Imbalances between the levels of tissue factor and tissue factor pathway inhibitor in ARDS patients

INTRODUCTION: To evaluate the pathogenetic role of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and neutrophil elastase in acute respiratory distress syndrome (ARDS), as well as to test the hypothesis that TFPI levels modified by neutrophil activation are not sufficient to prevent TF-dependent intravascular coagulation, leading to sustained systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), which determine the prognosis of these patients. MATERIALS AND METHODS: The study subjects consisted of 55 patients with trauma and sepsis who were divided into three groups according to the Lung Injury Score. Ten normal healthy volunteers served as control. Plasma levels of TF, TFPI, and neutrophil elastase were measured on the day of injury or the day of diagnosis of sepsis (day 0) and days 1 through 4. The number of SIRS criteria that the patient met and the disseminated intravascular coagulation (DIC) score is determined daily. RESULTS: Patients (15) developed ARDS, 23 were at risk for but did not develop the syndrome, and 17 patients were without risk for ARDS. TF and neutrophil elastase levels in ARDS patients were persistently higher than those in other two groups and control subjects. However, the TFPI levels showed no difference among the three groups, which retained normal or slightly elevated levels compared to the control subjects. DIC scores did not improve and SIRS continued during the study period in patients with ARDS. The ARDS patients showed higher numbers of dysfunctioning organs and associated with poorer outcome than the other two groups. CONCLUSION: Systemic activation of the TF-dependent pathway not adequately balanced by TFPI is one of the aggravating factors of ARDS. High levels of neutrophil elastase released from activated neutrophils may explain the imbalance of TF and TFPI. Persistent DIC and sustained SIRS contribute to MODS, determining the prognosis of ARDS patients.     

Inhibitory effects of LDL-associated tissue factor pathway inhibitor

Introduction

Tissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI_1–161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays.

Materials and Methods

LDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4 μM phospholipids or 0.5 nM factor Xa (FXa) and 4 μM phospholipids, respectively.

Results

TFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI_1–161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height.

Conclusions

Our results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI_1–161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.     

Alternatively spliced isoforms of tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is the major regulator of tissue factor (TF)-induced coagulation. It down regulates coagulation by binding to the TF/fVIIa complex in a fXa dependent manner. It is predominantly produced by microvascular endothelial cells, though it is also found in platelets, monocytes, smooth muscle cells, and plasma. Its physiological importance is demonstrated by the embryonic lethality observed in TFPI knockout mice and by the increase in thrombotic burden that occurs when heterozygous TFPI mice are bred with mice carrying genetic risk factors for thrombotic disease, such as factor V Leiden. Multiple TFPI isoforms, termed TFPIα, TFPIβ, and TFPIδ in humans and TFPIα, TFPIβ, and TFPIγ in mice, have been described, which differ in their domain structure and method for cell surface attachment. A significant functional difference between these isoforms has yet to be described in vivo. Both human and mouse tissues produce, on average, approximately 10 times more TFPIα message when compared to that of TFPIβ. Consistent with this finding, several lines of evidence suggest that TFPIα is the predominant protein isoform in humans. In contrast, recent work from our laboratory demonstrates that TFPIβ is the major protein isoform produced in adult mice, suggesting that TFPI isoform production is translationally regulated.     

Structure and biology of tissue factor pathway inhibitor.

Human tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.     

伴糖尿病的脑梗死患者血清组织因子与组织因子途径抑制物水平的研究

目的 探讨伴糖尿病的脑梗死患者血清组织因子(Tissue factor,TF)、组织因子途径抑制物(Tissuefactor pathway inhibitor,TFPI)水平的变化及其临床意义.方法 采用酶联免疫吸附法(ELISA)测定30例门诊体检者(正常对照组)和60例急性脑梗死患者(分为伴糖尿病脑梗死和不伴糖尿病脑梗死两组)血清TF及TFPI水平,并于发病后72h进行头部CT检查,计算梗死灶大小,同时进行神经功能缺损评分.结果 (1)脑梗死组较正常对照组血清TF及TFPI水平明显升高(P＜0.05).(2)伴糖尿病脑梗死与不伴糖尿病脑梗死相比,前者血清TF及TFPI的水平升高更明显(P＜0.05).(3)脑梗死体积、临床神经功能缺损评分均与血清TF、TFPI水平无明显相关性.结论 血清TF及TFPI水平与脑梗死的严重程度无明显相关性,但伴糖尿病脑梗死确实存在更为严重的凝血功能障碍.     

