Sources of Tissue Factor Bjarne Østerud, Ph.D.,1,2 and Eirik Bjørklid, Ph.D.2

ABSTRACT

Tissue factor (TF) exhibits a distinct nonuniform tissue distribution. Thus, high levels are found in highly vascularized organs such as the lung, brain, and placenta; intermediate levels in the heart, kidney, intestine, testes, and uterus; and low levels in the spleen, thymus, and liver. Several cell types are known to express TF constitutively, such as astrocytes in the brain, epithelial cells enveloping organs and body surfaces, adventitial fibroblasts and pericytes, and cardial myocytes in the heart. Smooth muscle cells in the media of the vessel wall and monocytes/macrophages contain small amounts of TF, which is enhanced substantially upon activation of the cells. Endothelial cells probably do not express TF. The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed has been challenged recently by the observation that blood of healthy individuals may form TF-induced thrombi under conditions entailing shear stress and activated platelets, corroborating the notion of blood-borne TF. Accordingly, small amounts of decrypted TF activity is detected in calcium ionophore-stimulated monocytes, and microparticles from plasma of healthy subjects possess TF-like activity subject to partial inactivation by anti-TF antibody. In addition to microparticles, plasma TF also comprises the soluble alternatively spliced human TF and truncated TF, both of which probably require factor VIIa to be physiologically active. Although it has been suggested that activated platelets possess active TF, the notion of TF as an integral platelet component is contested by more recent data. Rather, platelets may be very important in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets. KEYWORDS: Tissue factor, TF, blood-borne TF, monocytes, platelets

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n 1905, Morawitz proposed a three-step theory of blood clotting.1 Platelets were conceived as containing tissue thromboplastin (tissue factor [TF]) activity that was released at sites of damage to the vessel wall in vivo, or upon contact with some foreign surface in vitro. The released TF promoted conversion of the inactive precursor prothrombin to the active form thrombin in a calcium-dependent manner, the latter eventually cleaving fibrinogen to fibrin. The discovery of many additional clotting factors, beginning with the identification in 1947 by Owren2 of a

patient with a congenital lack of factor V (FV), was followed by a string of reports pertaining to deficiencies in FVII,3,4 FIX,5,6 and FX,7 revealing the inadequacy of this classical theory. Coon et al8 and Hjort9 showed that FVII formed a complex with TF in the presence of calcium. In 1964, the cascade10 and waterfall11 theories were launched, promoting the idea of TF-mediated conversion of FVII to an enzyme that may in turn activate FX. In a review, Nemerson12 put forth the notion of an extrinsic FX activator working much like the

Tissue Factor; Editor in Chief, Eberhard F. Mammen, M.D.; Guest Editor, Marcel Levi, M.D., Ph.D. Seminars in Thrombosis and Hemostasis, volume 32, number 1, 2006. Address for correspondence and reprint requests: Bjarne Østerud, Ph.D., Department of Biochemistry, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway. E-mail: [email protected]. 1Professor; 2 Department of Biochemistry, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Tromsø, Norway. Copyright # 2006 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. 0094-6176,p;2006,32,01,011,023, ftx,en;sth01126x.

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prothrombin-converting complex, and composed of FVII, TF, and calcium ions in complex. According to the current view, available TF forms a complex with FVII/FVIIa, which then converts FIX and FX into the corresponding active forms, FIXa and FXa, by way of limited proteolysis.13 Although association of FVIIa and TF is greatly enhanced in the presence of calcium ions and phospholipids,14,15 neither factor is absolutely essential for the interaction.15,16 This was also evident from experiments where FVII was allowed to interact with phospholipids (PLs) in the presence of Ca2þ, and then exposed to phospholipase C (PLC) or vehicle only. Despite the gross inactivation of PLs by PLC, 10 to 20% of FVIIa activity was retained.17 In contrast, FVIIa similarly recombined with PLs remained unaffected by the PLC treatment. When bound to TF, FVII can be activated by FXa, FIXa, FVIIa, or thrombin.18–20 Part of this activation requires membrane anchoring.21 Although the low amidolytic activity of FVIIa is enhanced up to 100fold in the presence of TF,22,23 membrane anchoring is not required for this to occur.24,25 In contrast, the activation of FIX and FX is highly dependent on membrane anchoring,26 and is supported by negatively charged phospholipid.27–29

DISTRIBUTION OF TF An early assessment of the gross distribution of TF in the organism was accomplished by testing various tissues for overall TF activity. Thus Astrup30 found evidence for high levels of TF activity in brain, lung, and kidney. Detailed immunohistochemical analysis of TF distribution in tissues has more recently become possible using increasingly available mono- and polyclonal antibodies specific for TF. Notably, the brain, lung, kidney, and placenta have proven to be very rich in TF. The realization that TF of blood vessels is localized predominantly in adventitia, well shielded from the vessel lumen, prompted the launching of an envelope theory of TF. In theory, TF constitutes a hemostatic envelope encapsulating the vascular bed; rupture of the integrity of the envelope would trigger the clotting process instantly.31 However, it has become evident more recently that whole blood, even in healthy subjects, may already contain active TF, as demonstrated by TF-induced thrombus formation when blood of healthy individuals is allowed to pass along a collagen-coated slide ex vivo.32 Such circulating TF is generally considered to derive from circulating monocytes, which may release TFbearing microparticles (MPs) to the plasma. This topic is discussed further on page 16 under ‘‘TF in MPS of Plasma.’’

