Hospital Practice
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Why Do Hemophiliacs Bleed? George J. Broze Jr. To cite this article: George J. Broze Jr. (1992) Why Do Hemophiliacs Bleed?, Hospital Practice, 27:3, 71-86, DOI: 10.1080/21548331.1992.11705381 To link to this article: http://dx.doi.org/10.1080/21548331.1992.11705381
Published online: 17 May 2016.
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Date: 16 June 2016, At: 18:09
IPIHIY§llOlOGY llN MIElDllCllNIE
Why Do Hemophiliacs Bleed?
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G E 0 R G E J. B R 0 Z E, JR.
Washington University, St. Louts
A revised hypothesis of coagulation integrates all the factors known to be involved into a single pathway that is initiated by factor Vlla/tissue factor and in which "contact" factors are not required. A key point is that the initial hemostatic response must be "consolidated" by the progressive local generation of factor Xa and thrombin.
The dramatic phenotype of hemophilia led to its recognition in the second century A.D., when Jewish infants were exempted from circumcision if their brothers had died after that operation. The Xlinked pattern of inheritance of hemophilia was described by John Otto in 1803, but it was not until 1893 that Sir Almroth Wright showed that hemophilia represented a defect in blood coagulation. The most famous hemophilia kindred is that of Queen Victoria of England. Victoria was the source of the hemophilia gene that ultimately coursed through the royal families in Europe. Perhaps the most historically noteworthy of these royal hemophiliacs was Grand Duke Alexis, the son of Czar Nicholas IT and Czarina Alexandra of Russia. Since the time of the czar, advances in biochemistry and molecular biology have vastly expanded our knowledge of the physical, chemical, and functional properties of the proteins involved in blood coagulation. All the known factors involved in coagulation have been purified and characterized, their cDNAs isolated and cloned, and the chromosome location and organization of their genes determined. Despite this, however, a clear understanding of the overall process of in vivo coagulation has remained elusive, and existing theories of coagulation do not explain the clinical information derived from patients with specific coagulation factor deficiencies.
Although we could now treat Grand Duke Alexis effectively, we could not until very recently have explained to the csar why his son bled. Contemporary physicians knew that the Grand Duke was deficient in a factor that is necessary for normal hemostasis. The question to be addressed in this article is why such a deficiency leads to severe hemorrhage.
Theories ofBlood Coagulation Researchers in the early 1800s discovered that exposure of blood to tissues induces clotting. The responsible entity was initially called tissue thromboplastin and later, tissue factor. In 1905, a two-step theory of blood coagulation was proposed: Tissue factor acts on prothrombin to produce thrombin, and thrombin converts fibrinogen to fibrin. The subsequent development of improved assays for thrombin generation and the identification of specific hereditary coagulation factor deficiencies (factors V. VII, and X}, in which plasma does not clot normally after the addition of tissue factor, led to
Dr. Broze is Professor of Medicine, Washington University School of Medicine, and Attending Physician, Division of Hematology/Oncology, Jewish Hospital of St. Louis. This is the third article In a series on the molecular biology of coagulation disorders.
In cooperation with the American Physiological Society Hospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE modifications of that theory. Nevertheless, the thesis that tissue factor initiates coagulation remained widely accepted during the next half century. Initial studies suggesting the existence of an alternative coagulation pathway that did not require tissue factor were ignored or rationalized to conform to the prevailing theory. Mounting evidence-particularly the further definition of hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency)-eventually forced areassessment of the hemostatic mechanism. Tissue factor-induced coagulation could not explain the bleeding in hemophiliacs since their blood did not appear to clot normally after the addition of tissue factor. In 1964, the"cascade"theoryof R. G. McFarlane and "waterfall" theory of E. W. Davie and 0. D. Ratnoff separated the known coagulation factors into two pathways, the intrinsic and the extrinsic, that converge at the activation
of factor X, with the subsequent generation of thrombin proceeding through a single, common pathway. As additional information became available (for example, the identification offactor V. factor VIII, and tissue factor as cofactors rather than enzymes), the theories were modified, but the general concept of two separate pathways for the generation of thrombin became dogma. An integral part of both the cascade and waterfall hypotheses was the amplification of coagulation that is achieved through sequential activation of protease zymogens to active enzymes. In the intrinsic pathway, exposure of the contact factors (factor XII, high molecular weight kininogen, and prekallikrein) in plasma to a surface led to the activation offactor XI and consequent activation of factor IX, which, in the presence of factor VIII, cleaved factor X to Xa. In the extrinsic pathway, coagulation was triggered by the exposure of tis-
Figure 1. Tissue factor is expressed by only certain cell types that are not exposed to plasma unless vascular endothelium is disrupted. This is illustrated by immunohistochemical staining of a sectioned human saphenous vein. The lumen is at the top. Endothelial cells are not stained by specific antibody, and only light staining is discernible in scattered cells of the tunica media. The adventitia, however, stains strongly for tissue factor. (Reprinted from Wilcox JN et al, Proc Natl Acad Sci (USA) 86:2839, 1989)
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sue factor to plasma factor VII and consequent direct activation offactor X. The intrinsic pathway was considered to be critical for hemostasis since it required the presence of factors VIII and IX, the factors missing in hemophilia. The extrinsic, or tissue factor-induced, pathway was relegated to a minor, ancillary role in coagulation. The segregation of known coagulation factors into intrinsic, extrinsic, and common pathways and the availability of simple in vitro tests for coagulation initiated by contact (partial thromboplastin time) and tissue factor (prothrombin time) were invaluable in the laboratory diagnosis of clinical bleeding disorders. In hemostasis laboratories today, rabbit brain tissue is typically used as the source of tissue factor for the prothrombin time assay; specific types of dirt (e.g., kaolin or silica) are used to accelerate contact activation in the activated partial thromboplastin time. The past 10 years has wit~ nessed resurgent interest in tissue factor-induced coagulation and a renewed appreciation of its pivotal role in the initiation of coagulation. It was noted that patients deficient in one of the contact factors required for the initiation of intrinsic coagulation are asymptomatic, whereas individuals deficient in factor VII bleed abnormally. Furthermore, in 1977, B. 0sterud and S. I. Rapaport showed that factor VIIa/tissue factor can activate factor IX of the intrinsic pathway as well as factor X. But the puzzle remained: If tissue factor plays the predominant role in the initiation of hemostasis, and factor Vlla/tissue factor complex is a potent activator of factor X, why, then, do hemophilia patients bleed? R. Biggs and colleagues provided a clue to
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PHYSIOLOGY IN MEDICINE the resolution of this dilemma by showing that when plasma was induced to clot by the addition of small amounts of tissue factor, the presence of factor VIII and factor IX enhanced coagulation. The result contrasted with that of the standard prothrombin time assay, in which relatively large quantities oftissue factor are used to initiate coagulation, and in which plasma deficient in factor VIII or factor IX is indistinguishable from normal plasma. The properties of a recently rediscovered and characterized endogenous inhibitor of tissue factor-induced coagulation led us to propose a revised theory of coagulation that can reconcile those in vitro results and explain the in vivo requirement for intact extrinsic and intrinsic (factors VIII, IX, XI) pathways for normal hemostasis. The inhibitor is associated with the lipoproteins in plasma, directly inhibits activated factor X, and, in a factor Xa-dependent fashion, inhibits the factor Vlla/tissue factor complex. This inhibitor has been called antithromboplastin, anticonvertin, tissue factor inhibitor, extrinsic pathway inhibitor, and lipoprotein-associated coagulation inhibitor. In July 1991, a subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis proposed the currently used term, tissue factor pathway inhibitor (TFPI).
