Endothelial Tissue Factor Pathway Inhibitor Research Articles

PF798 ANTITHROMBOTIC EFFECT OF SULFATED NON‐ANTICOAGULANT HEPARIN WITHOUT COMPROMIZING HEMOSTASIS

Background:The pathophysiology of thrombosis comprises a complex interplay of factors associated with vascular endothelial activation, platelet activation and adhesion. Blood coagulation process initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. Although heparin can be administered therapeutically to prevent and treat thrombosis, the effects of unfractionated heparin are more unpredictable, Low molecular weight heparin (LMWH) was recommended for prophylaxis and treatment of deep vein thrombosis as it significantly shortened both duration of thromboembolic crisis and hospitalization by ∼40%. Safety concerns associated with the narrow therapeutic index (bleeding risks) of LMWH are a major barrier to dose escalation/optimization of treatments. Here, we used the novel sulfated non‐anticoagulant LMWH, referred to as S‐NACH, which has a similar activity of LMWH but devoid of systemic anti‐factor Xa and IIa activity. Also, the sulfated form enhanced S‐NACH binding to the vascular endothelial cells and potentiate the action of tissue factor pathway inhibitors (TFPI‐1 and 2).Aims:To determine if S‐NACH exerts antithrombotic activity without compromizing hemostasis (bleeding) side effects that are associated with the clinical use of LMWHs. In the present study we investigated the effects of S‐NACH on clot kinetics in comparison to LMWH using thrombelstography (TAG) and examined its in vitro and in vivo effects on vascular endothelial release capacity of TFPI.Methods:We conducted both in vitro and in vivo experiments using our compound (S‐NACH) to test its efficacy on coagulation properties in comparison to LMWH (enoxaparin). We compared the effects on the coagulation time, time to clot initiation and thrombus strength level in vitro, with and without HUVEC endothelial cell line coated into cups used for the TEG analysis. TFPI‐1 and TFPI‐2 levels were also estimated in both in vitro and in vivo models at different doses of S‐NACH. In addition, the effect of S‐NACH on factors Xa and IIa was assessed in mice and rabbits.Results:TEG assay have shown that, increasing concentrations of S‐NACH (0.1 – 10 ug) did not affect the clotting time (R) and maximum amplitude of the clot formation (MA), while LMWH (enoxaparin) was associated with marked increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. Using HUVEC cell line coated cups; both S‐NACH and LMWH were associated with increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) (Table 1). This is due to the ability of HUVEC cell to release of TFPI‐1 and TFPI‐2. In the in vivo study, administration of enoxaparin inhibited both factors Xa and IIa, while administration of S‐NACH did not affect the activities of these coagulation factors. However, S‐NACH caused 3 folds greater tissue factor pathway inhibitor (TFPI) release from human endothelial cells and in mice versus enoxaparin (Figure 1).Summary/Conclusion:S‐NACH is devoid of anti‐thrombin binding and inhibition of systemic anti‐thrombin‐dependent coagulation factors such as factor Xa and factor IIa but has an optimal releasing capacity of vascular endothelial TFPI that is associated with antithrombotic activities without any effect on hemostasis.image

Angiotensin II promotes the anticoagulant effects of rivaroxaban via angiotensin type 2 receptor signaling in mice

Rivaroxaban is an oral direct factor Xa inhibitor approved for the treatment of stroke and systemic thromboembolism in patients with non-valvular atrial fibrillation. Despite its efficacy, rivaroxaban therapy results in adverse effects and complications, such as bleeding. Angiotensin II (AngII) is implicated in many cardiovascular conditions, such as hypertension and heart failure. In this study, we investigate whether AngII influences anticoagulant effects of rivaroxaban by using an experimental mouse model with type 2 diabetes mellitus and advanced glycation end product (AGE)-exposed human umbilical vein endothelial cells (HUVECs). We found that AngII promoted the anticoagulant effects of rivaroxaban in KKAy mice. The combination of rivaroxaban and AngII enhanced in vivo tissue factor pathway inhibitor (TFPI) activity and induced TFPI expression and activity in AGE-exposed HUVECs. Angiotensin type 2 receptor (AT2R) and Mas antagonists attenuated the AngII-enhanced anticoagulant action of rivaroxaban in vivo, and abolished the increased endothelial TFPI expression and activity. However, angiotensin type 1 receptor (AT1R) antagonist exerted no effects. Additionally, combination of rivaroxaban and AngII induced aortic AT2R and Mas expression. Our data suggest that the anticoagulant effects of rivaroxaban are promoted by AngII via AT2R and Mas signaling. These findings are significant for the clinical administration of rivaroxaban.

