Plasma tissue factor and tissue factor pathway inhibitor levels in patients with disseminated intravascular coagulation

We measured the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with disseminated intravascular coagulation (DIC) to examine the relationship between TFPI and vascular endothelial cell injury. Plasma TF (273 ± 90 pg/ml) and TFPI (252 ± 125 ng/ml) levels were significantly increased in patients with DIC compared with non-DIC patients. Plasma TF antigen level was significantly increased in pre-DIC patients (285 ± 85 pg/ml), while the plasma TFPI level (152 ± 54 ng/ml) was not markedly increased in such a state. The plasma TF/TFPI ratio was high in the pre-DIC patients (2.10 ± 0.90), and low in the DIC patients (1.40 ± 0.87) and healthy volunteers (0.84 ± 0.26). There was no significant difference between the DIC patients with a good outcome and those with a poor outcome in terms of plasma TF levels, although the plasma TFPI level in the DIC patients with a good outcome (289 ± 133 ng/ml) was significantly higher than that in those with a poor outcome (187 ± 75 ng/ml). During the clinical course of DIC, plasma TF antigen was increased first, and an increase of the plasma TFPI level followed the increase in plasma TF level. These findings suggest that plasma TFPI is released from vascular endothelial cells and it may reflect vascular endothelial cell injury. It is conceivable that TF and TFPI may play an important role in the onset of DIC. © 1996 Wiley-Liss, Inc.     

Plasma tissue factor and tissue factor pathway inhibitor levels in patients with disseminated intravascular coagulation

We measured the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with disseminated intravascular coagulation (DIC) to examine the relationship between TFPI and vascular endothelial cell injury. Plasma TF (273 ± 90 pg/ml) and TFPI (252 ± 125 ng/ml) levels were significantly increased in patients with DIC compared with non-DIC patients. Plasma TF antigen level was significantly increased in pre-DIC patients (285 ± 85 pg/ml), while the plasma TFPI level (152 ± 54 ng/ml) was not markedly increased in such a state. The plasma TF/TFPI ratio was high in the pre-DIC patients (2.10 ± 0.90), and low in the DIC patients (1.40 ± 0.87) and healthy volunteers (0.84 ± 0.26). There was no significant difference between the DIC patients with a good outcome and those with a poor outcome in terms of plasma TF levels, although the plasma TFPI level in the DIC patients with a good outcome (289 ± 133 ng/ml) was significantly higher than those with a poor outcome (187 ± 75 ng/ml). During the clinical course of DIC, plasma TF antigen was increased first, and an increase of the plasma TFPI level followed the increase in plasma TF level. These findings suggest that plasma TFPI is released from vascular endothelial cells and it may reflect vascular endothelial cell injury. It is conceivable that TF and TFPI may play an important role in the onset of DIC. Am. J. Hematol. 55:169–174, 1997. © 1997 Wiley-Liss, Inc.     

Orchestration of coagulation protease signaling by tissue factor

Protease-activated receptors (PARs) are vascular sensors for signaling of the trypsinlike coagulation serine proteases that play key roles in cardiovascular medicine. In the initiation phase of coagulation, tissue factor (TF) orchestrates the assembly of VIIa with substrate X, forming a ternary complex in which product Xa is generated. The resulting TF-VIIa-Xa complex is an efficient activator of PAR1 and PAR2. TF initiation of the coagulation cascade is thus intimately linked to inflammatory cell signaling. Inflammation is an increasingly appreciated component of the vulnerable atherosclerotic plaque. Targeting inflammatory cell signaling events of the coagulation system may become an important aspect of efforts to improve antithrombotic therapy.     

Initiation of coagulation by tissue factor carriers in blood

Fibrin can be generated in blood vessels in the absence of substantial damage to the vessel wall. According to novel observations, fibrin formation within the vessel lumen could be initiated by intravascular (blood borne) tissue factor (TF), a central starter protein of blood coagulation. We have recently detected TF in platelets, principally allowing coagulation to be initiated within the developing thrombus. While TF is stored in intraplatelet compartments under resting conditions, it is rapidly translocated to the cell surface in response to platelet activation, accumulating on filopodia. Platelet TF might be acquired from extracellular sources via transfer of TF positive microparticles and/or, potentially, be generated through translational mechanisms. Exocytotic microparticles, regular blood components, share the ability of the activated platelets to coexpose TF and phosphatidylserine, allowing the assembly of the entire coagulation system on a single membrane surface. Nonetheless, the procoagulant activity of the microparticles, when present alone, is limited. However, the vesicular TF might be uncovered by platelet-microparticle interactions. Thereby, the recruitment of platelets and microparticles to the site of vascular injury could synergistically trigger fibrin generation. In summary, by utilizing differential pathways, activated platelets are mandatory for the TF-mediated coagulation start in blood.     

The tissue factor pathway in disseminated intravascular coagulation.

