Tumor microenvironment mediated by suppression of autophagic flux drives liver malignancy.

The physiological role of autophagy in the catabolic process of the body involves protein synthesis and degradation in homeostasis under normal and stressed conditions. In hepatocellular carcinoma (HCC), the role of tumor microenvironment (TME) has been concerned as the main issue in fighting against this deadly malignancy. During the last decade, the crosstalk between tumor cells and their TME in HCC extensively accumulated. However, a deeper knowledge for the actual function of autophagy in this interconnection which involved in supporting tumor development, progression and chemoresistance in HCC is needed but still largely unknown. Recent studies have shown that coagulants tissue factor (TF) and factor VII (FVII) has a pathological role in promoting tumor growth by activating protease-activated receptor 2 (PAR2). Autophagy-associated LC3A/B-II formation was selectively suppressed by FVII/PAR2 signaling which mediated by mTOR activation through Atg7 but not Atg5/Atg12 axis. The coagulant-derived autophagic suppression seemed potentiate a vicious circle of malignancy in producing more FVII and PAR2 which facilitate in vivo and in vitro tumor progression of HCC and the investigations are consistent with the clinical observations. In this review, we briefly summarize the current understanding of autophagy and discuss recent evidence for its role in HCC malignancy.

[Mechanism of cross talk between tissue factor/active coagulation factor VII and epidermal growth factor receptor signalings in colon cancer cells in culture].

OBJECTIVE: To preliminarily verify the cross talk between tissue factor/active coagulation factor VII (TF/FVIIa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture. METHODS: FVIIa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/FVIIa pathway, qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG), ligands of EGFR on mRNA and protein levels, respectively. After knocking down expression of TF by TF-targeted siRNA transfection, FVIIa was treated and mRNA expressions of AREG and EREG were detected to see whether the FVIIa-induced effects were dependent on TF. Expressions of mRNA of TF and FVII were detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells. RESULTS: After TF/FVIIa pathway was activated, for HT-29 cells, expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group, gene expressions of AREG and EREG were 0.55±0.09 vs.0.99 ±0.09, 0.67±0.10 vs.1.02±0.02, protein expressions of EREG were 0.54±0.09 vs.1.04±0.13, all P<0.05. For LoVo cells, expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group, gene expression of AREG were 1.87±0.39 vs. 0.93±0.23, protein expressions of AREG and EREG were 3.09±0.73 vs.1.11±0.21, 1.53±0.19 vs.0.97±0.23, all P<0.05. The regulating effect of AREG and EREG mRNA expression by FVIIa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression. For HT-29 cells, activation of EGFR pathway induced no significant TF mRNA expression, FVII mRNA expression was not detected. However,for LoVo cells, activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FVII, expressions were 1.53±0.23 vs.1.00±0.23, 53.20±6.08 vs.1.00±0.15, all P<0.05. CONCLUSION: In colon cancer cell LoVo, when activated, TF/FVIIa pathway and EGFR pathway could interact through upregulating the other pathway's effectors, and mutant KRAS might play a critical role in the two pathways' cross talk.

Activity of factor VII in patients with isolated blunt traumatic brain injury: association with coagulopathy and progressive hemorrhagic injury.