Fluvastatin Upregulates the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are cholesterol-lowering drugs with a variety of pleiotropic effects including antithrombotic properties. Tissue factor pathway inhibitor (TFPI), which is produced predominantly in endothelial cells and platelets, inhibits the initiating phase of clot formation. We investigated the effect of fluvastatin on TFPI expression in cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with fluvastatin (0-10μM). The expression of TFPI mRNA and antigen were detected by RT-PCR and western blotting, respectively. The effects of mevalonate intermediates, small GTP-binding inhibitors, and signal transduction inhibitors were also evaluated to identify which pathway was involved. A luciferase reporter assay was performed to evaluate the effect of fluvastatin on TFPI transcription. The stability of TFPI mRNA was estimated by quantitating its levels after actinomycin D treatment. Fluvastatin increased TFPI mRNA expression and antigen in HUVECs. Fluvastatin-induced TFPI expression was reversed by co-treatment with mevalonate or geranylgeranylpyrophosphate (GGPP). NSC23766 and Y-27632 had no effect on TFPI expression. SB203580, GF109203, and LY294002 reduced fluvastatin-induced TFPI upregulation. Moreover, fluvastatin did not significantly affect TFPI promoter activity. TFPI mRNA degradation in the presence of actinomycin D was delayed by fluvastatin treatment. Fluvastatin increases endothelial TFPI expression through inhibition of mevalonate-, GGPP-, and Cdc42-dependent signaling pathways, and activation of the p38 MAPK, PI3K, and PKC pathways. This study revealed unknown mechanisms of the anticoagulant effect of statins and gave a new insight to its therapeutic potential for the prevention of thrombotic diseases.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.

Melatonin stimulates release of tissue factor pathway inhibitor from the vascular endothelium

We previously found an association between the circadian variation of free tissue factor pathway inhibitor (TFPI) and melatonin in able-bodied males and in men with complete cervical spinal cord injuries. We therefore examined whether melatonin modifies production and/or secretion of TFPI in endothelial cells. We sampled supernatants from cultures of primary human umbilical vein endothelial cells (HUVECs) and of human coronary artery endothelial cells (HCAECs), that had been exposed to varying doses (0-300 pg/ml) of melatonin for 0.5-24 h. We then measured the protein concentrations of free TFPI, tissue factor and plasminogen activator inhibitor type 1 (PAI-1). We also measured endothelial TFPI, tissue factor and PAI-1 transcripts using quantitative real-time PCR. Melatonin dose dependently increased free TFPI levels about 25-30-fold in supernatants of both HUVEC and HCAEC, and independent of incubation duration. In contrast, TF and PAI-1 remained unaltered upon increasing doses of melatonin. Neither TFPI mRNAs nor tissue factor mRNAs nor PAI-1-mRNAs were changed in cell cultures added melatonin. The ratio of free TFPI in cell supernatants to free TFPI in cell lysates about doubled upon addition of melatonin, indicating that melatonin increased release from intracellular storages of free TFPI or from membrane-bound free TFPI. Our data indicate that melatonin stimulates vascular endothelial cells to secrete TFPI without altering transcription of the TFPI gene. If melatonin increases TFPI release in a similar fashion in vivo as in vitro, this could have potential clinical implications in both prophylaxis and treatment of thromboembolic events.

Different Effects of Enoxaparin, Nadroparin, and Dalteparin on Plasma TFPI During Hemodialysis

Low-molecular-weight heparins (LMWHs) are an alternative to unfractionated heparin (UFH) for anticoagulation during hemodialysis (HD). We performed a prospective randomized crossover study of the effect of enoxaparin, nadroparin, and dalteparin on some hemostatic factors, including tissue factor pathway inhibitor (TFPI), in patients with maintenance HD. Plasma levels (immunoassays) of total TFPI, platelet-derived growth factor-AB (PDGF-AB), and prothrombin fragment 1 + 2 (PF 1 + 2) were evaluated pre-HD, after 10 (T10) and 180 (T180) minutes of HD in 21 patients, who completed a 3-period (for 2 months each) crossover study in 6 groups (Latin-square design). The baseline TFPI, PDGF-AB, and PF 1 + 2 levels were comparable under all LMWH treatments. Tissue factor pathway inhibitor levels, compared with the baseline, significantly increased (all P < 10(-4)), whereas PDGF-AB levels remained stable at each interval during enoxaparin, nadroparin, and dalteparin anticoagulated HD. Interestingly, TFPI increment at T10 was the highest, dose-dependent, and accompanied by PF 1 + 2 decrease under enoxaparin administration. The switch from enoxaparin to nadroparin and dalteparin used as anticoagulants had no long-term effect on the baseline total TFPI and PF 1 + 2 levels in chronically HD patients. Only short-term, overdialytic differences were noticed, indicating a single bolus of enoxaparin (0.75 mg/kg) as the most potent stimulus for endothelial TFPI.