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TF IN THE VESSEL WALL TF in the Endothelium In vivo findings do not concur with the notion of an important role for TF in activated endothelium per se, and are at odds with results emerging from corresponding in vitro studies. Thus endothelial cells (ECs) isolated from human umbilical veins were shown to generate TF activity when exposed to lipopolysaccharide (LPS), thrombin, interleukin (IL) -1b, tumor necrosis factor alpha (TNFa), and so on (for review, see Camerer et al33). To make ECs available for in vitro studies, exposure to relatively harsh treatment such as collagenase digestion is required, in a process normally taking 4 days. The cells adapt by acquiring characteristics rather deviant from those of the original resident ECs; the proclivity for TF expression apparently is one such attribute. Thus, saphenous veins and internal mammary arteries from coronary bypass patients failed to show endothelial TF expression.34 Furthermore, when we subjected isolated saphenous veins to perfusion with buffers containing LPS or thrombin, no induction of TF activity was detected in the ECs,35 whether on intact endothelium or in subsequently isolated ECs. We also failed to find any TF exposure in ECs from rabbits subjected to Schwartzman reaction by the intravenous injection at the 24-hour interval of two doses of LPS.36 Corroborative evidence was found in two more recent studies on rabbits and rats, in which dual immunohistochemical staining for TF and von Willebrand factor revealed no detectable endothelial TF antigen in the rabbits,37 and TF mRNA was found in the mononuclear cells but not in the ECs around the hepatic vein of rats subjected to LPS treatment. In apparent contradiction to the latter three studies, one study claimed an increase in procoagulant activity (PCA) identified as TF in thoracic aorta harvested from rabbits subjected to endotoxin injection.38 Conceivably, this PCA may be of nonendothelial origin, for example, arising from TF-bearing MPs of monocyte origin that also contain P-selectin glycoprotein ligand1 (PSGL-1), facilitating their binding to activated endothelium via the exposed receptor P-selectin. After lethal doses of Escherichia coli had been given intravenously to baboons, a thorough immunohistochemical examination revealed the absence of TF antigen in the endothelium of all tissues of the animal apart from the spleen39; the latter observation was probably due to monocyte-derived TF-rich MPs bound to the endothelium. In accordance with these observations, immunohistochemical studies on normal human saphenous vein and internal mammary artery samples did not reveal TF mRNA or TF protein in any of the vascular tissues analyzed.39 Recent reports of TF in the endothelium of sickle cell anemia patients along with the detection of

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blood-borne TF-rich MPs of EC and monocyte origin40 have rekindled the notion that ECs may synthesize TF. However, in a study of TF expression in sickle cell transgenic mice, Solovey et al41 found by immunostaining that endothelial TF expression was confined almost exclusively to the pulmonary veins. Recently, it has been reported by Del Conde et al42 that TF-rich microvesicles from monocytes not only bound to activated platelets, but also fused with them, in effect transferring both proteins and lipids to the platelet membrane. Thus, by inference, in sickle cell anemia patients a scenario can be envisioned whereby TF-rich MPs bind and fuse with activated ECs as well as with MPs derived from activated endothelium. One may therefore question the origin of the TF-rich MPs in sickle cell anemia patients. Most probably, the TF is derived from the monocytes that are activated in these patients.40 Thus, emerging from a whole range of studies is the notion that the proclivity for TF expression in ECs propagated in vitro, as reported by many, is but a cellular trait acquired during the isolation and culturing processes, and hence of an artifactual nature. Our opinion is that in the absence of any injury to the vessel wall, there is no exposure to blood of extralumenal TF except when monocytes become activated, which may lead to the tethering of TF-bearing MPs at the endothelial surface. However, under conditions in which the endothelial barrier becomes compromised (for example due to EC damage), leading to enhanced barrier permeability, plasma factors may rapidly gain access to TF already present in subendothelial strata, and henceforth thrombin generation is initiated. Such is the state that may pervade the microcirculation of several organs of patients subject to disseminated intravascular coagulation (DIC) syndrome.