In most instances, tissue factor (TF) exposed to the circulation is the sole culprit underlying the initiation of disseminated intravascular coagulation (DIC), although notable exceptions because of a more direct activation of the coagulation system, by snake venoms, for example, do occur. Peripheral monocytes and subendothelial structures are the potential sources of such TF; in the former, TF emerges on the cell surface on synthesis induction and in the latter it becomes available subsequent to permeability changes or damage to the endothelium. Subendothelial TF is constitutively present in fibroblasts, pericytes, and macrophages and at a higher than normal level in tumor-associated macrophages. This scenario of coagulation activation probably describes the principal events underlying emerging acute DIC states under pathophysiological conditions such as abruptio placentae, septic abortion, amniotic fluid embolization, and pregnancy toxemia. Under disease conditions associated with DIC, the continuous exposure to excess TF typically exhausts the available tissue factor pathway inhibitor (TFPI), leading to rampant thrombin generation, persistent feedback activation of factor XI (FXI) by the generated thrombin, and hence virtually uncheckable ongoing fibrin generation (DIC). Recently, it was shown that patients subject to meningococcal sepsis had comparatively large amounts of mainly monocyte-derived circulating TF-containing microparticles. Because phosphatidylserine (PS) is exposed on such particles, in addition to TF, they probably contribute crucially to DIC during meningococcal sepsis. Although endothelial cells (EC) have been shown to express large amounts of TF in vitro, this observation hardly relates to the situation in vivo, where, in contrast, synthesis and exposure of EC TF is very limited and not likely to be of any significance in emerging and ongoing DIC.     

Signaling of the tissue factor coagulation pathway in angiogenesis and cancer

Activation of coagulation precedes or coincides with angiogenesis in wound healing and postischemic tissue regeneration. Advanced cancer is associated with a hypercoagulable state, and tissue factor expression by cancer cells has received widespread attention because of its significant contribution to the pathogenesis of cancer progression and metastasis. Our recent work demonstrates that tissue factor-mediated cellular signaling is relevant to cancer angiogenesis. Here we review the molecular mechanisms of tissue factor pathways in angiogenesis and tumorigenesis with emphasis on the intriguing role for tissue factor cytoplasmic domain signaling.     

Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor

Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF. VIIa. Xa. TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.     

Disulfide isomerization switches tissue factor from coagulation to cell signaling

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF.VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys186-Cys209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys186 retains TF.VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF.PAR2 complex and TF.VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF.VIIa signaling can be specifically inhibited with minimal effects on coagulation.     

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)--factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF--VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF--VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF--VIIa--X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF--VIIa--Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF--VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.     

Activation of factor VII bound to tissue factor: a key early step in the tissue factor pathway of blood coagulation.

Whether the factor VII/tissue factor complex that forms in tissue factor-dependent blood coagulation must be activated to factor VIIa/tissue factor before it can activate its substrates, factor X and factor IX, has been a difficult question to answer because the substrates, once activated, back-activate factor VII. Our earlier studies suggested that human factor VII/tissue factor cannot activate factor IX. Studies have now been extended to the activation of factor X. Reaction mixtures were made with purified factor VII, X, and tissue factor; in some experiments antithrombin III and heparin were added to prevent back-activation of factor VII. Factor X was activated at similar rates in reaction mixtures containing either factor VII or factor VIIa after an initial 30-sec lag with factor VII. In reaction mixtures with factor VII a linear activation of factor X was established several minutes before cleavage of 125I-labeled factor VII to the two-chain activated molecule was demonstrable on gel profiles. Adding antithrombin III and heparin blocked activation of factor X by factor VII/tissue factor but not by factor VIIa/tissue factor. When the antithrombin III and heparin were added 1 min after the other reagents, factor VII/tissue factor activation of factor X was not blocked. These data suggest that factor VII/tissue factor cannot activate measurable amounts of factor X over several minutes. Overall, our results support the hypothesis that a rapid preferential activation of factor VII bound to tissue factor by trace amounts of factor Xa is a key early step in tissue factor-dependent blood coagulation.     

Magnesium and manganese ions accelerate tissue factor-induced coagulation independently of factor IX.

The purpose of the present study was to assess the effect of magnesium and manganese ions on tissue factor (TF)-induced coagulation and the possible role of factor IX therein. When magnesium chloride or manganese chloride were added in low concentrations to normal human plasma, the human (recombinant) TF-induced coagulation time was shortened. At higher concentrations, magnesium and manganese prolonged the TF-induced coagulation time. Maximum shortening of the coagulation time was obtained at a concentration of 0.5 mmol/l Mn or 2 mmol/l Mg in plasma. Shortening of the TF-induced coagulation time by magnesium and manganese was also observed in factor IX-deficient plasma. A comparison was made between TF preparations from human, rabbit, and bovine brain. The accelerating effect of magnesium was greater with human than with rabbit brain TF. Using bovine brain TF, the clotting time was not shortened by magnesium. Activated factor X-induced coagulation of normal plasma was not accelerated by magnesium. From these experiments, it is inferred that activation of factor X by factor VII-TF can be accelerated by magnesium and manganese ions independently of factor IX.     

Effects of hyperglycemia and hyperinsulinemia on the tissue factor pathway of blood coagulation

Tissue factor (TF) is the primary initiator of blood coagulation. Circulating TF procoagulant activity (TF-PCA) is associated with blood cells and microparticles and is elevated in patients with type 2 diabetes mellitus. Combined hyperinsulinemia and hyperglycemia and to a lesser degree selective hyperinsulinemia for 24 hours in healthy volunteers increased circulating TF-PCA, monocyte TF surface expression and mRNA, plasma thrombin generation, and coagulation factors VII and VIII activities, suggesting that the coagulation system had been activated. In addition, platelet CD40L and platelet-monocyte aggregates increased, indicating platelet activation. Somatostatin abolished these changes. We conclude that hyperinsulinemia, but particularly the combination of hyperinsulinemia and hyperglycemia, creates a prothrombotic state and may, in addition, be proinflammatory and proatherogenic by virtue of the actions of CD40L and TF.     

Recombinant nematode anticoagulant protein c2 and other inhibitors targeting blood coagulation factor VIIa/tissue factor

Originally isolated from a haematophagous hookworm, recombinant nematode anticoagulant protein c2 (rNAPc2) is an 85-amino acid protein with potent anticoagulant properties. Unlike conventional anticoagulants that attenuate blood coagulation via inhibition of thrombin or activated factor X (FXa) at the downstream portion of the cascade, rNAPc2 is a potent inhibitor of the activated factor VII/tissue factor complex (FVIIa/TF), the key physiological initiator of blood coagulation. Its mechanism of action requires prerequisite binding to circulating FXa or zymogen factor X (FX) to form a binary complex prior to its interaction and inhibition of membrane-bound FVIIa/TF. The binding of rNAPc2 to FX results in an elimination half-life of longer than 50 h following either subcutaneous or intravenous administration. Recombinant NAPc2, like other inhibitors of FVIIa/TF including tissue factor pathway inhibitor (TFPI) and active site-blocked FVIIa (ASIS, FFR-rFVIIa or FVIIai), may have a promising role in the prevention and treatment of venous and arterial thrombosis, as well as potential efficacy in the management of disseminated intravascular coagulopathies because of their potent and selective inhibition of FVIIa/TF.     

Elevated plasma tissue factor antigen level in patients with disseminated intravascular coagulation

The plasma tissue factor (TF) antigen level was measured in patients with disseminated intravascular coagulation (DIC). The plasma TF antigen was detected In normal volunteers, and it was significantly higher in DIC patients than in non-DiC patients. However, in some patients with DiC, the plasma TF antigen level was within the normal range. The plasma TF antigen level in patients with DIC significantly decreased after therapy, but it was not correlated with organ failure or outcome. The plasma TF antigen level in patients with DIC was not correlated with other hemostatic markers. The plasma TF antigen level tended to be higher in DIC patients with nonlymphoid leukemia than in those with lymphoid tumor. TF might be implicated in the occurrence and progression of DIC. © 1994 Wiley-Liss, Inc.     

Initiation of the tissue factor pathway of coagulation in the presence of heparin: control by antithrombin III and tissue factor pathway inhibitor

Activation of factor X by both the unactivated tissue factor:factor VII complex (TF:VII) and the activated tissue factor:factor VIIa complex (TF:VIIa) has been studied in the presence of tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and heparin. At near-plasma concentrations of TFPI, ATIII, and factor X, factor X activation that occurs in response to TF:VII is essentially abolished in the presence of heparin (0.5 micromol/L). This effect requires both inhibitors, acting on different targets: (1) ATIII, which in the presence of heparin blocks the activation of TF:VII, and (2) TFPI, which inhibits the TF:VIIa that is generated. In the absence of ATIII, TFPI alone with heparin reduces but does not abolish factor X activation. Conversely, in the absence of TFPI, ATIII + heparin reduces but does not abolish TF:VIIa generation and allows continuing activation of factor X. These results indicated that when the unactivated TF:VII complex is the initiating stimulus, heparin-dependent reduction in the rate and extent of factor X activation requires both ATIII and TFPI. In contrast, if TF:VIIa is used to initiate activation, only TFPI is involved in its regulation.     

Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity

The integrity of the γ-carboxylic glutamic acid (GLA) residues of coagulation factor VII are thought to be essential for both the interaction of factor VII with its cell-surface lipoprotein receptor tissue factor and for the activated protein to manifest its serine protease activity. During the course of transiently expressing recombinant human factor VII in monkey COS cells it was noted that the factor VII synthesized in the absence of added vitamin K had < 20% of expected procoagulant activity yet retained 65% of its binding activity to recombinant human tissue factor. Similar results were obtained when vitamin K was omitted from human 293 cell cultures permanently expressing recombinant factor VII. In contrast, both transient and permanent expression of factor VII in human 293 cell cultures containing physiological concentrations of vitamin K resulted in the synthesis of fully functional factor VII. Furthermore, factor VII in plasma samples from 24 patients undergoing warfarin therapy bound quantitatively to tissue factor whereas factor VII procoagulant activity averaged 65% of normal. Thus, data from both in vitro and in vivo situations indicated that factor VII molecules with suboptimal GLA content retained most of their ability to bind tissue factor but exhibited reduced procoagulant activity.