Tissue Factor Pathway of Coagulation Human tissue factor is a 45,000 molecular weight, integral membrane protein, which acts as a cofactor to enhance the proteolytic activity of activated factor VII (factor VIla) toward its substrates, factor IX and factor
X. Tissue factor is a member of the cytokine-interferon receptor superfamily of cell surface proteins. The extracellular domains of the receptors in this superfamily are predicted to consist of two barrel-like structures that meet at an angle. The V-shaped trough formed by fj-sheet surfaces of each barrel is thought to represent the factor VIla binding site. Unlike the cytokine-interferon receptors, however, tissue factor apparently is not involved in transmembrane signaling. It has been shown in immunohistochemical studies of normal human tissues that the cellular expression of tissue factor is selective. It is absent from cells normally in contact with plasma (blood cells and the endothelium of vessels). Brain, lung, and placenta stain strongly for tissue factor, as do peripheral nerves, autonomic ganglia, the epithelium of the skin and mucosa, and the vascular adventitia (Figure 1). Monocytes and endothelial cells lack tissue factor activity when they are quiescent but express tissue factor in response to agents that may be physiologically relevant (Table 1). The monocyte response to certain stimuli requires or is enhanced by the presence ofT lymphocytes. It is likely that many of the agents listed in Table 1 are elaborated in a variety of clinical diseases, particularly those associated with inflammation. Increased levels of tissue factor have been detected ex vivo in peripheral blood monocytes and tissue macrophages from animals and humans with certain pathologic states (Table 2). Thus, the local coagulation and propensity to systemic thrombosis that accompany such diseases may be due to the aberrant production of tissue factor by these cells, which normally
Table 1. Agents Affecting Tissue Factor Expression in Monocytes and Endothelial Cells in Vitro Induction Endotoxin lnterleukin-1 Thrombin Activated complement Immune complexes Tuftsin Allogeneic stimulation Native and modified lipoproteins Adherence Cytotoxic agents Tumor cell exposure Vascular permeability factor Viral and bacterial infections and phagocytosis Streptokinase-dependent plasma factor
Enhancement T-lymphocyte collaboration CD11 b/CD18 receptor occupancy Tumor necrosis factor Platelet activating factor Inhibitors of calmodulin/protein kinase C High glucose concentrations
Inhibition Glucocorticoids Heparin Dipyridamole lloprost Pentoxifylline
are not thrombogenic. Human factor VII is a singlechain glycoprotein of about 50,000 molecular weight that is present in plasma at trace concentrations (500 ng'ml). It is synthesized predominantly by the liver, although stimulated monocytes-macrophages may provide Hospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE an important source at local sites. Factor VII can be activated to a two-chain, disulfide-linked form, factor VIla, through limited proteolytic cleavage by factor Xa, factor IXa, factor Xlla, or thrombin. Factor VIla also can activate factor VII. Factor VII and the substrates of factor Vlla/tissue factor, factor IX and factor X, are vitamin K-dependent proteins. Each contains a number of ~carboxy glutamic acid (Gla) residues
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Table 2. MonocyteMacrophage Generation of Tissue Factor ex Vivo Peripheral Blood Monocytes Inflammatory bowel disease Systemic lupus Trauma and surgery Unstable angina Renal transplant rejection Parenteral nutrition solutions Meningococcal infections Shwartzman reaction Diabetes Cancer Tissue Macrophages Spleen Systemic lupus Atherogenic diet Kidney Transplant rejection Glomerulonephritis Neoplastic tumors Endocarditis Atheromatous plaques Bronchoalveolar lavage fluid Sarcoidosis Granulomatous pneumonitis Respiratory distress syndrome Cancer Asbestos exposure Ozone
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near its amino terminus. The post-translational modification of these glutamic acid residues is mediated by a carboxylase enzyme that requires reduced vitamin K as an obligate cofactor. The reaction produces y-carboxyglutamic acid residues and converts reduced vitamin K to vitamin K epoxide. The oral anticoagulant warfarin inhibits the regeneration of reduced vitamin K from vitamin K epoxide. Calcium ion binding mediated by the Gla residues in vitamin K-dependent proteins induces a conformational change that leads to the expression of membrane and cofactor binding properties. Thus, the binding of factor VIla to the tissue factor on the surface of cells requires calcium ions and the presence of the Gla domain. Similarly, the Gla domains offactor IX and factor X are needed for the optimal interaction between these substrates and the catalytic factor Vlla/tlssue factor complex. Adjacent to the Gla domain, each of these coagulation factors has an epidermal growth factor (EGF) domain, which contains two modules that are homologous to epidermal growth factor. The EGF domain appears to play an important role in mediating protein-protein interactions. For example, it is critical for binding of factor VIla to tissue factor. The catalytic domains of factor VII, factor IX, and factor X are similar in structure to the prototype serine proteases, trypsin and chymotrypsin. Proteolytic activation of the zymogen forms of these factors induces a conformational change that converts them from inactive precursors to active enzymes. In the case of factor IX and factor X, the activation process proteolytically removes a portion of the protein called the activation peptide.
Factor IX and factor X are true zymogens in that they lack enzyme activity; whether zymogen factor VII possesses intrinsic catalytic activity is controversial. Zymogen factor VII and factor VIla bind to tissue factor in the presence of calcium ions with equal affinities, and tissue factor binding dramatically affects their coagulant activities. Factor VII bound to tissue factor, as opposed to that in solution, is rapidly and preferentially cleaved to factor VIla by trace concentrations of factor Xa. FUrthermore, binding to tissue factor enhances the enzymatic activity of factor VIla several thousandfold. The catalytic factor Vlla/tissue factor complex is a potent activator of both factor X and factor IX. Most, but not all, investigators have found factor X to be the preferred substrate for factor Vlla/tissue factor in vitro (Figure2).
In comparison to other activated coagulation factors, factor VIla is remarkably stable in plasma, remaining in the circulation nearly as long as zymogen factor VII. No potent inhibitor offactor VIla has been described, and antithrombin III, even in the presence of heparin, inhibits factor VIla at only slow rates. Thus, physiologic regulation of tissue factor-initiated coagulation does not appear to be mediated at the level of the enzyme factor VIla. Instead, TFPI, which targets the catalytic factor Vlla/tissue factor complex, provides the endogenous means for regulating this pathway of coagulation.
History ofthe Tissue Factor Pathway Inhibitor In 1947, L. Thomas and C. L. Schneider independently showed that incubation of crude tissue (continues)
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Figure 2. According to current concepts, the earliest event in coagulation is formation of a protein complex at a site of vessel injury. The complex consists of factor VIla bound to tissue factor (left). Factor X (or IX) then binds to the complex (middle) and becomes activated (right). As schematlzed, tissue factor is believed to have two extracellular domains that form a V-shaped, ligandbinding trough, anchored by a transmembrane segment
(continued)
thromboplastin with serum prevented the lethal disseminated intravascular coagulation that follows thromboplastin infusion in animals. The inhibitory effect of serum required the presence of calcium ions, the inhibitor appeared to bind to the thromboplastin, and calcium ion chelators could reverse the inhibition. Later, P. F. Hjort showed that the serum inhibitor, which he called convertin, recognized the factor VIIa/Ca2 +/tissue factor complex rather than factor VIla
and a cytoplasmic tall. Both factor VIla and factor X have globular catalytic, epidermal growth factor-like, and ~carboxyglutamlc acid (Gia) domains. Calcium binding by the Gla domain is required for factor VIla to bind tissue factor, and for factor X's association with factor Vlla/tissue factor. Note that a portion of factor X known as the activation peptide Is proteolytically released with the factor's activation to factor Xa.