TF in Adventitia Investigation of the vessel wall revealed the most pronounced expression of TF in the adventitia, where fibroblasts showed intense TF protein staining and mRNA hybridization.34 In contrast, in the media fewer cells contained TF mRNA, as indicated by in situ hybridization, and no TF protein could be detected. This study confirmed the earlier observations of Drake et al31 and Fleck et al43 that TF in the blood vessel wall was localized predominantly to cells in the adventitia. Thus smooth muscle cells in the media did not express detectable TF, whereas pericytes surrounding capillaries and adventitial fibroblasts surrounding small and large vessels stained strongly positive.43 The confinement of TF almost exclusively to the adventitia led to the concept that normally distributed TF represents a hemostatic envelope ready to activate coagulation whenever vascular integrity is disrupted.31

Diseased Vessel Wall In contrast to the normal vessel wall, the necrotic cores of atherosclerotic plaques were characterized by extensive TF protein localization in the extracellular matrix, particularly surrounding some cholesterol clefts. Additional staining for TF was seen in the macrophage foam cell regions of many of the atherosclerotic plaques examined. Such foam cell–rich regions were often found underneath the fibrous cap and adjacent to the necrotic cores. Interestingly, just as with normal vessels, no TF mRNA or protein was detected in the endothelium lining the vascular surface of the small vessels within the plaques.34 Restenosis is a serious complication of coronary angioplasty that involves the proliferation and migration of vascular smooth muscle cells (SMCs) from the media to the intima. The assembly of SMCs in the intima is associated with a significant increase in TF synthesis. Interestingly, inhibition of TF-mediated coagulation by tissue factor pathway inhibitor (TFPI) or active site inactivated FVIIa markedly attenuates or reduces restenosis in animal models.44,45

Distribution of TF in Organs TF exhibits a nonuniform tissue distribution, and richly vascularized organs such as the lung, brain, and placenta have been known to possess large amounts of TF. This was confirmed in the immunohistochemistry studies by Drake et al31 and Fleck et al,43 showing that these organs as well as peripheral nerves and autonomic ganglia stained strongly for TF. More recent studies on murine TF gene expression have revealed intermediate levels of TF in the kidney, intestine, testes, and uterus, and low levels in the spleen, thymus, and the liver.46–50 Furthermore, epithelia of skin, mucosa, and glomeruli also stained, whereas skeletal muscle did not and cardiac muscle stained only faintly.43 Cell-type–specific distribution of TF in the various organs was documented by the expression of astrocytes in the brain, alveolar cells in the lung, epithelial cells surrounding organs and at body surfaces, adventitial fibroblasts and pericytes in the blood vessel wall, and cardiomyocytes. TF activity has also been tested in various organs, including arterial walls of rabbits.36 This study confirmed the high concentrations of TF in the lungs, given that the TF activity of the lungs was six-fold higher than TF activity of kidneys and approximately 25-fold higher than TF activity of the spleen. Examination of the TF activity in homogenized arterial walls showed a 10-fold higher TF activity of the carotid compared with the femoral artery, whereas the TF activity of aorta and renal arteries were respectively four-fold and two-fold lower than that in the carotid. The high level of TF activity in the carotid artery may be of great significance if the same distribution of TF occurs in man, given that the carotid arteries are known to be subject to frequent thrombotic events.

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BLOOD-BORNE TF In recent years the envelope paradigm of TF expression and function has been challenged by the demonstration of blood-borne TF or circulating TF (cTF).32 The occurrence of circulating TF-containing MPs,51–53 elevated levels of TF antigen measured in patients with acute coronary syndromes,54,55 neutrophil-associated TF, and platelet-associated TF are all phenomena that raise new questions regarding the role and potential of TF in inflammation, thrombosis, and hemostasis. A thrombusforming potential has been demonstrated for nonfunctional or encrypted TF present in MPs from monocytes and neutrophils in blood from healthy subjects, and the term blood-borne TF was recently introduced for such TF.32 This first report on blood-borne TF has aroused quite a bit of interest in the prospect of detecting TF in blood cells, an area imbued with considerable confusion presumably due to the different techniques, test systems, and reagents used. We provide an overview of the current knowledge of blood-borne TF and its potential to generate thrombin in circulating blood.

Monocytes as a Source of Blood-Borne TF TF synthesis in monocytes was first reported in 1976.56 Substantial TF-type procoagulant activity was measured in isolated monocytes subjected to LPS stimulation. In contrast, monocytes circulating in healthy blood were considered unlikely to possess TF due to the perceived hazard of thrombogenesis. At that time, the regulation of TF-triggered coagulation by TFPI was not known. Although constitutive expression of encrypted TF was reported in a monocytic cell line,57 few data supported the idea of the presence of TF in resting monocytes of whole blood. The concept of blood-borne TF raised the question of the origin of TF incorporated into fibrin thrombi formed on the collagen-covered slides. Recent reports on blood-borne TF have focused largely on MP-associated TF, and rather little on the presence or absence of TF in circulating monocytes. Recently, Butenas et al58 failed to detect significant TF antigen in resting monocytes (0.2% of mononuclear cells stained for TF) and TF activity was not detectable in their assay system. We found that in blood of healthy individuals, only 1.5% of the circulating monocytes are positive for CD14 and express TF (only CD14-positive monocytes express TF).59 However, after stimulating freshly isolated monocytes for 10 minutes with the Ca ionophore A23187, significant TF activity could be detected, particularly in cells from so-called high responders60 (Breimo and Østerud, unpublished data). Thus, the apparent TF in resting monocytes of blood is encrypted, and as such will not induce thrombin generation in the blood. Most likely, the TF activity is too low to play any thrombogenic role in the absence of activated platelets. At a site of platelet adherence to the

