Identification and characterization of murine alternatively spliced tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that initiates coagulation and plays a critical role in regulating hemostasis and thrombosis. We have recently reported a naturally occurring, soluble form of human tissue factor (asTF) generated by alternative splicing. This splice variant has a novel C-terminus with no homology to that of the full-length TF (flTF), lacks a transmembrane domain, and is active in the presence of phospholipids. Mouse models offer unique opportunities to examine the relative importance of flTF and asTF in mediating thrombosis, the response to arterial injury, and ischemic damage. To that end, we have identified and characterized murine asTF (masTF). Like the human splice variant, masTF lacks a transmembrane domain and has a unique C-terminus. We have generated antibodies specific to masTF and murine flTF (mflTF) to examine the expression of both forms of TF. masTF antigen is widely and abundantly expressed, with a pattern similar to that of mflTF, in adult tissues, in experimentally induced thrombi, and during development. These studies demonstrate that masTF contributes to the pool of total TF and may thus play an important role in mediating TF-dependent processes.

Inhibition of tissue factor limits the growth of venous thrombus in the rabbit.

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.

Tissue factor on the loose.

The enzymatic complex of tissue factor (TF) and the blood coagulation factor VIIa is generally considered to be the initiator of coagulation. Coagulation that occurs at the site of luminal injury to an artery is, along with platelet deposition, the cause of arterial thrombosis, which is the leading cause of death in Western society. Under pathological conditions the intima, the neointima and the atherosclerotic plaque contain active TF. Therefore the initiation of thrombosis is believed to be due to TF present in the wall of the pathologically changed artery. This classical view of thrombosis has been challenged. In this article we review the evidence for the presence of TF activity in various tissues outside the vessel wall, in extracellular form, in encrypted form, and even in plasma. We found TF expression in a variety of cells in culture after growth factor or cytokine stimulation. This TF was often also present in the extracellular matrix, and in addition we found latent TF on the outside of unbroken smooth muscle cells. Freeze-thawing the cells or detergent lysis could activate this TF. We also found TF activity in native whole blood and in plasma. Inhibition of this circulating TF prevented formation of thrombi on collagen-coated glass slides in an ex vivo perfusion system. Furthermore, in a thrombosis model in which rat aorta was injured, TF was found on the intimal surface of the injured aorta. TF activity was measured in a flow chamber, and it was shown that all measurable activity was extracellular. We conclude that blood-borne TF plays a major role in thrombosis. Encryption of TF present in circulation could be a mechanism that prevents thrombosis. Alternatively, circulating TF may be active but below the threshold required for the initiation of blood coagulation.

Circulating tissue factor and thrombosis.

Tissue factor (TF) on circulating microparticles has recently received much attention as a factor in myocardial infarction. We have developed systems by which we have been able to investigate the thrombogenic potential of blood-borne TF. Thrombi develop when native human blood is passed over either collagen-coated glass slides or over pig arterial media. These thrombi immunostain for TF even when the substrate contains none. Moreover, we have demonstrated that the deposited TF is active because the thrombi contain fibrin; fibrin deposition and thrombotic mass are both inhibited by the inclusion of a potent TF-inhibitor in the perfusions. We have also shown that leukocyte-derived particles attach to platelets in a reaction mediated by adhesion proteins.

Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor.

We describe thrombogenic tissue factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF(+) particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF(+) leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.

Release of active tissue factor by human arterial smooth muscle cells.

Tissue factor (TF), the initiator of coagulation, is thought to function predominantly at the cell surface. Recent data have suggested that active TF is present extracellularly in atherosclerotic plaques, the arterial wall, and the blood. This study was conducted to determine whether smooth muscle cells (SMCs), a major source of arterial TF, could generate extracellular TF. Active TF accumulated in the medium of cultured human SMCs, representing approximately 10% of that measured in the underlying cells at 24 hours. Platelet-derived growth factor, phorbol ester, and tumor necrosis factor-alpha caused approximately 3-fold increases in TF activity in the medium. Release of TF into the medium was dependent on the presence of the TF transmembrane domain but not the cytoplasmic domain. Antibodies to TF precipitated most of the activity from the culture medium, whereas antibodies to the beta(1)-integrin subunit precipitated approximately 33% of the activity. Treatment with detergent or phosphatidylserine:phosphatidylcholine did not increase activity, suggesting that all TF released by SMCs was in the appropriate lipid milieu and not encrypted. Western blotting showed that the medium contained full-length TF protein. Fluorescent cytometry showed that extracellular TF was present largely in particles < or =200 nm, which had a density of 1.10 g/mL. We hypothesize that active extracellular TF found in the injured arterial wall and atherosclerotic plaques derives, in part, from SMC microparticles.

A different view of thrombosis.

