Dose-dependent release of endogenous tissue factor pathway inhibitor by different low molecular weight heparins.

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous and subcutaneous injections of heparins, and may thus contribute to the antithrombotic effect of heparins. A previous study suggested different abilities of various low molecular weight heparins (LMWH) to release endogenous TFPI, but the dose-response relationship was not determined. In the present study, the dose-response relationship for escalating doses of two LMWHs, dalteparin and enoxaparin, on the release of endogenous TFPI was investigated. Six healthy male participants were given 50, 100 and 200 U/kg dalteparin and 0.5, 1.0 and 2.0 mg/kg enoxaparin as a single subcutaneous injection. The study was a randomized, cross-over design with a 1-week wash-out period between each injection. Peak free TFPI antigen and TFPI activity were detected after only 1 h, whereas anti-activated factor X (anti-FXa) and anti-activated factor II (anti-FIIa) activities were detected after 2-6 h. Putative therapeutic equivalent doses of dalteparin and enoxaparin gave similar release of endogenous TFPI, but dissimilar effects on anti-FXa and anti-FIIa activities.     

Physiological function of tissue factor pathway inhibitor and interaction with heparins

Tissue factor pathway inhibitor (TFPI) is now recognized as a major physiological anticoagulant. Its main role is to modulate factor VIIa/tissue factor catalytic activity. Another important role is to potentiate the effect of heparins. TFPI is released from the vascular endothelium after injection of either unfractionated heparin (UFH) or low-molecular-weight heparins (LMWHs), which may then provide high concentrations of TFPI at sites of tissue damage and ongoing thrombosis. In dilute prothrombin-time-based assays, released TFPI contributes approximately one-third to the anticoagulant effect of heparin, the remaining being accounted for by antithrombin. Released TFPI, but not plasma TFPI, contains the basic carboxy-terminal tail which is important for the anticoagulant effect. UFH and LMWH exert differential effects on intravascular TFPI. UFH, but not LMWH, given in therapeutic doses, is associated with a progressive depletion of TFPI, which is associated with a strong rebound activation of coagulation after cessation of treatment. Such depletion may explain the apparent superior efficacy of LMWH observed in clinical trials.     

Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins

Unfractionated heparin (UFH) has procoagulant activity in antithrombin/heparin cofactor II (HCII)‐depleted plasma. UFH prevents tissue factor pathway inhibitor alpha (TFPIα) from inhibiting the procoagulant enzyme complex, prothrombinase, providing a possible mechanism for its procoagulant activity. The procoagulant potential of UFH and various low molecular weight heparins (LMWHs) were characterized for TFPIα dependence, using thrombin generation assays performed with antithrombin/HCII‐depleted plasma. UFH, the LMWHs enoxaparin and dalteparin, and the low anticoagulant LMWH 2‐O, 3‐O desulphated heparin (ODSH) all promoted thrombin generation, but fondaparinux did not, and this activity was blocked by a TFPIα antibody. UFH, enoxaparin, and dalteparin were anticoagulant in reactions containing 1–2% normal plasma. In prothrombinase activity assays, UFH, enoxaparin, dalteparin and ODSH blocked prothrombinase inhibition by TFPIα, while again fondaparinux did not. In both the plasma and purified assays, LMWHs displayed greater procoagulant potential than UFH, even when normalized to saccharide concentration. These biochemical data reveal that UFH and LMWHs, but not fondaparinux, block prothrombinase inhibition by TFPIα, thereby producing their paradoxical procoagulant activity observed in the absence of antithrombin/HCII. The findings may help to understand the complex pathophysiology and treatment of patients that are simultaneously bleeding and clotting, such as those with disseminated intravascular coagulation.     