or tissue factor alone. About the same time, Biggs and her colleagues reported that coagulation was delayed and incomplete after the addition of low concentrations of tissue factor to plasma from patients with hemophilia. The advent of the intrinsic pathway's recognition, however, diverted interest away from the possible connection between Biggs's work and the thromboplastin inhibitor. Experiments performed 25 years later showed that plasma lipoproteins inhibit the catalytic activity of the factor VIIa/tissue
factor complex and that anticonvertin activity is present in two high molecular weight peaks identified by gel ffitration of plasma. In 1982, Richard Marlar and John Griffin showed that when coagulation was induced by small amounts of tissue factor, plasma lacking either factor VIII or factor IX activated much less factor X than did normal plasma. Subsequently, other investigators noted that the activation offactor IX and factor X in normal plasma was also incomplete after the addition of tissue factor and that this apparent inHospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE hibition of factor VIIa/tissue factor enzymatic activity was directly related to the presence of factor X or brief treatment of the plasma with factor Xa. It was the 1985 report of Natalie Sanders and colleagues from the laboratory of Samuel Rapaport at the University of California, San Diego, that revitalized interest in the inhibitor. They showed that the apparent inhibition of tissue factor-directed coagulation required the presence of not only factor X but also an inhibitor present in the total lipoprotein fraction of plasma
after density centrifugation. Studies from several groups confirmed those results and went on to show that chelation of calcium ions with EDTA reversed the inhibition, with release of functionally active factor VIla and tissue factor. Thus, the rediscovered inhibitor appeared to be identical to the anticonvertln studied by Hjort in 1957. With the initial purification of TFPI in 1987, my colleagues and I at the Jewish Hospital of St. Louis showed that TFPI not only inhibits the factor VIIa/tissue factor complex in a factor Xa-de-
pendent fashion but also inhibits factor Xa directly. Thus, in retrospect, the same inhibitor was probably the subject of studies that had described a "fast-acting'' factor Xa inhibitor that was carried by plasma lipoproteins and whose plasma concentration increased after in vivo administration of aheparin analogue. We proposed that the inhibition of the factor VIIa/tissue factor complex by TFPI involved the formation of a stoichiometric complex containing factor VIla, tissue factor, factor Xa, and the inhibitor.
1FPI Structure
Figure 3. Structurally, the most prominent components of tissue factor pathway inhibitor are three tandem domains homologous to Kunitz-type protease inhibitors. Each domain has an active-site Inhibitory cleft (not shown). The first domain interacts with factor Vlla/tissue factor complex, the second domain with factor Xa. Function of the third Kunitz domain is unknown.
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TFPI eDNA was subsequently isolated and cloned from placental and endothelial cell eDNA libraries through a collaboration between T.-C. Wun and his colleagues at the Monsanto Company and Thomas J. Girard in our group at the Jewish Hospital. The primary structure of TFPI predicted by eDNA sequence is shown in Figure 3. The mature molecule has an acidic aminoterminal region followed by three tandem domains with homology to Kunitz-type protease inhibitors and a basic carboxylterminal region. Kunitz-type inhibitors appear to act by the standard mechanism: The inhibitor resembles a good substrate, but after the enzyme binds, cleavage between the P 1 and P 1' amino acid residues at the active-site cleft of the inhibitor occurs only very slowly or not at all. The P 1 residue is an important determinant of the specificity of these inhibitors, and alterations of the residue in the P 1 position can profoundly alter their inhibitory activity. The presence ofKunitz-type domains in the TFPI molecule is consistent with its dual inhibitory specificities-that is, factor Xa
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PHYSIOLOGY IN MEDICINE and factor Vlla/tissue factor. Studies in which the P 1 residue of each Kunitz domain in TFPI was individually altered showed that the second Kunitz domain mediates factor Xa binding and inhibition, whereas the first Kunitz domain is necessary for inhibition of the factor VIla/ tissue factor complex. Alteration ofthe P 1 residue in the third Kunitz domain does not affect either of these functions ofTFPI, and its physiologic role is not currently known. Post-translational modifications in the tissue factor pathway inhibitor molecule include at least partial phosphorylation of serine-2 and N-linked glycosylation. TheN-linked oligosaccharides in TFPI expressed by certain cells (e.g., endothelial, renal) in vitro are sulfated. The effect of these modifications on TFPI function is not known.
TFPI Inhibition ofFactorXa TFPI produces direct inhibition of factor Xa by binding at or near the serine active site of the enzyme. Factor Xa-TFPI interaction has 1: 1 stoichiometry, does not require the presence of calcium ions, and can be reversed by treatment with the detergent dodecyl sodium sulfate or high concentrations of the serine protease inhibitor benzamidine, which binds to the active site in factor Xa. TFPI is also a potent inhibitor of trypsin, but this inhibitory effect is unlikely to be physiologically important. The second Kunitz domain in the TFPI molecule is not alone in its interaction with factor Xa. For example, optimal inhibition of factor Xa requires the basic carboxyl-terminal region of TFPI, and proteolytic cleavage by human leukocyte elastase between
the first and second Kunitz domains dramatically reduces the ability of TFPI to inhibit factor Xa. TFPI is a potent inhibitor of factor Xa, and the prolongation of the prothrombin time produced by the addition of exogenous TFPI to plasma is predominantly due to its effect on factor Xa, rather than to an effect on the factor Vlla/tissue factor complex.
sent than when it is present. Inhibition of factor Vlla/tissue factor by TFPI independent of factor Xa is of uncertain physiologic relevance, but it could be important when TFPI is used as atherapeutic agent and plasma levels of TFPI are many times that of normal plasma.
TFPI Inhibition of Factor VI/a/Tissue Factor
TFPI circulates bound to low density, high density, and very low density lipoproteins. Several forms of TFPI are present in plasma: predominant proteins with a molecular weight of 34,000 and 40,000 and less abundant forms of higher apparent molecular weight. The size heterogeneity appears to be due in part to the formation of mixed disulfide complexes between TFPI and apollpoproteinA-11. The major form ofTFPI in LDL is 34,000, that in HDL is 40,000, and VLDL contains both 34,000 and 40,000 molecular weight forms. The disparity in size between the TFPI in LDL and recombinant TFPI expressed by tissue culture cells (42,000) may in part reflect differences in glycosylation. The mechanism by which TFPI associates with lipoproteins is not known. Approximately 10% of the total blood TFPI is carried by platelets and released after stimulation by thrombin. Thus, at a wound, where platelets aggregate, the quantity of TFPI released from platelets could be substantial. Indeed, the concentration of TFPI in blood escaping from a superficial laceration (template bleeding time) reaches levels severalfold higher than in venous blood obtained simultaneously by venipuncture. Although the increase is presumably related to the release of platelet TFPI, the contribution of other cells, per-
The proposed mechanism for the factor Xa-dependent inhibition of factor Vlla/tissue factor by TFPI involves the formation of a quaternary factor Xa-TFPI-factor Vlla/tissue factor complex. This inhibitory complex could result either from the initial binding of TFPI to factor Xa with the subsequent binding of the factor XaTFPI complex to factor Vlla/tissue factor or from binding of TFPI to a preformed factor Xafactor Vlla/tissue factor complex (Figure 4). The quaternary complex explains both the need for the persistent presence of factor Xa for effective factor Vlla/tissue factor inhibition and the requirement for active factor Xa, since active site-inactivated factor Xa does not bind to TFPI. A chimeric molecule containing only the amino-terminal portion of factor Xa and the first Kunitz domain of TFPI directly inhibits factor Vlla/tissue factor. Thus, the binding offactor Xa to TFPI may serve to juxtapose those domains on separate molecules, thereby producing a complex that binds to factor Vlla/tissue factor with high affinity. Factor Xa is not absolutely required for TFPI's inhibition of factor Vlla/tissue factor, but 100fold greater concentrations of TFPI are required for an equivalent effect when factor Xa is ab-
TFPiinVivo
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Figure 4. Tissue factor pathway Inhibitor appears to exert its effect by forming a quaternary complex with factor Xa and factor Vlla/tissue factor. In one possible mechanism, the inhibitory complex could resuH from TFPI's binding of preformed factor Xs-VIIa/tissue factor (above); aHerna-
haps including endothelial cells, cannot be excluded. Another source of readily available TFPI exists in vivo, as plasma TPFI levels increase twofold to fourfold after the infusion of heparin. The effect appears to be related to the release of TFPI from the endothelium, where the inhibitor may be bound to heparan sulfate or to other glycosaminoglycans at the endothelial cell surface. 82
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tively, TFPI could first bind to factor xa, and that complex could then bind to Vlla/tissue factor (below). In either case, TFPI's first Kunltz domain (marked 1) would bind the catalytic site of factor VIla, and its second Kunltz domain (marked 2) would bind the catalytic site of factor xa.
