIa/TF activity (218). Surprisingly, a decrease of about 15% in the plasma level of factor X was found both in the rabbits that were immunodepleted of TFPI and those that were not, even when given factor Xa-phospholipid that produced only minimal evidence of DIC. It is well known that factor Xa-phospholipid complexes can induce DIC in a number of animal models (219-222). Using higher doses of factor Xa-phospholipid than in the previous experiments, Warn-Cramer and Rapaport (223) studied the effect of infusion of factor Xa-phospholipid complexes on the plasma levels of a number of coagulation factors. A substantial decrease in the plasma levels of factors VIII, V, XII, XI, X, and fibrinogen was observed and interpreted as the result of the factor Xa-phospholipid-induced generation of thrombin. This might trigger the feedback activation of the intrinsic coagulation, resulting in a consumption of factor X and the other coagulation factors involved, independent of the factor VIIa/TF pathway. The plasma TFPI increased promptly following the

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infusion of the factor Xa-phospholipid and then gradually fell (218,223), a pattern somewhat similar to what is seen following an injection of heparin (224). The role of TFPI in the defense against endotoxin-induced DIC was further emphasized by the findings that large doses of a recombinant human TFPI given to rabbits suppressed the fibrinogen consumption induced by the injection of rabbit brain thromboplastin (225). Similar results were obtained with recombinant two-domain human TFPI in rabbits infused to protect against endotoxin-induced DIC (226). Reduced mortality from E. coli septic shock was documented in baboons following administration of TFPI (227). This effect was seen even when TFPI was administered after induction of DIC. The doses used in all these experiments were rather high, 3-6 mg/kg. Also, substantially less fibrin precipitates in lungs, kidney, and adrenals were seen in the animals that had received TFPI. Interestingly, significantly lower levels of interleukin-6 were found in the TFPI-treated animals. In a study by Bregengard et al. (228), recombinant factor VIIa inactivated by chemical modification of the active center with dansyl-glu-gly-arg-chloromethylketone was given to endotoxin-treated rabbits. A dose-dependent effect on the consumption of factor VIII, fibrinogen, and platelets occurred with doses of inactivated factor VIIa up to 200 pg/kg. Significant effects were, however, found already at a dose of 40 pg/kg, which also completely abolished fibrin precipitates in the kidneys. The same inactivated factor VIIa has also been given to baboons in the model described by Taylor et al. (229). Preliminary results seem encouraging and the study is under publication. The importance of the factor VII/TF-dependent system in the development of DIC induced by endotoxin, thus, is emphasized by the preventive effect of anti-TF IgG (213), monoclonal Fab fragments against factor VII/factor VIIa (230), TFPI (226,227), and inactivated factor VIIa (228). It is, however, still not clarified to what extent the mitigating effect on the fibrin precipitation influences the development of a fulminant septic shock and its course. Thromboembolic Disease As pointed out above, the hemostatic process is initiated by the formation of the factor VIIa/TF-complex. After the first factor Xa molecules are formed, the factor VIIa/ TF-dependent coagulation is inhibited by TFPI. Inhibition of factor Xa as well as of thrombin is known to prevent the development of thromboembolic disease (23 1,232). The most commonly used drugs in these situations are heparin and low molecular weight (LMW) heparins (233-236). The plasma level of TFPI was found to increase sharply following a bolus intravenous injection of heparin (54,237). Heparin is thought to release TFPI bound to glycosaminoglycans on the luminal surface of vascular endothelium, as it has been shown to do with lipoprotein lipase (238). The pool of TFPI bound to the endothelium seems to exceed the plasma pool substantially. It was also shown that repeated injections of a LMW-heparin did not exhaust the TFPI pool, but gave the same peak and elimination curve each time (150,239). It has been speculated whether the antithrombotic effect of heparin may be linked to the release of TFPI with a strong factor Xa-inhibiting effect (240). However, so far, no patient with deep venous thrombosis (DVT) has been found to have a lack of, or a defective TFPI (237). The same group also failed to find any effects on the plasma levels of TFPI by venous occlusion. Furthermore, no differences between the TFPI levels in plasma were observed in patients with or without phlebographically verified DVT. Such patients also showed a normal pattern of TFPI following a heparin injection (241).