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vessel wall (e.g., after surgery), events leading to TF decryption may conceivably occur, and at the same time TF expression in the membranes of monocytes may be upregulated due to the surgery.61 These events may at least partially account for the enhanced risk of thrombosis postoperatively.

REGULATION OF TF EXPRESSION IN MONOCYTES: ROLE OF PLATELETS AND GRANULOCYTES Platelets are well known to play an essential role in hemostasis, through their sealing effect at the injured vessel wall and in their activated form through promoting thrombin generation by providing a catalytic surface for the pivotal enzyme complexes tenase, prothrombinase as well as FVIIa-TF. In addition, activated platelets release FVa,62 which is thought to be important in the initial phase of thrombin generation and in the feedback activation of FVII by FXa.63 However, many years ago platelets were suggested to be part of leukocyte-associated TF expression. Thus, in 1974, Niemetz and Marcus64 proposed that platelets enhance the procoagulant activity of white blood cells. This was later confirmed in monocyte cell cultures, in which isolated platelets added to monocytes enhanced LPS-induced TF activity.65,66 Furthermore, 12-hydroxyeicosatetraenoic acid, a platelet product, was claimed to enhance LPS-induced TF activity in adherent monocytes.65 The platelet effect was confirmed in our whole blood model system. By removing most of platelets from blood by centrifugation, followed by recombination of a platelet-poor blood cell fraction with either platelet-rich plasma (PRP) or platelet-poor plasma (PPP), and subsequent stimulation with LPS, a significant enhancing effect by the platelets on LPS-induced TF activity in monocytes was observed.67 The significance of the platelet effect was corroborated by the observation that the great variability in LPS-induced TF activity in monocytes of different individuals was at least partly due to platelets. Thus, recombining a platelet-poor blood cell fraction from high responders with platelets from low responders led to a marked reduction in LPS-induced TF activity.67 Conversely, a significant increase in the activity was observed when platelets isolated from high responders were recombined with platelet-poor blood cells from low responders. It should be emphasized that granulocytes were also present in this test model. In an extension of this model, granulocytes and mononuclear cells were separated, and subsequently recombined with PPP or PRP followed by incubation with LPS alone or in combination with TNFa or phorbol 12-myristate 13-acetate (PMA). Experimental data from this test model revealed a minor effect of platelets on LPS induction of TF activity in monocytes.68 In contrast, when granulocytes were also present

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along with platelets, a several-fold increase in TF activity was found. This was in accordance with the observation that exogenously added platelet-activating factor (PAF) enhanced LPS-induced TF activity in monocytes, provided that both granulocytes and platelets were present.69 A PAF antagonist was shown to suppress LPS-induced TF activity in monocytes. Although PAF is known to activate platelets, its effect in this system was probably targeted to potent activation of granulocytes by PAFs, and subsequent activation of platelets by granulocytes. The platelet/ granulocyte effect in these systems was entirely dependent on P-selectin–PSGL-1 interaction. It is well established that granulocyte-derived cathepsin G is a strong platelet agonist.70 Accordingly, evidence was obtained that inhibition of cathepsin G caused a significant reduction in LPS-induced TF activity in monocytes.71 However, further studies must be performed to clarify whether the effect on LPS-induced TF activity is mediated solely by cathepsin G or whether an unknown mechanism may be involved. The importance of platelets and granulocytes in LPS-induced TF activity in monocytes was established using TNFa and PMA as stimulatory agents. Although neither of these agonists induced TF activity by themselves, they both enhanced several fold LPS-induced TF activity in a platelet- and granulocyte-dependent manner. This was shown to be due to stimulation of both granulocytes and monocytes.68 In a follow-up to this study we examined whether the platelet effect was mediated by upregulation of TF synthesis or whether it just reflected an upregulation in procoagulant activity of TF already present. To this end, blood that had been depleted partially in platelets (10% of original remaining) was recombined with either PPP or PRP, followed by stimulation with LPS (5 ng/mL) alone, or with LPS in combination with TNF or the phorbol ester PMA. Under neither of these conditions did the presence of platelets affect TF antigen synthesis. TNF and PMA enhanced the TF activity two- to threefold compared with LPS alone. Although platelets had no effect on the LPS-induced TF antigen in the presence or absence of TNF or PMA, TF activity was on average 190 to 240% higher in the presence of platelets than in their absence.72 This probably reflects a crucial role of platelets in the decryption of TF activity in monocytes. In contrast, platelet lysate not only enhanced LPS-induced TF activity by approximately three-fold, but this was accompanied by a substantial increase in LPS-induced TF antigen. Although the above-mentioned studies have been done ex vivo with LPS as stimulating agent, we believe that the level of TF expression in vivo may reflect the activation conditions of the blood cells, particularly the platelets (for example, as seen in patients with unstable angina). The platelet effects may be mediated through