Site Directed Delivery of Chemotherapy and Non-Anticoagulant Sulfated Heparin in Breast Cancer

There is substantial literature support for the use of low molecular weight heparins (LMWH) for treating coagulation disorders in cancer patients. Recent prospective and retrospective clinical trials have also demonstrated that they provide significant advantages in terms of progression-free and overall survival in certain cancers and in certain subgroups of patients. LMWH treatments are often associated with increased bleeding, constituting a dose-limiting effect. We have developed novel non-anticoagulant heparin (NACH) compounds that have minimal effects on hemostasis (El-Naggar and Mousa, US Patents 6,908,907 B2, (2005), and 10, 667,216, (2003). We have evaluated them for efficacy vs. tumor growth and metastasis and have begun to investigate the mechanisms involved in anti-tumor activities. Modified sulfated LMWH with weak or no anticoagulant activities were still highly effective in inhibiting angiogenesis and metastasis, demonstrating that anticoagulation is not essential for attenuation of angiogenesis or metastasis. The modified heparins were characterized with respect to their ability to release endothelial tissue factor pathway inhibitor (TFPI) and inhibit selectin-mediated interactions, molecular components that have been shown to modulate tumor growth, tumor angiogenesis and metastasis. One of these modified heparin compounds that showed significant activity in a selectin-mediated tumor cell adhesion assay was also highly effective in reducing tumor burden in mice with MC-38 colon carcinoma and B16-BL6 melanoma (>70%) and in reducing the number of metastatic foci (>65%) in these animals. We also investigated the efficacy of NACH compounds on growth factor-induced angiogenesis in a mouse Matrigel model in which new vessel growth was quantified by measuring hemoglobin concentration extracted from the Matrigel plug. Values are Means ± SEM. Matrigel alone: 0.57± 0.12; bFGF + Matrigel: 7.27 ± 1.18; NAC-S-S: 0.86 ± 0.10. This sulfated compound which demonstrated no anticoagulant activity in aPTT and TEG assays, reduced capillary formation to baseline levels. These data demonstrate that non-anticoagulant heparin compounds exhibit a profile of anti-tumor activities without disrupting normal hemostasis. Site-directed therapy with non-anticoagulant heparins (NACH) and chemotherapy would allow for optimization of treatments in the tumor microenvironment. In studies that are currently underway, we are targeting the sites of tumor neovascularization using a biodegradable nanoparticulate system made up of a blend of MPEG-PLGA (methoxy-polyethyleneglycol-poly (lactide-co-glycolide) and maleimide-PEG-PLGA. These nanoparticles have their surfaces conjugated to alpha-v beta-3 antibody and contain chemotherapeutic agent Doxorubicin, with or without NACH. Preliminary data indicate that repeated administration of sulfated non-anticoagulant heparin compound at 10 mg/kg S.C. daily for up to 14 days in conjunction with doxorubicin caused no bruising at the injection site, whereas Enoxaparin showed injection site bruising in >50% of the mice. The use of NACH agents that are co-encapsulated with chemotherapeutic agents could minimize the toxic side effects of the chemotherapy while delivering a combination of effective therapeutic agents directly to the tumor.

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. Guggulsterone inhibited TNF-alpha-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-alpha-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-alpha-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.

Molecular weight dependent tissue factor pathway inhibitor release by heparin and heparin oligosaccharides

Heparin and low molecular weight heparins exert their vascular effects by mobilizing tissue factor pathway inhibitor (TFPI) from the vascular endothelium into the blood circulation. We compared the influence of molecular weight on the TFPI release by heparin and its fractions in a non-human primate model. Primates were treated with unfractionated heparin, a low molecular weight heparin (gammaparin), or a heparin-derived oligosaccharide mixture (C3). Endothelial TFPI release was determined using both immunologic and functional assays. After intravenous administration, all agents significantly increased TFPI levels ( p < 0.05) in a dose dependent manner. The increase produced by unfractionated heparin and gammaparin was greater than that by C3 at an equal dosage ( p < 0.05). With subcutaneous injection, all agents produced less TFPI release. Repeated administration of heparin-derived oligosaccharides gradually increased TFPI release. A 1.89 fold increase in TFPI levels was observed 4 days after C3 treatment (2.5 mg/kg). Our findings indicated that TFPI release is dependent on the molecular weight of heparin and its derivatives. Heparin oligosaccharides exert their vascular effects through increased TFPI release after long-term repeated administration.