Which cell type is responsible in vivo for the maintenance of plasma TFPI levels is not known, but the endothelium is an attractive candidate, given its mass and location. Unlike levels of tissue plasminogen activator (t-PA) and von Willebrand factor, which are also synthesized in endothelial cells, TFPI levels in plasma do not rise after infusion of desmopressin acetate nor do they rise after venous occlusion.
The broad range of plasma TFPI concentrations in normal subjects has a mean of approximately 100 ng'ml. Modest alterations in the plasma level ofTFPI have been described in certain clinical conditions, but the physiologic relevance of the differences between plasma TFPI concentrations in health and disease is difficult to interpret. The severalfold increase in (continues)
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(continued)
plasma TFPI after heparin treatment suggests that TFPI in plasma represents only a fraction of the total TFPI that can be readily mobilized. This raises the possibility that other physiologic or pathologic mechanisms may similarly affect the concentration of TFPI in plasma or at local sites. The TFPI presumably bound at the surface of the endothelium and in contact with plasma is probably a major contributor to overall TFPI action in vivo, but that is not reflected by the plasma TFPI concentration. Although low levels of plasma TFPI are occasionally seen in patients with septicemia or disseminated intravascular coagulation, their TFPI concentrations are more often normal. The progression of disseminated intravascular coagulation in the face of a normal plasma level of TFPI is consistent with the fact that at physiologic concentrations, TFPI inhibits factor VIla/ tissue factor effectively only after factor Xa has been generated. Thus, TFPI dampens but does not prevent the coagulation process as long as generation of tissue factor continues. The depletion of endogenous TFPI sensitizes rabbits to the disseminated intravascular coagulation induced by tissue factor or endotoxin, and the infusion of high, therapeutic concentrations of TFPI into animals ameliorates the intravascular coagulation induced by tissue factor. Although TFPI deficiency might be expected to cause a prothrombotic phenotype, no person with TFPI deficiency has yet been identified. The low plasma levels of TFPI found in abetalipoproteinemic patients appear simply to reflect the absence of LDL, which is a carrier of TFPI. Their
total TFPI, as estimated from plasma TFPI levels after heparin infusion, is the same as that in normal persons.
Revised Hypothesis of Blood Coagulation A revised formulation of blood coagulation shown in Figure 5 takes into account the feedback inhibition of factor VIIa/tissue factor by TFPI. In this hypothesis, coagulation ensues when
damage to blood vessels allows the exposure of blood to the tissue factor produced constitutively by cells beneath the endothelium. The factor VII or factor VIla present in plasma then binds to tissue factor, and the factor VIIa/tissue factor complex activates limited quantities of factor X and factor IX. With the generation of factor Xa, the inhibitory effect ofTFPI becomes manifest, preventing further production of Xa and
Figure 5. A revised hypothesis of coagulation postulates a single pathway whose initiating event is exposure of tissue factor to factor VIla (or VII) at a site of vessel injury (A). The resulting Vlla/tissue factor complex generates small amounts of factors IXa and Xa (B). Tissue factor pathway inhibitor (TFPI) then prevents further Vlla/tissue factor activation of IX and X (C). Only the action of factors IXa and VIlla (D) can generate the additional factor Xa (E) needed to sustain coagulation. In this regard, factor XI activation by thrombin (F) and Xla autoactivation (G) may generate additional factor IXB (H). Hospital Practice March 15. 1992
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PHYSIOLOGY IN MEDICINE factor IXa by factor Vlla/tissue
Seattle and David Gailani in my
factor Xa and thrombin. The
factor. Additional factor Xa can
laboratory showed that throm-
need for sustained production
be produced only through the alternative pathway involving factor IXa and factor VIlla. The presence of a variable but usually mild bleeding diathesis in persons with factor XI deficiency (sometimes called hemophilia C) implies that under certain conditions, the initial quantity of factor IXa produced by factor VIIa/tissue factor is insufficient and additional factor IXa generated by factor XIa is needed for normal hemostasis.
bin can activate factor XI and that, in the presence of a polyanion (e.g., dextran sulfate, heparin, or sulfatides), this process is amplified through the autoactivation of additional factor XI by factor Xla (Figure 5). Evidence that this actually occurs in vivo has not yet been presented.
of factor Xa and thrombin for normal hemostasis has been shown in vivo and presumably reflects the influence of inhibitors of the coagulation proteases and the competing process offibrinolysis. Thus, hemophilia patients bleed because the factor Xa generated through the action of factor VIIa/tissue factor, and dampened by TFPI, is insufficient to sustain hemostasis and must be amplified through the action of factor IXa and factor VIlla. Similarly, factor Xla is required to supplement the factor IXa formed by factor VIIa/tissue factor, which is limited by the presence of TFPI. As a corollary, we suggested in 1988 that effective inhibition ofTFPI could ameliorate the hemorrhagic manifestations of hemophilia.
The mechanism for the activation of factor XI is not clear, but the model predicts that rather than being involved in the initiation of coagulation, as suggested by the cascade-waterfall hypothesis, factor XI would be activated "late" in the coagulation process, after factor Xa and thrombin have been generated through the action of factor VIIa/tissue factor. Recently, Koji Naito in the laboratory of Kazuo Fujikawa in
The revised hypothesis of blood coagulation differs from the cascade-waterfall model in two major respects. First, it integrates all of the factors known to be involved in coagulation into a single pathway that is initiated by factor VIIa/tissue factor and that does not require the contact factors (factor XII, prekallikrein, and high molecular kininogen). Second, in the revised model, the hemostatic process does not end with the initial generation of factor Xa and thrombin. Instead, the initial hemostatic response must be "consolidated" by the progressive local generation of
Selected Reading Braze GJ Jr et al: The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: Insightinto its possible mechanism of action. Blood 71 :335. 1988 Girard T J et al: Functional signiflcance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 338:518, 1989 Braze GJ Jr, Girard T J, Novotny WF: Perspectives in Biochemistry: Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 29:7539. 1990 Repke D et al: Hemoph111a as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous flow reactor. Proc Natl Acad Sci USA 8 7:7623, 1990 Gailani D, Braze GJ Jr: Factor XI activation in a revised model of blood coagulation. Science 253:909, 1991 Massie RK: Nicholas and Alexandra. Atheneum. New York. 1968
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TFPI-induced feedback inhibition of factor VIIa/tissue factor can explain the clinical need for both the extrinsic and intrinsic (factors VIII, IX, and XI) coagulation pathways and is consistent with in vitro results showing deficient factor Xa production in hemophiliac plasma induced to clot by small quantities of tissue factor. In addition, data at present are consistent with the view that in normal hemostasis; factor VIIa/tissue factor is responsible for initial factor Xa generation, which provides sufficient thrombin to induce the local aggregation of platelets and the activation of the critical cofactors, factor V and factor VIII. That persistent hemostasis requires the continued production of additional factor Xa through the action of factor IXa and factor VIlla is consistent with the delayed onset of bleeding frequently seen in hemophilia. D
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Why Do Hemophiliacs Bleed? George J. Broze Jr. To cite this article: George J. Broze Jr. (1992) Why Do Hemophiliacs Bleed?, Hospital Practice, 27:3, 71-86, DOI: 10.1080/21548331.1992.11705381 To link to this article: http://dx.doi.org/10.1080/21548331.1992.11705381
Published online: 17 May 2016.