the PAF and thromboxane A2 receptors, given that antagonists to these receptors together with a protease inhibitor blocked approximately 77% of LPS-induced TF activity of monocytes in whole blood73(for review, see Eilertsen and Østerud74).

LOCALIZATION OF TF IN CELLS It is well established that TF activity associated with stimulated cells is strongly encrypted. Thus, in activated monocytes examined for TF availability only 10 to 15% of the total TF activity accessible upon lysis proved available on intact cells, even though 75% of the antigen had emerged on the surface.75 More recently, three pools of TF antigen constituting the total TF were quantified in smooth muscle cells following lysis of the cells with boctyl glucoside.76 It was estimated that approximately 20% of the total cellular TF is available on the surface, approximately 30% is intracellular, and some 50% is latent. The intracellular material was found to be associated with sufficient membrane material to be biologically active if released.77 Thus, by subjecting TF-bearing cells to a succession of freeze–thaw cycles, calcium ionophore, or PMA, TF activity increased markedly without TF m-RNA or protein being significantly affected.78,79 Studies on the intracellular and surface distribution of LPS-induced monocyte TF by the use of flow cytometry and in-cell Western assays revealed a larger population of TF-positive monocytes in high responders (32.0
3.5%) versus low responders (11.2
1.2%).59 In-cell Western assay showed higher accumulation of TF in monocytes from low responders because LPS induced a 3.7-fold increase of total TF levels in low responders versus a 1.5-fold increase in high responders. In contrast, in response to LPS stimulation, monocytes from high responders exhibited a four-fold induction of surface TF, whereas monocytes from low responders only had a minor increase in surface TF levels. To explain the encryption phenomenon, a mechanism of clustering of TF molecules in the membrane has been suggested, whereby the activity remains latent and only reaches its full potential once the molecules are dispersed.77 Calcium ionophore has been shown to cause a 100-fold increase in the TF activity of intact cells.80 This effect was blocked by pretreating the cells with calmodulin inhibitors. It was hypothesized that the increase in TF activity upon decryption may stem from the exposure of an essential macromolecular substrate binding site in the TF-VIIa complex, as a direct consequence of some change in TF quaternary structure. More specifically, the suggested cryptic form was TF dimers, and the activity potential was believed to be unlocked by their conversion to monomers. In a recent study, it was concluded that the calcium ionophore effect in decrypting TF activity is not solely the result of

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increased availability of exposed phosphatidylserine (PS), and that the FVIIa/TF substrate specificity is not altered by the decryption process.81

Models for the Decryption of Monocyte TF by Platelets To account for the platelet effect observed in our ex vivo blood model, at least two possible explanations may be suggested. A mechanism based on some platelet– monocyte fusion event facilitating transfer to the monocyte of rate-limiting PS from the platelet partner would account for the typical asymmetry observed in the expression of TF antigen and activity. By acquiring PS from the platelet membrane, marginally active TF antigen present in the monocyte membrane would have its specific activity upregulated. Alternatively, the plateletdependent increase in TF activity may be linked somehow to the generation of monocyte-derived MPs with decrypted TF activity. Corroborative evidence for this notion is the observation that monocyte-derived TFcontaining MPs may be associated with platelets,42,71,82 suggesting a mechanism for platelet upregulation of TF by rapid supplementation of TF apoprotein with essential lipid at the MP level. Given that monocyte-derived MPs in addition to TF apoprotein also have PS exposed on their surface,83 they may also by themselves facilitate significant activation of FVII to VIIa, leading to generation of TF/FVIIa complexes, which in turn provide for binding of the substrates FIX and FX, and their rapid conversion to the corresponding active enzymes. Whatever the predominant mechanism, evidence is mounting for a role of platelets in decrypting the activity of TF in monocyte membranes. Interactions of monocytes with other blood cells therefore may not only affect the TF activity at the level of synthesis or degradation, but also at the level of activity encryption or decryption. TF activity expression in blood may be regulated by the profusion of MPs derived from platelets and monocytes, which is possibly one of the most efficient and important ways to decrypt monocyte-derived TF activity in vivo. Monocyte shedding of MPs with membraneassociated procoagulant activities was provoked by LPS stimulation of monocytes.83 These MPs had TF as well as PS (the active template in the coagulation enzyme complex) exposed on their surface, in addition to the adhesion molecules CD14, CD11a, and CD18. Recently, it was shown that LPS-stimulated whole blood generated low levels of TF-rich MPs after 6-hour incubation, and this was upregulated 100-fold by adding PMA to LPS.82 In contrast, platelets acquired TF activity after only a 2-hour incubation of whole blood with LPS alone. Thus, TF appeared to be transferred in its encrypted form from monocytes to platelets, probably due to the ability of activated platelets to bind or fuse