The present status of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is the factor Xa-dependent inhibitor of the factor VIIa/tissue factor complex. The plasma concentration of this 276 amino acid, 40 kDa glycoprotein is normally about 100 ng/ml. There are three intravascular pools of TFPI: 50-90% is on the endothelium, 10-50% is in plasma and less than 2.5% is in platelets. The TFPI in plasma is mainly associated with lipoproteins-only about 5% is free TFPI. The lipoprotein-associated TFPI seems to be of less anticoagulant effect than the free TFPI. Both unfractionated heparin, low-molecular-weight heparins and pentosan polysulphate induce release of TFPI after intravenous injection, whereas dermatan sulphate does not. The interactions with TFPI account for a considerable amount of the anticoagulant effect of heparin. Studies have shown increased TFPI levels in plasma from patients with advanced malignancy and in subjects with fatal DIC or septicaemia. The reason for this is unknown. For measuring the anticoagulant activity of TFPI in plasma, end-point or antigen assays may be less useful than the clotting assay with dilute tissue factor. Animal studies indicate that the main physiological role of TFPI is the inhibition of small amounts of tissue factor. TFPI is probably essential for a normal haemostatic balance.     

Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor

Tissue factor (TF) plays an important role in hemostasis, inflammation, angiogenesis, and the pathophysiology of atherosclerosis and cancer. In this article we uncover a mechanism in which protein S, which is well known as the cofactor of activated protein C, specifically inhibits TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The stimulatory effect of protein S on FXa inhibition by TFPI is caused by a 10-fold reduction of the K(i) of the FXa/TFPI complex, which decreased from 4.4 nM in the absence of protein S to 0.5 nM in the presence of protein S. This decrease in K(i) not only results in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it also brings the TFPI concentration necessary for effective FXa inhibition well within range of the concentration of TFPI in plasma. This mechanism changes the concept of regulation of TF-induced thrombin formation in plasma and demonstrates that protein S and TFPI act in concert in the inhibition of TF activity. Our data suggest that protein S deficiency not only increases the risk of thrombosis by impairing the protein C system but also by reducing the ability of TFPI to down-regulate the extrinsic coagulation pathway.     

Cocaine unbalances endothelial tissue factor and tissue factor pathway inhibitor expression

Cocaine consumption can lead to myocardial infarction. Tissue factor (TF) has been implicated in acute coronary syndromes, and the balance of TF and tissue factor pathway inhibitor (TFPI) determines initiation of thrombus formation. This study was designed to investigate the effect of cocaine on endothelial TF and TFPI expression. Cocaine (10(-8)-10(-5) mol/l) increased thrombin-induced TF expression by 24% at 10(-7) mol/l (P < 0.001) without affecting basal TF expression. In contrast, cocaine reduced endothelial TFPI expression by 47% at 10(-7) mol/l (P < 0.01). Moreover, thrombin impaired endothelial TFPI expression, and cocaine (10(-8) mol/l) further reduced TFPI expression by 33% as compared to thrombin (P < 0.02). These effects occur at cocaine concentrations usually present in plasma of consumers. Given the importance of TF in the pathogenesis of acute coronary syndromes, TF induction in conjunction with TFPI suppression may be relevant for the increased frequency of myocardial infarction observed in cocaine consumers.     

Preeclampsia: the role of tissue factor and tissue factor pathway inhibitor

Preeclampsia (PE) is a multi-system disorder of human pregnancy, whose etiology remains poorly understood. Preeclamptic women are known to have an increased hypercoagulable state that result in excess fibrin deposition in several organs, which compromises their function. Tissue factor (TF) is the main physiological initiator of blood coagulation and its activity is regulated by a specific inhibitor known as Tissue factor pathway inhibitor (TFPI). Based on the important role of TF and TFPI in hemostasis, we hypothesize that their levels may change in the severe PE contributing to exacerbate hypercoagulable state. Some studies have assessed the balance between TF and TFPI in preeclamptic women, but results are inconsistent. Therefore, the aim of this study was to examine these inconsistencies and to assess TF and TFPI plasma levels in three groups of age matched women; pregnant with severe PE (n = 60), normotensive pregnant (n = 50) and normotensive non-pregnant women (n = 50). There was not significantly different among the three groups for TF plasma levels; severe PE women: 338.4 pg/mL (248.1-457.6), normotensive pregnant women: 301.5 pg/mL (216.4-442.9) and normotensive non-pregnant women 393 pg/mL (310.3-522.9). TFPI plasma levels were higher in severe PE comparing to normotensive pregnant women and normotensive non-pregnant women, 115.8 ng/mL (75-149.8); 80.3 ng/mL (59.6-99.7) and 74.5 ng/mL (47.1-98.0), respectively No difference was found between normotensive pregnant women and normotensive non-pregnant women. As for gestational age, a significant difference in TFPI levels was found between severe PE and normotensive pregnant women up to the 33rd week of pregnancy (p = 0.001), and severe PE and non-pregnant women up to the 34th (p = 0.01). In summary, our results indicated that TF plasma levels did not vary in the studied groups, while TFPI plasma levels were significantly increased in severe PE compared to normotensive pregnant and normotensive non-pregnant women. So, our data do not explain the exacerbated hypercoagulability state observed in severe PE. Further studies evaluating genes expression, TF activity and antigen, total and free TFPI and TFPI-2, both in plasma and obstetric tissues, throughout the pregnancy in PE (mild and severe forms) are required.     