Platelet tissue factor pathway inhibitor modulates intravascular coagulation

Tissue factor pathway inhibitor (TFPI) is produced by megakaryocytes and is found internally within quiescent platelets but is not in α-granules. It is released in soluble form and expressed on the surface of platelets that are dual activated with thrombin plus collagen. Platelet TFPI is exclusively TFPIα, the most evolutionarily conserved TFPI isoform. It appears to be physiologically active as an inhibitor of tissue factor (TF) initiated FXa generation in vitro, and acts locally to dampen clot growth in a murine vascular injury model. These data suggest that platelet TFPI plays an important role in modulating TF activity within a growing clot thereby preventing formation of an occlusive clot.     

Tissue factor reduction and tissue factor pathway inhibitor release after heparin administration

Elevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU X 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%. p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001). After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/10(5) monocytes to 86.1 U/10(5) monocytes, 28-320 U/10(5) monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.     

Local tissue factor pathway inhibitor release in the human forearm

Nineteen healthy men received unilateral brachial artery infusions of either unfractioned heparin (0.3-100 IU/min), saline or the endothelium-dependent vasodilators substance P (2-8 pmol/min) and bradykinin (100-1000 pmol/min), and the endothelium-independent vasodilator sodium nitroprusside (2-8 micro g/min). Heparin caused a dose-dependent increase in plasma TFPI concentrations in both arms (ANOVA, p <0.0001). Estimated net forearm TFPI release was 7 +/- 16, 29 +/- 20 and 138 +/- 72 ng/100 mL tissue/min during 10, 30 and 100 IU/min of heparin respectively (ANOVA, p <0.0001). Compared to the systemic circulation, the forearm sensitivity to heparin induced TFPI release was 3.6-fold lower (166 +/- 67 ng/IU vs. 596 +/- 252 ng/IU: t-test, p = 0.004). Substance P, bradykinin and sodium nitroprusside all caused substantial dose-dependent increases in blood flow (ANOVA, p <0.001 for all) without affecting plasma TFPI concentrations. There are important regional differences in endothelial TFPI release, with the forearm circulation being relatively insensitive to heparin.     

Tissue factor and tissue factor pathway inhibitor levels during and after cardiopulmonary resuscitation

Disseminated intravascular coagulation frequently occurs after global ischemia and reperfusion due to cardiac arrest. The present study was performed to demonstrate the role of tissue factor for coagulation pathway activation, as well as to investigate the precise time course of tissue factor pathway inhibitor (TFPI) during and after cardiopulmonary resuscitation (CPR). Thirty-two of out-of-hospital cardiac arrest patients were classified into two groups, those who achieved return of spontaneous circulation (ROSC) (n=13) and those without ROSC (n=19). Ten normal healthy volunteers served as control subjects. Serial levels of tissue factor and TFPI were measured during and after cardiac arrest and CPR. In patients with ROSC, cardiac arrest and CPR led to persistent increases in the levels of tissue factor that peaked 6 hours after arrival at the Emergency Department. Tissue factor levels in patients without ROSC also showed marked elevations compared to those of the control subjects. In both groups, the levels of TFPI were significantly lower than those in the control subjects. However, we could not find differences in the levels of the two markers between the patients with ROSC and those without ROSC. In conclusion, we demonstrated persistent elevation of the tissue factor levels associated with low TFPI during and after CPR in patients with out-of-hospital cardiac arrest. These results indicate the activation of the extrinsic coagulation pathway without adequate TFPI generation, which may contribute to thrombin activation and fibrin formation after whole-body ischemia and reperfusion.     

Murine strain differences in hemostasis and thrombosis and tissue factor pathway inhibitor

Introduction

Differences among murine strains often lead to differential responses in models of human disease. The aim of the current study was to investigate whether differences exist among strains in models of hemostasis and thrombosis and whether these differences are reflected in differences in the tissue factor (TF) pathway.