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with such TF-rich MPs in an interaction dependent on P-selectin–PSGL-1, as recently reported by Del Conde et al.42 Recently, we found that LPS-stimulated blood of the high responders contained two-fold more TF-positive MPs than that of low responders.59 The higher availability of TF surface antigen on monocytes from high responders and TF-containing MPs might make these individuals more susceptible to thrombosis.

TF IN MPS OF PLASMA MPs are circulating cell fragments derived from cells undergoing activation or apoptosis. They are less than 1 mm in diameter and they have significant amounts of surface-exposed negatively charged PLs such as PS that are essential for thrombin generation. MPs also contain many membrane-derived proteins, such as major histocompatibility proteins and PSGL-1, that contribute to the regulation of host immunity and inflammatory responses.84–87 Many different cell types have been shown to shed MPs that contain proteins and cytoplasmic components that specifically derive from their cells of origin.88 Examination of plasma from fresh blood samples of healthy individuals using flow cytometry identified MPs originating from platelets, erythrocytes, granulocytes, and ECs.89,90 The same group showed that under physiological conditions, 80% of the MPs are derived from platelets. Interestingly, when the MPs were tested for coagulation activity, it was found that they supported thrombin generation via TF-independent pathways, given that antibodies against TF or FVII did not abolish the activity. An elevated level of MPs has been demonstrated in numerous diseases, such as acute coronary syndromes,90–92 sickle cell anemia,40 preeclampsia,93 thrombotic thrombocytopenia purpura,94 antiphospholipid antibody syndrome,95 cerebral vascular events96 and heparin-induced thrombocytopenia.97 An atherogenic role of MPs, probably mainly of monocytic origin, can be inferred indirectly from a study where high levels of shed apoptotic MPs were detected in extracts from atherosclerotic plaques.51 These MPs expressed both TF and PS. MPs derived from platelets have for many years been acknowledged to be rather thrombogenic.98,99 They are released from activated platelets and express functional adhesion receptors, including P-selectin, on their surface. Platelet MPs provide a catalytic surface incorporating PS, which accelerates coagulation,100–102 and they can attach to neutrophils and monocytes.103 Platelet MPs have been shown to be present under various disease conditions.52,90,98,104–108 The demonstration of TF-containing neutrophils in native human peripheral blood subjected to perfusion over collagen-coated glass slides32 opened a field

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for exploring how coagulation is initiated within the vasculature in the absence of vessel wall injury or even when the injury is superficial, given that the subsequent reactions of the coagulation cascade are performed most efficiently on the surface of activated platelets. Thus the demonstration that TF also circulates in the blood under normal conditions, both as a component of cell-derived MPs and as a soluble alternatively spliced form may account for the propagation of a thrombus to the vascular space even in the absence of vessel wall damage. The authors32 suggested that pre-existing blood-borne TF was deposited on platelets on the growing thrombus, thereby forming TF-platelet hybrids. This study was later extended in the same laboratory by showing that leukocyte derived TFpositive MPs may be transferred to platelets via interactions mediated by CD15 (part of PSGL-1) and P-selectin.109 This resulted in procoagulant platelet aggregates containing TF-rich MPs. Events or conditions such as apoptosis, acute coronary syndrome, sepsis, and lupus, as well as agonists such as endotoxin and proinflammatory cytokines, have been reported to elicit the release of TF-rich MPs.52,83,88,110–114 TF-rich MPs derived from hematopoietic cells are essential in thrombus propagation in the microvasculature system. The concept of blood-borne TF was corroborated recently by Chou et al115,116 in elegant experiments using intravital microscopy for studying thrombus formation in living mice. It was found that low-TF mice (1%) in contrast to wild-type mice developed very small platelet thrombi lacking TF or fibrin. Furthermore, wild-type and low-TF mice were then given transplantations of bone marrow from wild-type or low-TF mice to produce chimera. Arterial thrombi in wild-type bone marrow/low-TF chimeric mice showed decreased platelet thrombus size but normal TF and fibrin levels, whereas low-TF bone marrow/ wild-type chimera had decreased thrombus size and decreased TF and fibrin levels. It was therefore concluded that blood-borne TF associated with MPs derived from hematopoietic cells contributes to thrombus propagation in the microvascular system.