BACKGROUND: Given the importance of factor VII (FVII) in extrinsic pathway of coagulation cascade, we sought to elucidate the relationship between FVII and traumatic brain injury-induced coagulopathy and progressive hemorrhagic injury (PHI). METHODS: Eighty-one patients with isolated traumatic brain injury, 16 years or older, were recruited between 2010 and 2012. Blood was collected on arrival in the emergency department and analyzed with activated partial thromboplastic time, international normalized ratio, platelet count, and activity of FVII. Coagulopathy was defined as thrombocytopenia (platelet count < 120,000/µL) or elevated international normalized ratio of greater than 1.2 or prolonged activated partial thromboplastin time greater than 40 seconds at admission. PHI was present when the follow-up computed tomographic scan reported any increase in size or number of the hemorrhagic lesions. Logistic regression examined the risks for coagulopathy and PHI. RESULTS: Mean (SD) FVII activity in patients with coagulopathy was 85.69% (34.88%), which was significantly lower than patients without coagulopathy (99.57% [29.37%], p = 0.04). Isolated traumatic brain injury patients with FVII activity less than 77.5% have an odds ratio for coagulopathy of 5.52 (95% confidence interval, 1.82-16.68; p = 0.03) relative to patients with FVII activity of 77.5% or greater. Mean (SD) FVII activity in patients with PHI was 70.76% (18.21%), which was significantly lower than patients without PHI (105.76% [32.27%], p < 0.001). A stepwise logistic regression analysis identified FVII less than 77.5% (odds ratio, 4.53; 95% confidence interval, 1.62-12.67; p = 0.004) as a predisposing risk factors independently associated with the presence of PHI. The overall mortality rate in the study population was 7.4% (n = 6). The plasma FVII in death patients (91.44% [47.19%]) was slightly lower than that in survival patients (92.01% [32.04%]). However, there was no statistical difference between the two groups (p = 0.95). CONCLUSION: A decrease of FVII activity significantly contributes to the coagulopathy and PHI in patients with isolated traumatic brain injury. LEVEL OF EVIDENCE: Prognostic study, level III.

[Identification and functional analysis of a novel missense mutation Ser250Phe underlying congenital coagulation factor â ¦ deficiency].

OBJECTIVE: To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family. METHODS: The FⅦ gene (F7) was analyzed by DNA sequencing, and the FⅦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a FⅦ-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of FⅦ deficiency caused by the FⅦ-250 Phe mutation. RESULTS: The patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both FⅦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of FⅦ-250 Ser and FⅦ-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant FⅦ-250 Phe and wild type FⅦ-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. FⅦ-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant FⅦ-250 Phe could be normally synthesized in the cells but was inefficiently secreted. CONCLUSION: Compound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the FⅦ deficiency in this patient. The novel FⅦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.

Tissue factor signals airway epithelial basal cell survival via coagulation and protease-activated receptor isoforms 1 and 2.

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Are vasomotor symptoms associated with alterations in hemostatic and inflammatory markers? Findings from the Study of Women's Health Across the Nation.

OBJECTIVE: Emerging research suggests links between menopausal hot flashes and cardiovascular risk. The mechanisms underlying these associations are unclear due, in part, to the incomplete understanding of the physiology of hot flashes. We aimed to examine the longitudinal associations between hot flashes/night sweats and both inflammatory and hemostatic markers, controlling for cardiovascular risk factors and estradiol concentrations. METHODS: Participants in the Study of Women's Health Across the Nation (n = 3,199), a longitudinal cohort study, were aged 42 to 52 years at cohort entry. Women completed interviews (hot flashes, night sweats: none, 1-5, and ≥6 d in the past 2 weeks), physical measures (blood pressure, height, weight), and a blood draw (high-sensitivity C-reactive protein, plasminogen activator inhibitor-1, factor VIIc, tissue plasminogen activator antigen [tPA-ag], fibrinogen, glucose, serum estradiol) yearly for 8 years. Hot flashes/night sweats were examined in relation to each inflammatory/hemostatic marker in linear mixed models adjusting for demographic factors, cardiovascular risk factors, and medication use, as well as serum estradiol. RESULTS: Compared with experiencing no flashes, reporting hot flashes was associated with higher tPA-aglog (hot flashes 1-5 d: percent change, or % change [95% CI], 3.88 [2.22-5.58]; P < 0.0001; ≥6 d: % change [95% CI], 4.11 [1.95-6.32]; P < 0.001) and higher factor VIIclog (hot flashes ≥6 d: % change [95% CI], 2.13 [0.80-3.47]; P < 0.01) in multivariable models. Findings persisted after adjusting for estradiol. Findings for night sweats were similar but attenuated with adjustment. CONCLUSIONS: Frequent hot flashes were associated with higher factor VIIc and tPA-ag. Hemostatic pathways may be relevant to understanding hot flash physiology and links between hot flashes and cardiovascular risk.

Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice.

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

Milestones and perspectives in coagulation and hemostasis.