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.

Anti-metastatic effect of a non-anticoagulant low-molecular-weight heparin versus the standard low-molecular-weight heparin, enoxaparin

Low-molecular-weight heparins (LMWH) exhibit potent anticoagulant efficacy via their plasmatic effects on thrombin and factor Xa. These agents are also effective in releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of tissue factor, and exhibit significant anti-metastatic effects in experimental animal models. However, the potential for bleeding complications has slowed down the more widespread adoption of LMWH therapy in cancer patients. In this study, the effect of a non-anticoagulant form of LMWH (NA-LMWH) on experimental lung metastasis and tumor cell-induced platelet aggregation in vivo was compared to the LMWH enoxaparin. Using the B16 melanoma mouse model of metastasis, subcutaneous (s.c.) injection of NA-LMWH or enoxaparin (10 mg/kg), three hours before intravenous (i.v.) injection of metastatic melanoma cells, followed by daily doses for 14 days, reduced lung tumor formation by 70% (P < 0.001). I.v. injection of tumor cells resulted in a significant (50-62%, P < 0.01) fall in platelet counts. Pre-injection (i.v.) of enoxaparin completely abolished the tumor cell-induced thrombocytopenia, whereas NA-LMWH had no effect. Four hours after a single s.c. dose, enoxaparin but not NA-LMWH prolonged the clotting time three-fold and delayed the time to clot initiation more than 10-fold as measured by a Sonoclot analyzer and by thromboelastography, respectively. Enoxaparin but not NA-LMWH demonstrated a significant anticoagulant effect in mice. Both NA-LMWH and enoxaparin caused similar TFPI release from endothelial cells in vitro. These data provide evidence to support the potential of NA-LMWH as an anti-metastatic agent without any significant impact on coagulation.

Vascular Endothelial Tissue Factor Pathway Inhibitor Kinetics in Culture Following Exposure to DX-9065a—A Selective and Direct Factor Xa Inhibitor

Tissue factor (TF), a membrane-bound glycoprotein that initiates blood coagulation by allosteric activation of factor (f) VII, is regulated predominantly by tissue factor pathway inhibitor (TFPI). Because vascular endothelial cells synthesize and constitutively secrete TFPI and fXa may directly influence its cellular clearance, we sought to determine the effects of DX-9065a, a direct and selective fXa inhibitor, on TFPI kinetics in culture. Human umbilical vein endothelial cells were grown to confluence and incubated with unfractionated heparin (1.0 U/mL), enoxaparin (1.5 U/mL), or DX-9065a at low (10 ng/ml), moderate (30 ng/ml), or high (90 ng/ml) concentrations. Compared to control, increases in TFPI were seen with both unfractionated heparin (182% higher, p < 0.001) and enoxaparin (194% higher, p < 0.001). Low concentration DX-9065a did not increase TFPI levels above control (0.8% higher, p = 0.91). In contrast, moderate and high concentrations produced 124% higher (p < 0.001) and 198% higher (p < 0.001) TFPI concentrations than control, respectively. DX-9065a increases vascular endothelial cell TFPI concentrations in human tissue culture. Although the mechanism has yet to be established, decreased fXa availability may limit fXa-TFPI complex formation and its subsequent cellular uptake. Whether increased surface TFPI contributes to the overall anticoagulant profile of DX-9065a will require further investigation.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.

Inhibition of endothelial cell tube formation by the low molecular weight heparin, tinzaparin, is mediated by tissue factor pathway inhibitor.