TF Derived from the Blood Vessel Wall Drives Macrovascular Thrombosis In the model described in the previous section there was minimal exposure of the subendothelium, and hence only small amounts of TF were exposed at the laserinduced vessel wall injury. However, in this model it is also concluded that the initial TF expression at the vessel wall injury is important for generation of the first traces of thrombin to activate and adhere platelets to the injury site. In contrast, a regular vessel wall injury entails gross exposure of subendothelial structures and thereby TF is exposed to blood. In a follow-up study on macrovascular

thrombosis using transgenic mice, Day et al116 showed that transplantation of low-TF marrow (marrow generates myeloid cells with low TF) into wild-type mice did not suppress arterial or venous thrombosis. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. Furthermore, in vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. It was therefore concluded that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall rather than from blood leukocytes. In control experiments they showed that compared with wildtype mice, mice with severe TF deficiency demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. Although these experiments suggest that the vascular wall is the dominant source of thrombogenic TF in medium and large blood vessels, the authors concluded that their study did not rule out a minor role of leukocyte-derived TF in thrombus formation. Another important point also discussed in this study is whether there are any fundamental species differences between mice and humans regarding the degree to which the different pools of TF contribute to thrombus development.

TF Activity in MPs Isolated from Blood of Healthy Individuals The observation that blood circulating over a collagencovered slide resulted in platelet aggregates and deposited fibrin incorporating TF led to the conclusion that blood must possess TF that is either already active or is becoming active in the presence of activated platelets under shear stress. However, as already mentioned, Berckmans et al89 failed to detect TF activity in MPs of plasma from healthy individuals, which might not be expected based on the above studies of thrombus formation in blood of healthy individuals. This was confirmed recently by Butenas et al,58 who were unable to detect TF antigen or activity in MPs from plasma of healthy individuals. In contrast, Jin et al117 found that the TF activity associated with MPs in 100 mL of plasma amounted to the equivalent of 12.2 fmol of recombined TF. Similarly, using a very sensitive assay for TF activity, we have measured small amounts of TF-like activity that was partially neutralized by antiTF antibodies (Breimo and Østerud, unpublished data). Thus, it is likely that the observed thrombus formation in various models is a reflection of shear stress–induced TF activity in circulating MPs or even circulating monocytes possessing encrypted TF. In the absence of shear stress conditions, blood-borne TF is probably unable to induce any thrombus, given that the level of TF activity is extremely low58 (Breimo and Østerud, unpublished data).

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Forms of TF in Plasma In addition to the TF associated with white cells or MPs, the presence of soluble TF in plasma has been suggested. Thus when TF protein in particulate-free plasma obtained by high-speed centrifugation was subjected to immunocapturing, relipidation, and quantification by FXa generation in the presence of FVIIa, patients with poorly controlled type 2 diabetes mellitus, smokers, and untreated hyperlipidemic subjects all had elevated levels of immunocaptured TF in their plasma, whereas the levels were reduced in patients showing improvements in glycemic control.118 It should be emphasized that the immunocaptured TF may not be active in plasma because it was relipidated before testing for TF activity. The soluble TF in plasma is probably a blend deriving mostly from monocytes and extravascular TFexpressing cells and truncated by, for example, enzymatic cleavage. A substantial part of total TF in plasma has been suggested to be constituted by an alternatively spliced human tissue factor (asHTF), containing most of the extracellular domain of TF but lacking a transmembrane domain and terminating with a unique peptide sequence.119 asHTF does not require integral incorporation into a phospholipid structure to be active, but becomes active simply by direct combination with phospholipids. However, the amount of asHTF in plasma is approximately 0.5 pM, whereas approximately 40 nM of the asHTF was used to test for its effect on plasma clotting, amounting to a concentration 80,000fold above physiologic levels. The clotting activity of this TF species is apparently similar to that observed for the extracellular domain of regular TF.21 It can be anticipated that MPs tend to be associated with circulating cells or activated endothelium. This notion notwithstanding, reports have emerged during the last decade showing elevated TF antigen in plasma, notably in DIC patients. One such study reported that 40% of the DIC patients were expressing high plasma TF levels.120 Probably, the high values were due mostly to the presence of truncated soluble TF devoid of biological activity, given that TF antigen did not correlate with hemostatic markers typically associated with DIC, such as prothrombin fragment 1 þ 2, thrombin-antithrombin complex, FDP, D-dimer, or fibrinogen. Interestingly, serial monitoring of plasma TF antigen revealed that most patients having elevated TF antigen at the moment of presentation of DIC showed plasma TF changes running roughly in parallel with the progression of DIC. This may reflect a worsening of the primary disease, leading to an increase in TF release into the circulation and the ensuing aggravation of the DIC condition. Although significant elevation of plasma TF antigen has been detected in cases of DIC associated with certain types of cancer,121 no such elevation was observed in similar cases associated with other types of cancer. Nor

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did patients without DIC have any significant elevation of TF, except in four cases of cancer with later-onset episodes of DIC. The apparent discrepancies emanating from these studies are probably rooted largely in the diversity of DIC states, evident, for example, in the extent of damage to the microvasculature walls. Furthermore, the result may be influenced by poor standardization and specificity for TF antigen in the assays, probably leading to falsely high levels in some cases58; the lesson is that data from such antigen measurements should be interpreted with caution.

TF IN NEUTROPHILS Considering all the reports and conflicting evidence whether TF may be associated with neutrophils, it is our opinion that the phenomenon may be explained by the generation and dispersal of TF-rich MPs derived from monocytes (for reviews, see Østerud122 and Nakamura et al123). So far we have failed to detect any encrypted or inducible TF activity in neutrophils using test systems incorporating optimal amounts of phospholipids. Whether the discrepancy between our studies and those of others could be explained by differences in attention to the relative contribution in the assay of PS exposed on the surface of activated neutrophils remains to be determined. Although human neutrophils most probably do not synthesize TF, under pathophysiological conditions featuring aberrant profiles of circulating MPs, such as severe sepsis or unstable angina, the circulating neutrophils may acquire functional TF via binding interactions with the MPs. Indeed, when TF-rich MPs are exposed to neutrophils, they become instantly attached to these cells.82 However, there are several reports on the presence of TF in neutrophils in animals undergoing sepsis.123 Thus, infiltrating neutrophils in the liver of rabbits with acute obstructive cholangitis, one of the most fatal causes of sepsis, was immunohistochemically positive using specific anti-TF antibodies.124 In our opinion, rabbits may very well express endogenously synthesized TF in their neutrophils, and there may be species differences regarding TF expression in neutrophils.

PLATELETS AS A SOURCE OF TF The association of TF with platelets was first suggested in a study in which platelets were required for the rapid appearance of TF belonging to the blood, and platelet conjugates were identified as major sites of TF presentation in the blood.125 An extension of this study revealed TF stored within a-granula and the open canalicular system of the platelets.126 Following activation with either collagen or thrombin, TF activity was

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exposed on the platelet membrane. Recently, other reports have corroborated the notion of the presence of TF in platelets. Thus Siddiqui et al127 claimed that collagen-stimulated platelets expressed TF activity, although they did not test whether this TF activity could be blocked by anti-TF antibodies, whereas Camera et al128 claimed the presence of functionally active, membrane-associated, immunoreactive TF in activated platelets of healthy individuals and detectable TF mRNA in unstimulated platelets. Very recently, Butenas et al58 challenged the many reports of blood-borne TF. In this study they failed to detect any TF antigen on blood mononuclear cells in the absence of LPS stimulation, nor was the presence of any TF antigen detected on platelets, whether in unstimulated or LPS-stimulated blood or on washed and activated platelets. They concluded that there is an absence of measurable amounts of active TF in blood and plasma from healthy individuals (< 20 fM), in apparent contradiction to other studies indicating the presence of up to 37 pM amounts of TF in plasma.129 Using a highly sensitive and specific assay for TF activity, we also failed to detect measurable TF activity in collagen-activated platelets (Breimo and Østerud, unpublished data).

DISCREPANCIES The discrepancies emerging from the various reports on the localization of TF in cells/MPs mainly are due to variations in methods and the usage of antibodies that do not meet stringent criteria for monospecificity. The latter shortcoming is probably the main reason for the apparent detection of TF protein in platelets, and many assays for TF activity are somewhat substandard regarding sensitivity and specificity. Another point relevant to MPs is the role of the anticoagulant used when collecting blood intended for their isolation. Because the MPs may be bound to activated platelets through P-selectin accessible on the platelet surface and to monocytes/ neutrophils through exposed PSGL-1 in a calciumdependent manner, citrate as anticoagulant may not be sufficient to separate the MPs from the cells. This might lead to a distinct underestimation of the MP concentration in blood. How can blood of healthy subjects trigger TFinduced thrombus formation as described earlier in this article when the TF activity measured in monocytes, MPs, or soluble TF in plasma is very low and as such probably insufficient to cause thrombus formation? Bogdanov et al130 have pointed out that one has to distinguish between the TF activity measured under nonflow conditions in vitro and the potential of bloodborne TF for participating in thrombus formation during conditions of flow. Indeed, the role of shear force was documented by the finding that fresh platelets did not

stain for TF, whereas following perfusion with TFpositive cells under physiologically relevant flow condition, platelet aggregates were markedly positive for TF.109 The blood-borne TF may originate from encrypted TF present in monocytes, which upon interaction with activated platelets becomes decrypted. Another alternative is that, for example, collagen-activated platelets function as a ‘‘vacuum cleaner’’ by adsorbing monocyte-derived MPs as well as soluble TF. The question arises whether this event under shear stress may convert the inactive TF into an active molecule by hybridization with the activated platelet membrane in a fusion process. It is our opinion that addressing these questions may be the most important challenge in the field of TF research in the near future.

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