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IPIHIY§llOlOGY llN MIElDllCllNIE
Why Do Hemophiliacs Bleed?
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G E 0 R G E J. B R 0 Z E, JR.
Washington University, St. Louts
A revised hypothesis of coagulation integrates all the factors known to be involved into a single pathway that is initiated by factor Vlla/tissue factor and in which "contact" factors are not required. A key point is that the initial hemostatic response must be "consolidated" by the progressive local generation of factor Xa and thrombin.
The dramatic phenotype of hemophilia led to its recognition in the second century A.D., when Jewish infants were exempted from circumcision if their brothers had died after that operation. The Xlinked pattern of inheritance of hemophilia was described by John Otto in 1803, but it was not until 1893 that Sir Almroth Wright showed that hemophilia represented a defect in blood coagulation. The most famous hemophilia kindred is that of Queen Victoria of England. Victoria was the source of the hemophilia gene that ultimately coursed through the royal families in Europe. Perhaps the most historically noteworthy of these royal hemophiliacs was Grand Duke Alexis, the son of Czar Nicholas IT and Czarina Alexandra of Russia. Since the time of the czar, advances in biochemistry and molecular biology have vastly expanded our knowledge of the physical, chemical, and functional properties of the proteins involved in blood coagulation. All the known factors involved in coagulation have been purified and characterized, their cDNAs isolated and cloned, and the chromosome location and organization of their genes determined. Despite this, however, a clear understanding of the overall process of in vivo coagulation has remained elusive, and existing theories of coagulation do not explain the clinical information derived from patients with specific coagulation factor deficiencies.
Although we could now treat Grand Duke Alexis effectively, we could not until very recently have explained to the csar why his son bled. Contemporary physicians knew that the Grand Duke was deficient in a factor that is necessary for normal hemostasis. The question to be addressed in this article is why such a deficiency leads to severe hemorrhage.
Theories ofBlood Coagulation Researchers in the early 1800s discovered that exposure of blood to tissues induces clotting. The responsible entity was initially called tissue thromboplastin and later, tissue factor. In 1905, a two-step theory of blood coagulation was proposed: Tissue factor acts on prothrombin to produce thrombin, and thrombin converts fibrinogen to fibrin. The subsequent development of improved assays for thrombin generation and the identification of specific hereditary coagulation factor deficiencies (factors V. VII, and X}, in which plasma does not clot normally after the addition of tissue factor, led to
Dr. Broze is Professor of Medicine, Washington University School of Medicine, and Attending Physician, Division of Hematology/Oncology, Jewish Hospital of St. Louis. This is the third article In a series on the molecular biology of coagulation disorders.
In cooperation with the American Physiological Society Hospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE modifications of that theory. Nevertheless, the thesis that tissue factor initiates coagulation remained widely accepted during the next half century. Initial studies suggesting the existence of an alternative coagulation pathway that did not require tissue factor were ignored or rationalized to conform to the prevailing theory. Mounting evidence-particularly the further definition of hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency)-eventually forced areassessment of the hemostatic mechanism. Tissue factor-induced coagulation could not explain the bleeding in hemophiliacs since their blood did not appear to clot normally after the addition of tissue factor. In 1964, the"cascade"theoryof R. G. McFarlane and "waterfall" theory of E. W. Davie and 0. D. Ratnoff separated the known coagulation factors into two pathways, the intrinsic and the extrinsic, that converge at the activation
of factor X, with the subsequent generation of thrombin proceeding through a single, common pathway. As additional information became available (for example, the identification offactor V. factor VIII, and tissue factor as cofactors rather than enzymes), the theories were modified, but the general concept of two separate pathways for the generation of thrombin became dogma. An integral part of both the cascade and waterfall hypotheses was the amplification of coagulation that is achieved through sequential activation of protease zymogens to active enzymes. In the intrinsic pathway, exposure of the contact factors (factor XII, high molecular weight kininogen, and prekallikrein) in plasma to a surface led to the activation offactor XI and consequent activation of factor IX, which, in the presence of factor VIII, cleaved factor X to Xa. In the extrinsic pathway, coagulation was triggered by the exposure of tis-
Figure 1. Tissue factor is expressed by only certain cell types that are not exposed to plasma unless vascular endothelium is disrupted. This is illustrated by immunohistochemical staining of a sectioned human saphenous vein. The lumen is at the top. Endothelial cells are not stained by specific antibody, and only light staining is discernible in scattered cells of the tunica media. The adventitia, however, stains strongly for tissue factor. (Reprinted from Wilcox JN et al, Proc Natl Acad Sci (USA) 86:2839, 1989)
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sue factor to plasma factor VII and consequent direct activation offactor X. The intrinsic pathway was considered to be critical for hemostasis since it required the presence of factors VIII and IX, the factors missing in hemophilia. The extrinsic, or tissue factor-induced, pathway was relegated to a minor, ancillary role in coagulation. The segregation of known coagulation factors into intrinsic, extrinsic, and common pathways and the availability of simple in vitro tests for coagulation initiated by contact (partial thromboplastin time) and tissue factor (prothrombin time) were invaluable in the laboratory diagnosis of clinical bleeding disorders. In hemostasis laboratories today, rabbit brain tissue is typically used as the source of tissue factor for the prothrombin time assay; specific types of dirt (e.g., kaolin or silica) are used to accelerate contact activation in the activated partial thromboplastin time. The past 10 years has wit~ nessed resurgent interest in tissue factor-induced coagulation and a renewed appreciation of its pivotal role in the initiation of coagulation. It was noted that patients deficient in one of the contact factors required for the initiation of intrinsic coagulation are asymptomatic, whereas individuals deficient in factor VII bleed abnormally. Furthermore, in 1977, B. 0sterud and S. I. Rapaport showed that factor VIIa/tissue factor can activate factor IX of the intrinsic pathway as well as factor X. But the puzzle remained: If tissue factor plays the predominant role in the initiation of hemostasis, and factor Vlla/tissue factor complex is a potent activator of factor X, why, then, do hemophilia patients bleed? R. Biggs and colleagues provided a clue to
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PHYSIOLOGY IN MEDICINE the resolution of this dilemma by showing that when plasma was induced to clot by the addition of small amounts of tissue factor, the presence of factor VIII and factor IX enhanced coagulation. The result contrasted with that of the standard prothrombin time assay, in which relatively large quantities oftissue factor are used to initiate coagulation, and in which plasma deficient in factor VIII or factor IX is indistinguishable from normal plasma. The properties of a recently rediscovered and characterized endogenous inhibitor of tissue factor-induced coagulation led us to propose a revised theory of coagulation that can reconcile those in vitro results and explain the in vivo requirement for intact extrinsic and intrinsic (factors VIII, IX, XI) pathways for normal hemostasis. The inhibitor is associated with the lipoproteins in plasma, directly inhibits activated factor X, and, in a factor Xa-dependent fashion, inhibits the factor Vlla/tissue factor complex. This inhibitor has been called antithromboplastin, anticonvertin, tissue factor inhibitor, extrinsic pathway inhibitor, and lipoprotein-associated coagulation inhibitor. In July 1991, a subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis proposed the currently used term, tissue factor pathway inhibitor (TFPI).
Tissue Factor Pathway of Coagulation Human tissue factor is a 45,000 molecular weight, integral membrane protein, which acts as a cofactor to enhance the proteolytic activity of activated factor VII (factor VIla) toward its substrates, factor IX and factor
X. Tissue factor is a member of the cytokine-interferon receptor superfamily of cell surface proteins. The extracellular domains of the receptors in this superfamily are predicted to consist of two barrel-like structures that meet at an angle. The V-shaped trough formed by fj-sheet surfaces of each barrel is thought to represent the factor VIla binding site. Unlike the cytokine-interferon receptors, however, tissue factor apparently is not involved in transmembrane signaling. It has been shown in immunohistochemical studies of normal human tissues that the cellular expression of tissue factor is selective. It is absent from cells normally in contact with plasma (blood cells and the endothelium of vessels). Brain, lung, and placenta stain strongly for tissue factor, as do peripheral nerves, autonomic ganglia, the epithelium of the skin and mucosa, and the vascular adventitia (Figure 1). Monocytes and endothelial cells lack tissue factor activity when they are quiescent but express tissue factor in response to agents that may be physiologically relevant (Table 1). The monocyte response to certain stimuli requires or is enhanced by the presence ofT lymphocytes. It is likely that many of the agents listed in Table 1 are elaborated in a variety of clinical diseases, particularly those associated with inflammation. Increased levels of tissue factor have been detected ex vivo in peripheral blood monocytes and tissue macrophages from animals and humans with certain pathologic states (Table 2). Thus, the local coagulation and propensity to systemic thrombosis that accompany such diseases may be due to the aberrant production of tissue factor by these cells, which normally
Table 1. Agents Affecting Tissue Factor Expression in Monocytes and Endothelial Cells in Vitro Induction Endotoxin lnterleukin-1 Thrombin Activated complement Immune complexes Tuftsin Allogeneic stimulation Native and modified lipoproteins Adherence Cytotoxic agents Tumor cell exposure Vascular permeability factor Viral and bacterial infections and phagocytosis Streptokinase-dependent plasma factor
Enhancement T-lymphocyte collaboration CD11 b/CD18 receptor occupancy Tumor necrosis factor Platelet activating factor Inhibitors of calmodulin/protein kinase C High glucose concentrations
Inhibition Glucocorticoids Heparin Dipyridamole lloprost Pentoxifylline
are not thrombogenic. Human factor VII is a singlechain glycoprotein of about 50,000 molecular weight that is present in plasma at trace concentrations (500 ng'ml). It is synthesized predominantly by the liver, although stimulated monocytes-macrophages may provide Hospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE an important source at local sites. Factor VII can be activated to a two-chain, disulfide-linked form, factor VIla, through limited proteolytic cleavage by factor Xa, factor IXa, factor Xlla, or thrombin. Factor VIla also can activate factor VII. Factor VII and the substrates of factor Vlla/tissue factor, factor IX and factor X, are vitamin K-dependent proteins. Each contains a number of ~carboxy glutamic acid (Gla) residues
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Table 2. MonocyteMacrophage Generation of Tissue Factor ex Vivo Peripheral Blood Monocytes Inflammatory bowel disease Systemic lupus Trauma and surgery Unstable angina Renal transplant rejection Parenteral nutrition solutions Meningococcal infections Shwartzman reaction Diabetes Cancer Tissue Macrophages Spleen Systemic lupus Atherogenic diet Kidney Transplant rejection Glomerulonephritis Neoplastic tumors Endocarditis Atheromatous plaques Bronchoalveolar lavage fluid Sarcoidosis Granulomatous pneumonitis Respiratory distress syndrome Cancer Asbestos exposure Ozone
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near its amino terminus. The post-translational modification of these glutamic acid residues is mediated by a carboxylase enzyme that requires reduced vitamin K as an obligate cofactor. The reaction produces y-carboxyglutamic acid residues and converts reduced vitamin K to vitamin K epoxide. The oral anticoagulant warfarin inhibits the regeneration of reduced vitamin K from vitamin K epoxide. Calcium ion binding mediated by the Gla residues in vitamin K-dependent proteins induces a conformational change that leads to the expression of membrane and cofactor binding properties. Thus, the binding of factor VIla to the tissue factor on the surface of cells requires calcium ions and the presence of the Gla domain. Similarly, the Gla domains offactor IX and factor X are needed for the optimal interaction between these substrates and the catalytic factor Vlla/tlssue factor complex. Adjacent to the Gla domain, each of these coagulation factors has an epidermal growth factor (EGF) domain, which contains two modules that are homologous to epidermal growth factor. The EGF domain appears to play an important role in mediating protein-protein interactions. For example, it is critical for binding of factor VIla to tissue factor. The catalytic domains of factor VII, factor IX, and factor X are similar in structure to the prototype serine proteases, trypsin and chymotrypsin. Proteolytic activation of the zymogen forms of these factors induces a conformational change that converts them from inactive precursors to active enzymes. In the case of factor IX and factor X, the activation process proteolytically removes a portion of the protein called the activation peptide.
Factor IX and factor X are true zymogens in that they lack enzyme activity; whether zymogen factor VII possesses intrinsic catalytic activity is controversial. Zymogen factor VII and factor VIla bind to tissue factor in the presence of calcium ions with equal affinities, and tissue factor binding dramatically affects their coagulant activities. Factor VII bound to tissue factor, as opposed to that in solution, is rapidly and preferentially cleaved to factor VIla by trace concentrations of factor Xa. FUrthermore, binding to tissue factor enhances the enzymatic activity of factor VIla several thousandfold. The catalytic factor Vlla/tissue factor complex is a potent activator of both factor X and factor IX. Most, but not all, investigators have found factor X to be the preferred substrate for factor Vlla/tissue factor in vitro (Figure2).
In comparison to other activated coagulation factors, factor VIla is remarkably stable in plasma, remaining in the circulation nearly as long as zymogen factor VII. No potent inhibitor offactor VIla has been described, and antithrombin III, even in the presence of heparin, inhibits factor VIla at only slow rates. Thus, physiologic regulation of tissue factor-initiated coagulation does not appear to be mediated at the level of the enzyme factor VIla. Instead, TFPI, which targets the catalytic factor Vlla/tissue factor complex, provides the endogenous means for regulating this pathway of coagulation.
History ofthe Tissue Factor Pathway Inhibitor In 1947, L. Thomas and C. L. Schneider independently showed that incubation of crude tissue (continues)
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Figure 2. According to current concepts, the earliest event in coagulation is formation of a protein complex at a site of vessel injury. The complex consists of factor VIla bound to tissue factor (left). Factor X (or IX) then binds to the complex (middle) and becomes activated (right). As schematlzed, tissue factor is believed to have two extracellular domains that form a V-shaped, ligandbinding trough, anchored by a transmembrane segment
(continued)
thromboplastin with serum prevented the lethal disseminated intravascular coagulation that follows thromboplastin infusion in animals. The inhibitory effect of serum required the presence of calcium ions, the inhibitor appeared to bind to the thromboplastin, and calcium ion chelators could reverse the inhibition. Later, P. F. Hjort showed that the serum inhibitor, which he called convertin, recognized the factor VIIa/Ca2 +/tissue factor complex rather than factor VIla
and a cytoplasmic tall. Both factor VIla and factor X have globular catalytic, epidermal growth factor-like, and ~carboxyglutamlc acid (Gia) domains. Calcium binding by the Gla domain is required for factor VIla to bind tissue factor, and for factor X's association with factor Vlla/tissue factor. Note that a portion of factor X known as the activation peptide Is proteolytically released with the factor's activation to factor Xa.
or tissue factor alone. About the same time, Biggs and her colleagues reported that coagulation was delayed and incomplete after the addition of low concentrations of tissue factor to plasma from patients with hemophilia. The advent of the intrinsic pathway's recognition, however, diverted interest away from the possible connection between Biggs's work and the thromboplastin inhibitor. Experiments performed 25 years later showed that plasma lipoproteins inhibit the catalytic activity of the factor VIIa/tissue
factor complex and that anticonvertin activity is present in two high molecular weight peaks identified by gel ffitration of plasma. In 1982, Richard Marlar and John Griffin showed that when coagulation was induced by small amounts of tissue factor, plasma lacking either factor VIII or factor IX activated much less factor X than did normal plasma. Subsequently, other investigators noted that the activation offactor IX and factor X in normal plasma was also incomplete after the addition of tissue factor and that this apparent inHospital Practice March 15, 1992
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PHYSIOLOGY IN MEDICINE hibition of factor VIIa/tissue factor enzymatic activity was directly related to the presence of factor X or brief treatment of the plasma with factor Xa. It was the 1985 report of Natalie Sanders and colleagues from the laboratory of Samuel Rapaport at the University of California, San Diego, that revitalized interest in the inhibitor. They showed that the apparent inhibition of tissue factor-directed coagulation required the presence of not only factor X but also an inhibitor present in the total lipoprotein fraction of plasma
after density centrifugation. Studies from several groups confirmed those results and went on to show that chelation of calcium ions with EDTA reversed the inhibition, with release of functionally active factor VIla and tissue factor. Thus, the rediscovered inhibitor appeared to be identical to the anticonvertln studied by Hjort in 1957. With the initial purification of TFPI in 1987, my colleagues and I at the Jewish Hospital of St. Louis showed that TFPI not only inhibits the factor VIIa/tissue factor complex in a factor Xa-de-
pendent fashion but also inhibits factor Xa directly. Thus, in retrospect, the same inhibitor was probably the subject of studies that had described a "fast-acting'' factor Xa inhibitor that was carried by plasma lipoproteins and whose plasma concentration increased after in vivo administration of aheparin analogue. We proposed that the inhibition of the factor VIIa/tissue factor complex by TFPI involved the formation of a stoichiometric complex containing factor VIla, tissue factor, factor Xa, and the inhibitor.
1FPI Structure
Figure 3. Structurally, the most prominent components of tissue factor pathway inhibitor are three tandem domains homologous to Kunitz-type protease inhibitors. Each domain has an active-site Inhibitory cleft (not shown). The first domain interacts with factor Vlla/tissue factor complex, the second domain with factor Xa. Function of the third Kunitz domain is unknown.
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TFPI eDNA was subsequently isolated and cloned from placental and endothelial cell eDNA libraries through a collaboration between T.-C. Wun and his colleagues at the Monsanto Company and Thomas J. Girard in our group at the Jewish Hospital. The primary structure of TFPI predicted by eDNA sequence is shown in Figure 3. The mature molecule has an acidic aminoterminal region followed by three tandem domains with homology to Kunitz-type protease inhibitors and a basic carboxylterminal region. Kunitz-type inhibitors appear to act by the standard mechanism: The inhibitor resembles a good substrate, but after the enzyme binds, cleavage between the P 1 and P 1' amino acid residues at the active-site cleft of the inhibitor occurs only very slowly or not at all. The P 1 residue is an important determinant of the specificity of these inhibitors, and alterations of the residue in the P 1 position can profoundly alter their inhibitory activity. The presence ofKunitz-type domains in the TFPI molecule is consistent with its dual inhibitory specificities-that is, factor Xa
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PHYSIOLOGY IN MEDICINE and factor Vlla/tissue factor. Studies in which the P 1 residue of each Kunitz domain in TFPI was individually altered showed that the second Kunitz domain mediates factor Xa binding and inhibition, whereas the first Kunitz domain is necessary for inhibition of the factor VIla/ tissue factor complex. Alteration ofthe P 1 residue in the third Kunitz domain does not affect either of these functions ofTFPI, and its physiologic role is not currently known. Post-translational modifications in the tissue factor pathway inhibitor molecule include at least partial phosphorylation of serine-2 and N-linked glycosylation. TheN-linked oligosaccharides in TFPI expressed by certain cells (e.g., endothelial, renal) in vitro are sulfated. The effect of these modifications on TFPI function is not known.
TFPI Inhibition ofFactorXa TFPI produces direct inhibition of factor Xa by binding at or near the serine active site of the enzyme. Factor Xa-TFPI interaction has 1: 1 stoichiometry, does not require the presence of calcium ions, and can be reversed by treatment with the detergent dodecyl sodium sulfate or high concentrations of the serine protease inhibitor benzamidine, which binds to the active site in factor Xa. TFPI is also a potent inhibitor of trypsin, but this inhibitory effect is unlikely to be physiologically important. The second Kunitz domain in the TFPI molecule is not alone in its interaction with factor Xa. For example, optimal inhibition of factor Xa requires the basic carboxyl-terminal region of TFPI, and proteolytic cleavage by human leukocyte elastase between
the first and second Kunitz domains dramatically reduces the ability of TFPI to inhibit factor Xa. TFPI is a potent inhibitor of factor Xa, and the prolongation of the prothrombin time produced by the addition of exogenous TFPI to plasma is predominantly due to its effect on factor Xa, rather than to an effect on the factor Vlla/tissue factor complex.
sent than when it is present. Inhibition of factor Vlla/tissue factor by TFPI independent of factor Xa is of uncertain physiologic relevance, but it could be important when TFPI is used as atherapeutic agent and plasma levels of TFPI are many times that of normal plasma.
TFPI Inhibition of Factor VI/a/Tissue Factor
TFPI circulates bound to low density, high density, and very low density lipoproteins. Several forms of TFPI are present in plasma: predominant proteins with a molecular weight of 34,000 and 40,000 and less abundant forms of higher apparent molecular weight. The size heterogeneity appears to be due in part to the formation of mixed disulfide complexes between TFPI and apollpoproteinA-11. The major form ofTFPI in LDL is 34,000, that in HDL is 40,000, and VLDL contains both 34,000 and 40,000 molecular weight forms. The disparity in size between the TFPI in LDL and recombinant TFPI expressed by tissue culture cells (42,000) may in part reflect differences in glycosylation. The mechanism by which TFPI associates with lipoproteins is not known. Approximately 10% of the total blood TFPI is carried by platelets and released after stimulation by thrombin. Thus, at a wound, where platelets aggregate, the quantity of TFPI released from platelets could be substantial. Indeed, the concentration of TFPI in blood escaping from a superficial laceration (template bleeding time) reaches levels severalfold higher than in venous blood obtained simultaneously by venipuncture. Although the increase is presumably related to the release of platelet TFPI, the contribution of other cells, per-
The proposed mechanism for the factor Xa-dependent inhibition of factor Vlla/tissue factor by TFPI involves the formation of a quaternary factor Xa-TFPI-factor Vlla/tissue factor complex. This inhibitory complex could result either from the initial binding of TFPI to factor Xa with the subsequent binding of the factor XaTFPI complex to factor Vlla/tissue factor or from binding of TFPI to a preformed factor Xafactor Vlla/tissue factor complex (Figure 4). The quaternary complex explains both the need for the persistent presence of factor Xa for effective factor Vlla/tissue factor inhibition and the requirement for active factor Xa, since active site-inactivated factor Xa does not bind to TFPI. A chimeric molecule containing only the amino-terminal portion of factor Xa and the first Kunitz domain of TFPI directly inhibits factor Vlla/tissue factor. Thus, the binding offactor Xa to TFPI may serve to juxtapose those domains on separate molecules, thereby producing a complex that binds to factor Vlla/tissue factor with high affinity. Factor Xa is not absolutely required for TFPI's inhibition of factor Vlla/tissue factor, but 100fold greater concentrations of TFPI are required for an equivalent effect when factor Xa is ab-
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Figure 4. Tissue factor pathway Inhibitor appears to exert its effect by forming a quaternary complex with factor Xa and factor Vlla/tissue factor. In one possible mechanism, the inhibitory complex could resuH from TFPI's binding of preformed factor Xs-VIIa/tissue factor (above); aHerna-
haps including endothelial cells, cannot be excluded. Another source of readily available TFPI exists in vivo, as plasma TPFI levels increase twofold to fourfold after the infusion of heparin. The effect appears to be related to the release of TFPI from the endothelium, where the inhibitor may be bound to heparan sulfate or to other glycosaminoglycans at the endothelial cell surface. 82
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tively, TFPI could first bind to factor xa, and that complex could then bind to Vlla/tissue factor (below). In either case, TFPI's first Kunltz domain (marked 1) would bind the catalytic site of factor VIla, and its second Kunltz domain (marked 2) would bind the catalytic site of factor xa.
Which cell type is responsible in vivo for the maintenance of plasma TFPI levels is not known, but the endothelium is an attractive candidate, given its mass and location. Unlike levels of tissue plasminogen activator (t-PA) and von Willebrand factor, which are also synthesized in endothelial cells, TFPI levels in plasma do not rise after infusion of desmopressin acetate nor do they rise after venous occlusion.
The broad range of plasma TFPI concentrations in normal subjects has a mean of approximately 100 ng'ml. Modest alterations in the plasma level ofTFPI have been described in certain clinical conditions, but the physiologic relevance of the differences between plasma TFPI concentrations in health and disease is difficult to interpret. The severalfold increase in (continues)
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(continued)
plasma TFPI after heparin treatment suggests that TFPI in plasma represents only a fraction of the total TFPI that can be readily mobilized. This raises the possibility that other physiologic or pathologic mechanisms may similarly affect the concentration of TFPI in plasma or at local sites. The TFPI presumably bound at the surface of the endothelium and in contact with plasma is probably a major contributor to overall TFPI action in vivo, but that is not reflected by the plasma TFPI concentration. Although low levels of plasma TFPI are occasionally seen in patients with septicemia or disseminated intravascular coagulation, their TFPI concentrations are more often normal. The progression of disseminated intravascular coagulation in the face of a normal plasma level of TFPI is consistent with the fact that at physiologic concentrations, TFPI inhibits factor VIla/ tissue factor effectively only after factor Xa has been generated. Thus, TFPI dampens but does not prevent the coagulation process as long as generation of tissue factor continues. The depletion of endogenous TFPI sensitizes rabbits to the disseminated intravascular coagulation induced by tissue factor or endotoxin, and the infusion of high, therapeutic concentrations of TFPI into animals ameliorates the intravascular coagulation induced by tissue factor. Although TFPI deficiency might be expected to cause a prothrombotic phenotype, no person with TFPI deficiency has yet been identified. The low plasma levels of TFPI found in abetalipoproteinemic patients appear simply to reflect the absence of LDL, which is a carrier of TFPI. Their
total TFPI, as estimated from plasma TFPI levels after heparin infusion, is the same as that in normal persons.
Revised Hypothesis of Blood Coagulation A revised formulation of blood coagulation shown in Figure 5 takes into account the feedback inhibition of factor VIIa/tissue factor by TFPI. In this hypothesis, coagulation ensues when
damage to blood vessels allows the exposure of blood to the tissue factor produced constitutively by cells beneath the endothelium. The factor VII or factor VIla present in plasma then binds to tissue factor, and the factor VIIa/tissue factor complex activates limited quantities of factor X and factor IX. With the generation of factor Xa, the inhibitory effect ofTFPI becomes manifest, preventing further production of Xa and
Figure 5. A revised hypothesis of coagulation postulates a single pathway whose initiating event is exposure of tissue factor to factor VIla (or VII) at a site of vessel injury (A). The resulting Vlla/tissue factor complex generates small amounts of factors IXa and Xa (B). Tissue factor pathway inhibitor (TFPI) then prevents further Vlla/tissue factor activation of IX and X (C). Only the action of factors IXa and VIlla (D) can generate the additional factor Xa (E) needed to sustain coagulation. In this regard, factor XI activation by thrombin (F) and Xla autoactivation (G) may generate additional factor IXB (H). Hospital Practice March 15. 1992
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PHYSIOLOGY IN MEDICINE factor IXa by factor Vlla/tissue
Seattle and David Gailani in my
factor Xa and thrombin. The
factor. Additional factor Xa can
laboratory showed that throm-
need for sustained production
be produced only through the alternative pathway involving factor IXa and factor VIlla. The presence of a variable but usually mild bleeding diathesis in persons with factor XI deficiency (sometimes called hemophilia C) implies that under certain conditions, the initial quantity of factor IXa produced by factor VIIa/tissue factor is insufficient and additional factor IXa generated by factor XIa is needed for normal hemostasis.
bin can activate factor XI and that, in the presence of a polyanion (e.g., dextran sulfate, heparin, or sulfatides), this process is amplified through the autoactivation of additional factor XI by factor Xla (Figure 5). Evidence that this actually occurs in vivo has not yet been presented.
of factor Xa and thrombin for normal hemostasis has been shown in vivo and presumably reflects the influence of inhibitors of the coagulation proteases and the competing process offibrinolysis. Thus, hemophilia patients bleed because the factor Xa generated through the action of factor VIIa/tissue factor, and dampened by TFPI, is insufficient to sustain hemostasis and must be amplified through the action of factor IXa and factor VIlla. Similarly, factor Xla is required to supplement the factor IXa formed by factor VIIa/tissue factor, which is limited by the presence of TFPI. As a corollary, we suggested in 1988 that effective inhibition ofTFPI could ameliorate the hemorrhagic manifestations of hemophilia.
The mechanism for the activation of factor XI is not clear, but the model predicts that rather than being involved in the initiation of coagulation, as suggested by the cascade-waterfall hypothesis, factor XI would be activated "late" in the coagulation process, after factor Xa and thrombin have been generated through the action of factor VIIa/tissue factor. Recently, Koji Naito in the laboratory of Kazuo Fujikawa in
The revised hypothesis of blood coagulation differs from the cascade-waterfall model in two major respects. First, it integrates all of the factors known to be involved in coagulation into a single pathway that is initiated by factor VIIa/tissue factor and that does not require the contact factors (factor XII, prekallikrein, and high molecular kininogen). Second, in the revised model, the hemostatic process does not end with the initial generation of factor Xa and thrombin. Instead, the initial hemostatic response must be "consolidated" by the progressive local generation of
Selected Reading Braze GJ Jr et al: The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: Insightinto its possible mechanism of action. Blood 71 :335. 1988 Girard T J et al: Functional signiflcance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 338:518, 1989 Braze GJ Jr, Girard T J, Novotny WF: Perspectives in Biochemistry: Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 29:7539. 1990 Repke D et al: Hemoph111a as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous flow reactor. Proc Natl Acad Sci USA 8 7:7623, 1990 Gailani D, Braze GJ Jr: Factor XI activation in a revised model of blood coagulation. Science 253:909, 1991 Massie RK: Nicholas and Alexandra. Atheneum. New York. 1968
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TFPI-induced feedback inhibition of factor VIIa/tissue factor can explain the clinical need for both the extrinsic and intrinsic (factors VIII, IX, and XI) coagulation pathways and is consistent with in vitro results showing deficient factor Xa production in hemophiliac plasma induced to clot by small quantities of tissue factor. In addition, data at present are consistent with the view that in normal hemostasis; factor VIIa/tissue factor is responsible for initial factor Xa generation, which provides sufficient thrombin to induce the local aggregation of platelets and the activation of the critical cofactors, factor V and factor VIII. That persistent hemostasis requires the continued production of additional factor Xa through the action of factor IXa and factor VIlla is consistent with the delayed onset of bleeding frequently seen in hemophilia. D