Methods

We examined baseline hemostatic parameters and the response to FeCl3-induced arterial thrombosis and a tail vein bleeding model in C57BL/6J (C57), 129S1/SvImJ (129S), and Balb/cJ (BalbC) mice. Finally, we examined TF and tissue factor pathway inhibitor (TFPI) activities in blood and expression in vascular tissue to determine whether these factors covary with a thrombotic phenotype.

Results

No differences were observed in PT or aPTT among strains. 129S mice had lower platelet counts (p < 0.001). BalbC had an increased rate of occlusion (mean occlusion time of 330 ± 45 sec) in a FeCl₃-induced model of thrombosis when compared to C57 (1182 ± 349 sec) or 129 S (1442 ± 281 sec) (p < 0.05). Similarly, BalbC demonstrated reduced blood loss in tail bleeding experiments when compared to C57 and 129S. Vascular expression of TF and TFPI content did not correlate with the thrombotic phenotype of BalbC. However, circulating TFPI activities were lower in BalbC compared to both C57 and 129S mice. When normalized to circulating TF activities, BalbC had lower circulating TFPI activity than C57 and 129S, and there was a significant correlation between tail bleeding and normalized TFPI activity (r = 0.67).

Conclusions

These data suggest that there are significant differences among strains in thrombosis and hemostasis and that circulating TFPI activity correlates with these differences.     

Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk

Introduction

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.

Methods

TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.

Results

TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p < 0.001). TF protein expression was also increased in endometroid carcinoma (P < 0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P < 0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P < 0.01). There was no difference in TFPI expression between the two groups.

Conclusion

TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.     

Enhancement of lipoprotein lipase activity by tissue factor pathway inhibitor

The effect of a basic synthetic peptide, representing the C-terminal region of tissue factor pathway inhibitor (TFPI - Lys254 - Met276), as well as that of the whole protein, on the activity of lipoprotein lipase (LPL) is described. The activity of bovine LPL was measured by chromogenic assay using a water-soluble chromogenic substrate, p-nitrophenyl butyrate. Five and 10 microM concentrations of the peptide increased Vmax of bovine LPL by 48.9% and 85.6% respectively as compared with the buffer control without affecting Km. Poly l-lysine, though positively charged did not have any effect, suggesting the importance of the amino acid sequence of the test peptide. On the other hand, 0.25, 0.5 and 1.0 mM n-butyric acid - a product of LPL catalysis in the chromogenic assay, when added to the incubation mixture decreased Vmax non competitively by 22.8%, 40.4% and 63% respectively as compared with buffer control, confirming the known product inhibition of LPL. A 100-fold molar excess of n-butyric acid produced inhibition of the LPL reaction as compared with the synthetic peptide which produced potentiation, suggesting a 1:100 stoichiometric interaction of the peptide with n-butyric acid. At a fixed concentration of 0.25 mM substrate, 10 nM full length recombinant TFPI, containing the basic C-terminal domain, increased velocity of LPL reaction by 39.4% as compared with buffer control. The same concentration of two-domain recombinant TFPI (TFPI1-160) had no effect. It is possible that negatively charged n-butyric acid is sequestered by the positively charged peptide or the basic region of recombinant full length TFPI. Relieving of product inhibition could then be a possible mechanism of the observed potentiation of bovine LPL activity by the basic peptide or full length recombinant TFPI. The 39.4% increase in reaction velocity of LPL catalysis produced by 10 nM full length recombinant TFPI was comparable to 38.9% increase produced by 5 microM of the basic peptide under the same conditions. A further increase of 78.7% was brought about by 10 microM concentration of the same peptide. The reason for about 500-fold increase in the potency of the whole protein as compared with that of the peptide is not clear. It is possible that in its tertiary conformational state, the whole protein is able to sequester product and relieve product inhibition more effectively than the short linear peptide. Rabbit polyclonal antiserum against the basic peptide partially inhibited LPL activity of human post heparin plasma, measured by radioenzymatic assay using triolein substrate. Since post heparin plasma contains full length TFPI, binding of the added antibody to its basic C-terminus and hence the relative unavailability of latter for product sequestration (oleic acid in this case) could explain the observed inhibition of human LPL activity by antibody against the peptide. Thus by enhancing lipase activity, full length TFPI may facilitate hydrolysis of triglyceride and concomitantly lower factor VII coagulant activity as demonstrated earlier, particularly after heparin injection when both TFPI and LPL are released in circulation.     