Hemostasis is traditionally defined as the physiologic process whereby bleeding is antagonized and possibly stopped to minimize blood loss. The first medical description of the clinical and inherited features of hemostasis can be dated back more than 1000 years, when Abu al-Qasim Khalaf ibn 'Abbas al-Andalusi al-Zahrawi' medical treatise provided some initial insights into this puzzling process. Since then, continuous and revolutionary scientific developments have contributed to decoding several aspects of this intricate but essential physiologic phenomenon, providing a reliable model to explain the leading mechanisms involved. Although the point at which bleeding stops is commonly referred to as "coagulation," blood coagulation is actually only one part of a two-part hemostatic process that develops through sequential steps referred to as primary and secondary hemostasis. Throughout its activation and development, the coagulation cascade is strictly regulated by a series of natural inhibitors, which prevent unnecessary and excessive clotting. The aim of this article is to provide a concise overview of the major discoveries and past and current perspectives in coagulation and hemostasis.

The use of recombinant activated factor VII in platelet-associated bleeding.

Recombinant activated factor VII (rFVIIa) was originally developed for the treatment of spontaneous and surgical bleeding of hemophiliacs with inhibitors. Along with the elucidation of its molecular mechanism of action, rFVIIa has been successfully used over the last few years in a wide range of non-hemophilic bleeding conditions. The aim of this review is to summarize the current clinical experience on the use of rFVIIa in the management of bleeding associated with congenital or acquired platelet disorders.

Hypercoagulability in chronic kidney disease is associated with coagulation activation but not endothelial function.

INTRODUCTION: Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease. MATERIALS AND METHODS: We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined. RESULTS: Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function. CONCLUSIONS: Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

Measurement of functional fibrinogen levels using the Thrombelastograph.

STUDY OBJECTIVE: To validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for functional fibrinogen. DESIGN: Correlation study of the Thromboelastograph assay with two conventional fibrinogen assays by the standard Clauss method. SETTING: Research laboratory of a university medical center. PARTICIPANTS AND INTERVENTIONS: Blood samples were obtained from 19 healthy volunteers. MEASUREMENT AND MAIN RESULTS: Thromboelastograph assays, using heparinized whole blood from 19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)-generated clot shear elasticity in dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus Hct gave linear correlations (r2 = 0.746) with Gf = 1258 - 17.8 x % Hct. A commercial collection of 19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X- or factor VII-deficient donors and one was from a coumadin-treated donor. There was a linear correlation of Activatorf Gf with functional fibrinogen (r2 = 0.940) with Gf = -730 + 9.21 x fibrinogen (mg/dL). CONCLUSION: Thrombelastography with Activatorf may be used to determine fibrinogen levels in whole blood.

Factor VII deficiency: defining the clinical picture and optimizing therapeutic options.

Factor VII (FVII) deficiency is the most frequent among rare congenital bleeding disorders, accounting for one symptomatic individual per 500,000 population, apparently without any racial/ethnic predilection. FVII deficiency prevalence in the general population is probably higher because of the presence of asymptomatic and poorly symptomatic individuals. In accordance with the role of FVII as part of the initiating complex of the extrinsic coagulation pathway, laboratory diagnosis is easy, because FVII deficiency is the only congenital bleeding disorder characterized by isolated prolonged prothrombin time. Molecular diagnosis is available, and a broad spectrum of mutations has been characterized in the FVII gene, which is located in chromosome 13. Clinical manifestations are heterogeneous, ranging from severe life-threatening haemorrhages, such as cerebral, gastrointestinal, and joint haemorrhages, to miscellaneous minor bleeding. The main clinical features in our database (International Registry on Congenital FVII Deficiency database, n = 515) are as follows: (i) the absence of a clear-cut and consistent correlation between bleeding symptoms and FVII clotting levels; (ii) an excess of symptomatic women compared with men; (iii) frequent surgery-related bleeding, which is often a diagnostic tool in previously asymptomatic individuals. Several therapeutic options are possible, including plasma-derived and recombinant products, but therapeutic schedules, optimal dosages, and administration times still have to be precisely defined, and clinical studies, including online registries such as the Seven Treatment Evaluation Registry, are actually ongoing to achieve in a better manner a safe, rational and standardized substitution treatment for this congenital disorder.