In this paper we discuss some of the physical difficulties associated with the classical view of thrombosis and conclude that there are problems associated with these concepts. We propose an alternative model based on our published observations that, during ex-vivo thrombus formation, the platelets, which normally do not stain for tissue factor, become tissue factor positive. These hybrid species are capable not only of propagating coagulation, but also of initiating it. Thus thrombi can grow without evoking long-range diffusion -- a very slow and inefficient mechanism, particularly in a flowing system.

Intimal tissue factor activity is released from the arterial wall after injury.

Tissue factor (TF), the initiator of coagulation, has been implicated as a critical mediator of arterial thrombosis. Previous studies have demonstrated that TF is rapidly induced in the normal rodent arterial wall by balloon injury, but is not associated with fibrin deposition. A second injury, however, performed 10-14 days after the first, is followed by small platelet-fibrin microthrombi. This study was undertaken to better localize active TF in balloon-injured rat arteries and to explore possible mechanisms underlying the apparent discrepancy between injury-induced TF expression and the lack of large platelet-fibrin thrombi. By immunohistochemistry, TF antigen was first detected in the media 24 h after injury to rat aortas, and subsequently accumulated in the neointima. Using an ex vivo flow chamber, no TF activity (Factor Xa generation) was found on the luminal surface of normal or injured aortas. Wiping the luminal surface with a cotton swab exposed TF activity in all vessels; levels were increased approximately 3-fold in arteries containing a neointima. The exposed TF activity was rapidly washed into the perfusate, rendering the luminal surface inactive. The loss of luminal TF into the circulation may attenuate thrombosis at sites of arterial injury.

Tissue factor, the blood, and the arterial wall.

Thrombogenic tissue factor (TF) on cell-derived microparticles is present in the circulating blood of patients with acute coronary syndromes. Recently, we reported that leukocytes transfer TF-positive particles to platelet thrombi, making them capable of triggering and propagating thrombus growth. This observation changes the original dogma that vessel-wall injury and exposure of tissue factor within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF-positive leukocyte particles is dependent on the interaction of CD15 and TF with platelet thrombi. The inhibition of TF transfer and TF activity suggests a novel therapeutic approach to the prevention of thrombosis that may prove to be effective in disorders associated with increased blood TF.

Regulation of the procoagulant response to arterial injury.

The last few years have provided increasing evidence to support a major role for TF in the initiation and propagation of thrombosis after acute arterial injury. Although thrombotic occlusion occurs in a small minority of patients undergoing acute coronary interventions or bypass surgery, mural thrombi are likely to be present in almost all cases. These thrombi may stimulate SMC and promote the development of intimal hyperplasia and luminal narrowing. The use of inhibitors of TF and factor VIIa, therefore, may not only be valuable for inhibiting thrombus formation associated with acute arterial interventions, but may also have benefit in attenuating intimal hyperplasia. Although this paper focuses on the role of TF in establishing a procoagulant state after arterial injury, the fibrinolytic system undoubtedly plays a role in balancing the effects of increased TF production in the arterial wall. This is underscored by the success of activators of fibrinolysis (tissue plasminogen activator, streptokinase, urokinase) in revascularization in the setting of acute myocardial infarction and is reviewed elsewhere. Likewise, local regulation of TFPI in the atherosclerotic plaque and injured vessel wall may be important in attenuating the effects of increased TF synthesis and accumulation. It has been assumed that the primary source of active TF after arterial injury is either SMC or invading macrophages and that active TF is anchored to the surface of these cells. Recent data have suggested that the majority of cell-associated TF is either encrypted on the cell surface or present in an intracellular pool. Arterial injury may, therefore, involve the de-encryption of surface TF or the release of intracellular TF. In addition, active vascular TF may be present in microparticles that are not anchored to the arterial wall and may be washed into the circulation. The procoagulant state may be further accentuated by the accumulation of bloodborne TF at sites of arterial injury and in developing thrombi. This TF is likely to arise from circulating leukocytes, including neutrophils and monocytes. These studies suggest that the cellular processing of TF may be an important target for inhibiting thrombotic complications associated with arterial injury and acute coronary events.

Blood-borne tissue factor: another view of thrombosis.

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused approximately 70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VIIai, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor Xa formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.

Cooperation between VEGF and TNF-alpha is necessary for exposure of active tissue factor on the surface of human endothelial cells.

This study was undertaken to characterize tissue factor (TF) induction, localization, and functional activity in cultured human umbilical vein endothelial cells (HUVECs) exposed to recombinant vascular endothelial growth factor (rVEGF) and recombinant tumor necrosis factor-alpha (rTNF-alpha). rVEGF (1 nmol/L) and rTNF-alpha (500 U/mL) synergistically increased TF mRNA, protein, and total activity, as measured in cell lysates. To examine surface TF expression, living cells were treated with antibody to TF and examined microscopically. Almost no staining was seen in control cells or cells treated with a single agent. In contrast, cells treated with both agonists showed intense membrane staining with surface patches, appearing as buds by confocal microscopy. To determine surface TF activity, studies were performed using a parallel-plate flow chamber, which allows detection of factor Xa generation on living cells. rVEGF and rTNF-alpha induced little surface TF activity (0.032+/-0.008 and 0.014+/-0.008 fmol/cm2, respectively). In combination, they significantly increased TF expression on the cell surface (0.429+/-0.094 fmol/cm2, P<0.05). These data indicate that the synergistic effect of rVEGF and rTNF-alpha is necessary to generate functional TF on the surface of endothelial cells. The requirement for multiple agonists to expose active TF may serve to protect endothelial cells from acting as a procoagulant surface, even under conditions of cell perturbation.