Effect of leukocyte proteinases on tissue factor pathway inhibitor.

The effect of human neutrophil elastase and cathepsin G on recombinant tissue factor pathway inhibitor (TFPI) was investigated. A weak inhibition by TFPI of both elastase (Ki = 0.4 microM) and cathepsin G (Ki = 0.1 microM) was observed. Neutrophil elastase rapidly cleaved TFPI at the Thr87-Thr88 bond situated at the link between Kunitz domains I and II. Cleavage of TFPI by cathepsin G was also observed, but the reaction was much slower and resulted in a number of fragments. Proteolytic cleavage by both elastase and cathepsin G resulted in destruction of inhibitor function with respect to TFPI's inhibition of factor Xa. Cleavage by neutrophil elastase was capable of restoring factor Xa amidolytic activity after its initial inhibition by TFPI. Inhibition of cathepsin G by TFPI was strongly augmented by stoichiometric amounts of factor Xa. However, the augmentation was temporary, presumably due to concomitant cleavage of TFPI by cathepsin G. These observations may have implications for the putative effect of neutrophil leukocyte stimulation on the regulation of the tissue factor-mediated coagulation pathway. Conversely, formation of a factor Xa/TFPI complex may reduce or modulate the proteolytic potential of stimulated leukocytes by temporary inhibition of cathepsin G.     

Changes in tissue factor and the effects of tissue factor pathway inhibitor on transient focal cerebral ischemia in rats

INTRODUCTION: To determine the contribution of tissue factor (TF) to focal cerebral ischemia/reperfusion injury, we investigated the changes in TF in rat brains with transient focal cerebral ischemia and also assessed the effect of TF pathway inhibitor (TFPI). MATERIALS AND METHODS: Spontaneous hypertensive rats were subjected to 90-min of middle cerebral artery occlusion (MCAO) and then were reperfused for up to 24 h. Immediately after MCAO, recombinant human TFPI (rhTFPI) (50 or 20 microg/kg/min) was administered by means of a continuous intravenous injection for 4.5 h. RESULTS AND CONCLUSIONS: TF immunoreactivity decreased or scattered in the ischemic area after reperfusion, however, an increased TF expression was observed in the microvasculature with the surrounding brain parenchyma and it peaked at 3 to 6 h, which coincided with the start of fibrin formation. On the other hand, total TF protein in ischemic area continued to exist and did not remarkably change until 24 h after reperfusion. At 24 h after reperfusion, the total infarct volume in the group treated with 50 microg/kg/min rhTFPI was significantly smaller than that in the controls (saline). Western blotting and immunohistochemical studies showed that rhTFPI treatment resulted in a decrease of fibrin in the ischemic brains and microvasculature. TF-mediated microvascular thrombosis is thus considered to contribute to focal cerebral ischemia/reperfusion injury. The continuous infusion of rhTFPI until a peak of TF-mediated microvascular thrombosis therefore attenuates the infarct volume by reducing fibrin deposition in the cerebral microcirculation.     

Protein S is a cofactor for tissue factor pathway inhibitor

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.     

Plasma tissue factor pathway inhibitor and tissue factor antigen levels after administration of heparin in patients with angina pectoris

The hypercoagulability is associated with expression of tissue factor in patients with angina. Tissue factor pathway inhibitor regulates the extrinsic coagulation pathway mediated by tissue factor. Plasma samples were obtained from 14 patients with angina pectoris and 9 with chest pain syndrome before and 5, 30, 60, and 120 minutes after administration of heparin (50 IU/kg). The tissue factor and prothrombin fragment 1+2 levels before administration were elevated in patients with angina pectoris and were reduced to the levels of chest pain syndrome after the administration. The free tissue factor pathway inhibitor levels after the administration were higher in patients with angina pectoris than in patients with chest pain syndrome. Plasma tissue factor pathway inhibitor levels correlated positively with plasma tissue factor and prothrombin fragment 1+2 levels. We showed that plasma-free TFPI levels after administration of heparin, which may indicate endothelial cell associated TFPI levels, increased in patients with angina pectoris compared with patients with chest pain syndrome. Increased endothelial cell associated TFPI was associated with hypercoagulability in patients with angina pectoris. These may help to explain the reduction in thrombotic risk associated with the use of heparin.