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Two independent studies have shown that TFPI had an antithrombotic effect. In one study, TFPI was shown to prevent the reocclusion following thrombolysis in dogs (242). The second study (243) was based on a rabbit thrombosis model in which the juglar vein was damaged by aethoxysklerol, and the thrombus was allowed to form during 30 min of restricted blood flow. Truncated recombinant TFPI lacking the C-terminal tail (TFPI,~250), at a dose of 0.25 mg/kg, reduced the mean thrombus weight significantly. Using the same model a two-domain form of recombinant TFPI (TFPIi-r6i), at a dose of 1 mg/kg, was found to have an antithrombotic effect similar to that of LMW-heparin (Tinzaparin) at a dose of 60 antifactor Xa IU/kg (244). Following the administration of TFPI (1 mg/kg) the peak level of antifactor Xa activity obtained in plasma was significantly higher and the antifactor IIa activity lower than after injection of LMW-heparin (60 anti-Xa III/kg). It also should be notified that this antifactor Xa activity was cleared from plasma more rapidly than antifactor Xa activity induced by LMW heparin. Thus, the two-domain TFPI molecule has the same antithrombotic effect as the fulllength molecule, in spite of a 50-fold higher anticoagulant activity demonstrated in vitro (77). The molecule is glycosylated at ASN 117 in the factorxa-inhibitory domain (KPI domain 2). A mutated, nonglycosylated TFPI (N117Q,TFPI,-16,) was recently shown to have a similar antithrombotic effect in the rabbit thrombosis model mentioned above when given in a dose of 0.8 mg/kg (245,246). The question whether the antithrombotic effect of TFPI in animal models is dependent on its antifactor Xa activity is still not fully clarified. Thus, the antithrombotic effect does not completely follow the antifactor Xa activity in plasma (244,246). Further studies are in progress to elucidate this in more detail. An antithrombotic effect of TFPI has also been demonstrated in a microcirculatory thrombosis model (247) and in a rabbit model in which a venous thrombus is induced in the caval vein (248). The truncated TFPI i _i6r has also been found to significantly decrease the deposition of platelets and fibrin in an arterio-venous dacron graft baboon model, an effect interpreted as an antifactor Xa effect (249). In the same baboon model, inactivated factor VIIa was ineffective in the dacron graft but was highly effective in deendothelilized carotid artery showing that the thrombus formation on the mechanically injured artery is factor VIIa/TF dependent (249). When fibrin deposition was studied in an ex viva model, using endotoxin stimulated endothelial cells perfused with nonanticoagulated blood, deposition was reduced effectively by preincubation with a combination of TFPI, factor VIIa, and factor Xa to levels observed with matrix from nonstimulated endothelial cells. In contrast, preincubation with TFPI and factor Xa reduced the fibrin deposition to a lesser extent. Neither alone, nor in combination with factor VIla had TFPI any effect on the fibrin deposition (250). Plasma Levels of TFPI in Various Diseases

The normal plasma concentration was reported by Novotny et al. (45) to be approximately 2.5 nM. Plasma TFPI levels did not vary in subjects sampled repeatedly over weeks to months and were unaffected by whether blood was drawn in the fasting or postprandial state (213). Slightly higher plasma levels were found in older individuals than in younger subjects (214,25 1,252) as well as in women during late pregnancy as compared to nonpregnant women (45,251,253). TFPI does not seem to increase after surgery and, thus, does not seem to be an acute phase reactant (45,251,254). Increased plasma levels were, however, found in patients with advanced cancer (255). Warfarin does not affect the plasma levels of TFPI (45,256).

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CONCLUSION

Recent successful treatment of hemorrhagic disorders with recombinant factor VIIa has led to a partial revision of current theories about the basic mechanisms of coagulation. The observation that a supplement of factor VIIa can compensate for deficiencies in factors VIII and IX and establish a temporal normalization of hemostasis has challenged classical theories and led to an increased emphasis on factor VII and the TF pathway. In contrast to classical models, current theories now consider TF-dependent coagulation as the primary pathway, and assumes that activation of factor X via factor XI and factor IX/VIII constitutes a coagulation amplification loop. Future attempts to formulate more comprehensive models of the coagulation process have to consider the fact that an increased plasma level of factor VIIa can compensate for a number of coagulation deficiencies, However, it is important to note that factor VIIainduced enforcement of coagulation is only obtained at a vastly increased factor VIIa plasma level. Crucial unsolved questions in this context remains to be answered. Does factor VIIa under these conditions exert its action by a TF-independent mechanism? Can normal TF plasma levels and reciprocal zymogen activation account for an on-going low level activation of coagulation? What is the exact role of TFPI; is it capable of neutralizing basal levels of TF at normal factor Xa and factor VIIa steady-state levels? What is the clearance mechanism of factor VIIa, TF, TFPI, and their complexes? Much evidence suggest that TF, its regulation and exposure to the blood, is of central importance to the control and regulation of hemostatic balance in health and disease. The same is true for the interaction of this receptor with its ligand, factor VII, and the interaction of the receptor-bound ligand with the factor Xa/TFPI complex. In fact, compared to other reactions of the coagulation cascade that may have a regulatory role, the reactions involving TF appears to be at the very heart of the hemostatic balance and, thus, to be the point at which a potential therapeutic treatment of a diseased state due to a an unbalanced hemostasis should be directed. The future will show to what extent this will be possible. Clinical studies have already shown that factor VIIa can normalize the hemostatic balance of hemophiliacs, and preclinical studies indicate that an inactive (active site-blocked) form of factor VII is a very efficient antithrombotic agent exerting its action by binding to TF. Other putative competitors of the TF/factor VII interaction includes antibodies towards TF or factor VII and various ligands (peptides or small organic compounds), with an effect on this interaction.

REFERENCES

1. DAVIE, E.W., FUJIKAWA, K. and KISIEL, W. The coagulation cascade, initiation, maintenance, and regulation. Biochemistry 30, 10363-10370, 1991. 2. OSTERUD, B. Factor VII and hemostasis. Blood Coag Fibrin01 2, 175-181, 1990. 3. RAPAPORT, S.I. and RAO, L.V.M. Initiation and regulation of tissue factor-dependent coagulation. Artcrioscler Thromb 12, 111-121, 1992. 4. BROZE, G.J. Tissue factor pathway inhibitor and the revised hypothesis of blood coagulation. Trends Cardiovasc Med 2, 72-77, 1992. 5. CARSON, SD. and BROZNA, J.P. The role of tissue factor in the production of thrombin. Blood Coag Fibrin01 4, 281-292, 1993.

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