La nueva cascada de la coagulación y su posible influencia en el difícil equilibrio entre trombosis y hemorragia / The new coagulation cascade and its possible influence on the delicate balance between thrombosis and hemorrhage

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A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Functional analysis of the genetic variability in the F7 gene promoter.

The FVII level is considered a risk factor for cardiovascular disease. Some of the polymorphic differences in the promoter of the F7 gene have been associated with variations in FVII levels. However, linkage disequilibrium among those polymorphisms has made it difficult to pinpoint the true functional variants, so contradictory results have often appeared among various studies. We provide new findings of the effect of the polymorphisms in the promoter region of F7. In vitro transfection of 15 plasmids containing different combinations of F7 promoter polymorphisms was performed in HepG2 cells. We found that allelic variants -323ins10 and -122C strongly reduced promoter activity and that allelic variant -402A significantly increased promoter activity. We report the effect of a novel variant (-2989A) that significantly increases F7 expression levels. However, this novel allelic variant is in strong linkage disequilibrium with the -323ins10 variant in our Spanish population, which has a clear dominant effect over the -2989A variant and completely masks its effect. Our results have important implications for mapping genes affecting complex diseases using association studies. That is, they imply that true functional variants should be chosen to confirm the analyses and to ensure that the results can be reproduced in other populations. In addition, our results suggest that it would be informative to screen for the -2989A variant in other populations, since it may well be a risk factor for cardiovascular disease in populations where it does not appear with the decanucleotide insertion.

Activation of cancer cell migration and invasion by ectopic synthesis of coagulation factor VII.

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.

Pro/con debate: does recombinant factor VIIa have a role to play in the treatment of patients with acute nontraumatic hemorrhage?

Perhaps it is not surprising that in the critical care environment, where lives are frequently on the line, off-label use of certain drugs is relatively common. In general, there are two camps of opinion on this type of utilization. One camp would suggest that potentially life saving products cannot ethically be withheld from patients who may benefit. The other camp would counter that it is inappropriate to administer products if the risk/benefit ratio has not been clearly defined in clinical trials. Off-label use of factor VII is debated in this issue of Critical Care for a patient with uncontrolled nontraumatic hemorrhage. Perhaps this product promotes additional discussion given that its ability to control bleeding can be dramatic, yet its costs and potential for complications high.

Thrombogenic risk factors for atherothrombosis.

Thrombosis superimposed on a disrupted plaque is the proximate event that triggers most acute ischemic syndromes and episodes of sudden cardiac death. A significant number of acute ischemic events occur in individuals without traditional atherosclerosis-related risk factors. In an attempt to pinpoint additional risk factors, researchers are examining the thrombotic cascade and the cellular components, plasma proteins, and endothelium-derived mediators, as well as their genetic polymorphisms, that may affect this system. This article enumerates a number of potential hemostatic risk factors and discusses the evidence linking them to atherothrombotic events.

Does endothelium agree with the concept of idiopathic hepatic vessel thrombosis?

AIM: To investigate the major steps of thrombogenesis and to identify the differences in these steps between idiopathic patient group and control group. METHODS: Fibrinogenesis was studied by measuring the activated factor VII, total and free levels of tissue factor pathway inhibitor (TFPI). The fibrinolysis step was investigated by determining the global fibrinolytic capacity. The endothelial function was assessed by measuring the levels of soluble adhesion molecules, namely soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1) and soluble E-selectin molecule. The exclusion criteria from "idiopathic" patient group were abdominal surgery, pregnancy, use of oral contraceptives, anti-phospholipid syndrome, Behçet's disease, cancer, myeloproliferative diseases. The congenital factors like mutations of factor-V Leiden and prothrombin, deficiencies of proteins C and S, antithrombin, hyperhomocysteinemia and hyperfibrinogenemia were ruled out. The total number of patients was reduced from 96 to 9 (7 with portal vein thrombosis, 2 Budd Chiari syndrome) by exclusion criteria. RESULTS: The levels of adhesion molecules sICAM-1, sVCAM-1, free TFPI levels and global fibrinolytic capacity were significantly different (P<0.05) in the patient group indicating an endothelial dysfunction and a lower fibrinolytic activity. CONCLUSION: These results show that this patient group should be tested by means of endothelial dysfunction and managed differently.