Heparin and low molecular weight heparins (LMWHs) have both antithrombotic and anti-angiogenic activities. The anti-angiogenic activity of LMWH may be associated with the release of endothelial tissue factor pathway inhibitor (TFPI), an important endogenous inhibitor of tissue factor/Factor VIIa (TF/fVIIa). To evaluate the effects of LMWH, tinzaparin, and TFPI in a model of angiogenesis-mediated processes, we compared the effects of tinzaparin, and recombinant TFPI in inhibiting either basic fibroblast growth factor-2 (FGF2)- or TF/fVIIa-induced endothelial cell tube formation in human umbilical vein endothelial cells (HUVEC). Our results show that tinzaparin and recombinant TFPI both blocked endothelial tube formation induced by either FGF2 or TF/fVIIa, in a concentration-dependent manner. Endothelial tube formation was only marginally inhibited by a potent and specific anti-Factor Xa, recombinant tick anticoagulant protein (rTAP). A monoclonal anti-TFPI anti-body reversed the inhibitory effects of either tinzaparin or recombinant-TFPI on HUVEC tube formation. Tinzaparin fractions in the range of 8,000 to 12,600 Da were most effective in stimulating the release of TFPI from HUVEC. These results suggest that the inhibitory effect of the LMWH tinzaparin on endothelial tube formation is associated with stimulation of the release of TFPI, but not to anti-Factor Xa activity.

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin

The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low-molecular-weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non-coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg-1) 4 h before intravenous administration of melanoma cells (2.0 x 105) markedly (89%) reduced lung tumor formation (3 +/- 2) compared with controls (31 +/- 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg-1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin-treated animals, despite a 4-fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg-1). Administration of tumor cells (2 x 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 +/- 37 vs. 498 +/- 94 x 106 mL-1, P < 0.01), but this was prevented by tinzaparin treatment (921 +/- 104 x 106 mL-1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.

Pharmacodynamic Properties of the Low Molecular Weight Heparin, Tinzaparin: Effect of Molecular Weight Distribution on Plasma Tissue Factor Pathway Inhibitor in Healthy Human Subjects

Low molecular weight heparins (LMWHs) stimulate the release of endothelial tissue factor pathway inhibitor (TFPI). An inhibitor of tissue factor-induced coagulation, TFPI may contribute to the efficacy of LMWHs. The release of TFPI may be affected by characteristics of LMWHs, such as molecular weight (MW) distribution. The authors measured plasma levels of TFPI after single, subcutaneous injections of tinzaparin (mean MW = 6500 Daltons [Da]; 3.5% of fractions < 2000 Da) or a tinzaparin-like LMWH (mean MW = 5650 Da; 18.3% of fractions < 2000 Da) in 30 healthy subjects. Maximum mean plasma levels of TFPI approached 200 to 230 ng/mL 0.8 hours after administration of either drug. Plasma levels of TFPI remained elevated compared with baseline values for 12 to 16 hours after drug administration. These results suggest that LMWH fractions < 2000 Da do not affect the release of TFPI.

Quantification of heparin-induced TFPI release: a maximum release at low heparin dose.

Heparin releases tissue factor pathway inhibitor (TFPI) from the endothelium and this release may decrease after repeated high dose heparin administration. The primary aim was to investigate and quantify this phenomenon during a short low dose heparin infusion. Also, the effects of heparin on tissue plasminogen activator (t-PA) were studied. Nine healthy, nonsmoking, male volunteers (range 19-23 years) received a continuous heparin infusion (2000 IU) over 40 min. The endothelial TFPI release rate was estimated from the total TFPI concentration profile using a pharmacokinetic model. Mean +/- s.d. total and free TFPI increased from 62.9 +/- 9.4/8.3 +/- 2.1 ng ml-1 at baseline to 237.2 +/- 40.9/111.0 +/- 19.9 ng ml-1 after 40 min infusion. The relationship between heparin concentration (anti-IIa activity) and TFPI concentration followed a maximum effect model and a clockwise loop (proteresis) was observed. The TFPI release rate rapidly increased to maximum of 200 +/- 45 micro g min-1 after 17.5 min heparin infusion but did not increase further although heparin concentrations further doubled. In contrast to TFPI, t-PA antigen decreased from 5.6 +/- 1.0 at baseline to 4.5 +/- 1.0 ng ml-1 at the end of infusion (t = 40 min) (difference of 1.1 ng ml-1 (95% confidence interval; 0.9, 1.3). Our application of concentration-effect models and pharmacokinetic principles to these haemostatic variables showed that endothelial TFPI release has a maximum that is already reached at low heparin dose, corresponding with an anti-IIa activity of 0.08 IU ml-1. The relationship between anti-IIa activity and TFPI release rate showed signs of acute tolerance (clockwise loop) indicating exhaustion of endothelial TFPI pools. These findings may be of importance for the heparin dose used in conditions such as unstable angina, in which the favourable effects of heparin have been ascribed to its ability to release TFPI.

Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

Thrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 microM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.