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor.

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.

Some thoughts about localization and expression of tissue factor.

It is likely that tissue factor (TF) evolved as a haemostatic protein and, as such, it is highly concentrated in vascular tissue. Most cell surface TF is latent and simple exposure of the cell surface to circulating procoagulant proteins is not sufficient to trigger coagulation. Recently, it has been shown that an intracellular pool of TF accumulates after stimulation of vascular smooth muscle cells with growth factors. We have estimated that 20% of cellular TF is available on the surface, 30% is intracellular and 50% is latent. Since the bulk of cell surface TF is latent, staining vessels for TF does not accurately reflect their haemostatic and thrombogenic potential. It has long been thought that, in vivo, initiation of haemostasis requires only disruption of the vascular wall. We have detected vesicular TF in arterial sections raising the possibility that this pool of TF initiates thrombosis and possibly haemostasis. Much progress has been made in investigating the role and mode of action of TF, but fundamental questions remain to be answered.

Hirudin reduces tissue factor expression in neointima after balloon injury in rabbit femoral and porcine coronary arteries.

BACKGROUND: Tissue factor (TF) is a transmembrane glycoprotein that, after binding to factor VII/VIIa, initiates the extrinsic coagulation pathway, resulting in thrombin generation and its sequelae. Thrombin has been shown to induce TF mRNA in endothelium, monocytes, and smooth muscle cells, further perpetuating the thrombogenic cycle. This study was designed to determine the effect of specific inhibition of thrombin by recombinant hirudin (r-hirudin) on TF distribution after balloon angioplasty in the cholesterol-fed rabbit femoral artery and porcine coronary artery models. METHODS AND RESULTS: Thirty-five femoral arteries from 32 cholesterol-fed New Zealand White rabbits and 84 coronary arteries from 55 Yorkshire-Albino swine were studied by use of a recently developed in situ method of TF localization based on digoxigenin labeling of recombinant factor VIIa (Dig-VIIa), with correlative studies of TF immunoreactivity by use of anti-rabbit (AP-1) or anti-human (sTF) antibodies. At sites of balloon angioplasty in rabbit femoral or pig coronary arteries (double or single injury), TF-antibody and Dig-VIIa staining were noted in association with endothelial cells, smooth muscle cells, and foam cells and within the fibrous tissue matrix primarily of the adventitia and neointima. Staining was significantly greater after balloon angioplasty than in vessels that had not undergone angioplasty but was similar after single and double balloon injury. Animals treated with r-hirudin (rabbits, 1 mg/kg bolus plus 2-hour infusion; pigs, 1 mg/kg bolus plus 0.7 mg x kg(-1) x d(-1) infusion for 14 days with implantable pump) had diminished TF-antibody and Dig-VIIa staining 28 days after balloon angioplasty compared with controls (bolus heparin only). This effect was more prominent on the neointima and was more striking in the porcine than the rabbit model. CONCLUSIONS: TF expression, persistent 1 month after balloon angioplasty in rabbit femoral arteries and porcine coronary arteries, is attenuated by specific thrombin inhibition with hirudin. These results suggest that thrombin inhibition, in addition to its effect on acute thrombus formation and its effect on luminal narrowing by plaque in experimental animals, may result in a prolonged reduction in thrombogenicity of the restenotic plaque through this effect on TF expression.

Factor Xa generation at the surface of cultured rat vascular smooth muscle cells in an in vitro flow system.

The purpose of the present investigation was to explore the effects of well-defined flow conditions on the activity of tissue factor (TF) expressed on the surface of cultured rat vascular smooth muscle cells. Cells were cultured to confluence on Permanox brand slides and stimulated to express TF by a 90 min incubation with fresh growth medium containing 10 percent calf serum. The stimulated cells were then placed in a parallel plate flow chamber and perfused with Hank's Balanced Salt Solution containing factor VIIa, factor X (FX), and calcium. The chamber effluent was collected and assayed for factor Xa (FXa) and the steady-state flux of FXa was calculated. The flux values were 68.73, 94.81, 139.75, 138.19, 316.82, and 592.92 fmole/min/cm2 at wall shear rates of 10, 20, 40, 80, 320, and 1280 s-1, respectively. The FXa flux depended on the wall shear rate to a greater degree than predicted by classical mass transport theory. The flux at each shear rate was three to five times less than that calculated according to the Leveque solution. These features of the experimental data imply nonclassical behavior, which may partially result from a direct effect of flow on the cell layer.

Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.