The haemostatic role of tissue factor pathway inhibitor

Under normal conditions the blood circulates freely within the confines of the vascular system, carrying oxygen, nutrients, and hormonal information around the body and removing metabolic waste. If blood gains access to extravascular sites, or the vasculature becomes pathologically challenged, hemostasis may be activated. This process is finely regulated by positive and negative feedback loops that modulate fibrin clot formation. Blood coagulation revolves around the activation and assembly of the components of the prothrombinase complex, which converts the inactive zymogen, prothrombin, into its active form, thrombin. This serine protease catalyzes the conversion of fibrinogen to fibrin, the structural scaffold that stabilizes platelet aggregates at sites of vascular injury. The extent of the hemostatic response is controlled by the action of inhibitory pathways, which ensure that thrombin activity and the spread of the hemostatic plug is limited to the site of vessel damage. This review article focuses on the major physiological regulator of tissue factor-induced coagulation, tissue factor pathway inhibitor, its expression, anticoagulant function, and its role in normal hemostasis.     

Fluid flow induces upregulation of synthesis and release of tissue factor pathway inhibitor in vitro

Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm(2) (minimal flow), 4.1 dyne/cm(2) (venous flow), and 19 dyne/cm(2) (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm(2) induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.     

The effect of leukocyte elastase on tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa-TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.     

Study of factor VII, tissue factor pathway inhibitor and monocyte tissue factor in noninsulin-dependent diabetes mellitus.

Changes in plasma tissue factor (TF)-activated factor VII (FVIIa) and plasma tissue factor pathway inhibitor (TFPI) in type II diabetes mellitus are assessed, vascular complicated and noncomplicated patients compared, and whether these novel hemostatic activity markers predict vascular complications in diabetic patients, improving risk assessment, is determined. Fifty type II diabetic patients and 20 healthy controls (age, sex and body mass matched) underwent medical history and examination, fasting plasma glucose level, glycosylated hemoglobin (HbA1c), lipid profile, hemostatic parameters, plasma TF activity, and TFPI and TF expression on blood monocytes. Mean TF, TF activity, TFPI, and FVIIa significantly increased among hyperlipidemic compared with normolipidemic diabetic patients, and normolipidemic diabetic patients compared with controls. Mean percentage TF-positive monocytes with and without lipopolysaccharide, plasma TF activity, TFPI and FVIIa were significantly higher among complicated than noncomplicated diabetic patients. Mean percentage TF-positive monocytes without and with lipopolysaccharide, plasma TF activity, plasma TFPI and FVIIa were higher among diabetic patients with macrovascular compared with microvascular complications. High significant correlation occurred between HbA1c, triglycerides and percentage TF-positive monocytes with and without lipopolysaccharide stimulation, plasma TF activity and both FVIIa and TFPI. High activity levels of plasma TF and FVIIa with increased circulating TF-positive monocytes occurred in type II diabetic patients, especially with vascular complications. Results reflect high procoagulant activity possibly involved in diabetic vascular complications. Elevated TFPI levels were observed, but were not sufficient to balance high procoagulant activity. Correlation of procoagulant activity markers with HbA1c reinforces the importance of optimal glycemic control in type II diabetes.     

Plasma thrombin activatable fibrinolysis inhibitor and tissue factor pathway inhibitor changes following sepsis.

Sepsis-induced systemic inflammation results in coagulation abnormalities that may be different in gram-positive and gram-negative infections. We used ciprofloxacin to induce a predominantly gram-positive Enterococcus faecalis polymicrobial sepsis in rats. Ciprofloxacin-untreated rats exhibited a predominantly gram-negative polymicrobial sepsis. Rats were subjected to 30% body surface area burn (B), cecal ligation puncture (CLP) with a 22-gauge needle, and B + CLP. Ciprofloxacin-treated B + CLP rats showed a significant decrease in plasma thrombin activatable fibrinolysis inhibitor (TAFI) levels compared with sham rats. However, plasma tissue factor pathway inhibitor (TFPI) levels decreased significantly in B, CLP, and B + CLP groups compared with sham rats. The ciprofloxacin-untreated group showed a significant decrease in plasma TAFI levels in CLP and B + CLP and plasma TFPI levels decreased in all 3 groups compared with sham rats. Histological changes in the liver and kidney included vascular congestion and parenchyma bleed following B + CLP in ciprofloxacin-untreated rats. These results suggest that plasma TAFI and TFPI levels differ depending on the type of bacteria involved in the septic process.     

Adrenomedullin augments the release and production of tissue factor pathway inhibitor in human aortic endothelial cells

OBJECTIVES: Adrenomedullin (AM) is a hypotensive and vasodilative peptide. It has been reported that AM plays several biological roles in cardiovascular and endocrine systems, however, effects on the blood coagulation system have not been examined. In this study, we examined its effect on tissue factor pathway inhibitor (TFPI), which is a potent inhibitor of tissue factor/factor VIIa complex-induced coagulation cascade, and is synthesized and constitutively secreted by endothelial cells (ECs). The aim of this study was to elucidate the effects of AM on release and production of TFPI in ECs. METHODS: Cultured human aortic ECs were incubated with AM (10(-14)-10(-6) M), and the antigen levels and activity of the free and total form of TFPI after AM exposure were measured in culture media using ELISA. Furthermore, receptor interaction and cellular signaling of AM were investigated. RESULTS: AM augmented TFPI release and production in a dose-dependent manner. The effect on TFPI production was inhibited by AM receptor antagonist (AM22-52), the monoclonal antibody against C-terminal region of AM, MAPKK inhibitor, and cAMP antagonist. CONCLUSION: These findings indicate that AM might play an important role in the modulation of anticoagulant properties in blood circulation.     

Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross-over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti-factor Xa activity and anti-factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre-injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.     

Clinical significance of expression of tissue factor and tissue factor pathway inhibitor in ulcerative colitis

AIM: To investigate the clinical significance of expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in ulcerative colitis (UC).METHODS: Thirty UC specimens taken by colonoscopy from patients with active UC treated at the Department of Pathology, Central Hospital Affiliated to Shenyang Medical College from February 2010 to January 2012 were included in an experimental group, and 30 normal colon tissue samples taken by colonoscopy from non-UC patients were included in a control group. Expression of TF and TFPI in UC and normal colon tissue samples was detected by immunohistochemistry.RESULTS: The positive rate of TF in UC was significantly higher than that in normal colon tissue (63% vs 33%, χ² = 5.41, P < 0.05). The positive rate of TFPI in UC was also significantly higher than that in normal colon tissue (43% vs 17%, χ² = 5.08, P < 0.05).CONCLUSION: Positive rates of TF and TFPI expression in UC are significantly higher than those in normal colon tissue. TF and TFPI may play an important role in the pathogenesis of UC.     

Effect of erythropoietin on tissue factor and tissue factor pathway inhibitor in uremic hemodialysis patients

Some parameters of extrinsic coagulation pathway, including concentration and activity of tissue factor and concentration of tissue factor pathway inhibitor, have been estimated in uremic hemodialysis patients. The impact of erythropoietin treatment on the extrinsic coagulation pathway has also been the aim of the study. Increased concentration of tissue factor pathway inhibitor--TFPI has been found both in dialysed and non-dialysed uremic patients. This finding may be the evidence of endothelial damage as well as the protective factor against thrombotic complications. Erythropoietin treatment seemed not to induce statistically significant changes in extrinsic coagulation pathway. Some results indicate that estimation of "truncated' and "full length" forms of TFPI may be more useful comparing to complete TFPI concentration.     

Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis.