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Fibrinolysis: the key to new pathogenetic mechanisms.

The fibrinolytic system includes a broad spectrum of proteolytic enzymes with physiological and pathophysiological functions in several processes, such as haemostatic balance, tissue remodeling, tumor invasion, angiogenesis and reproduction. The main enzyme of the plasminogen activator system is plasmin, which is responsible for the degradation of fibrin into soluble degradation products. The activation of plasminogen into plasmin is mediated by two types of activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). The activity of both is regulated by specific plasminogen activator inhibitors (PAIs). There are 3 types of PAIs described so far but the most important fibrinolytic inhibitor in vivo is PAI type 1 (PAI-1). Among others, the presence of metabolic syndrome and the -675 4G/5G promoter polymorphism are known to be modulators of PAI-1 levels. Besides their fibrinolytic profile, plasmin and plasminogen activators are implicated in tissue proliferation and cellular adhesion, as they can proteolytically degrade the extracellular matrix and regulate the activation of both growth factors and matrix metalloproteinases. By all these means, the fibrinolytic system is also involved in physiological processes, and in pathological situations such as thrombosis, arteriosclerosis, endometriosis and cancer. PAI 1 has been studied in different settings with thrombotic pathophysiology, such as coronary artery disease and ischaemic stroke. Controversial results have been published and concerns about study designs or presence of confounders have been claimed to be responsible of them. Recently, its involvement in adverse thrombotic events related to the modern drug-eluting coronary stents has renewed the interest of its study. PAI-1 also plays an important role in signal transduction, cell adherence, and migration. Indeed, studies of several types of cancers, including breast cancer, have shown that increased uPA and PAI-1 levels are associated with aggressive tumor behavior and poor prognosis. Endometriosis is defined by the presence of endometrial glands and stroma outside the uterus with marked ability to attach and invade the peritoneum. It is one of the most frequent benign gynecological diseases that affect women with pelvic pain or infertility during their reproductive age. Immune system disorders, genetic predisposition, altered peritoneal environment and endometrial alterations are believed to increase the susceptibility to endometriosis. The plasminogen activator system may be involved in this process, where local extracellular proteolysis plays a crucial role. Altered expression of several components of the fibrinolytic system in both eutopic and ectopic endometrium and peritoneal fluid of women with the disease has been implicated not only in the onset, but also in the progression of the endometriotic lesions.

Functionally active protein C inhibitor/plasminogen activator inhibitor-3 (PCI/PAI-3) is secreted in seminal vesicles, occurs at high concentrations in human seminal plasma and complexes with prostate-specific antigen

Protein C inhibitor (PCI) is a heparin-dependent serpin present in a native form in plasma at concentrations of 5 micrograms/mL. In vitro, PCI inhibits activated protein C (APC), thrombin, plasma kallikrein (KK) and urokinase-(uPA) and tissue-type plasminogen activator (tPA), and we have shown in vivo inhibition of APC, uPA and KK by PCI. In order to further characterize the physiological role of PCI, we have measured the level of PCI in several biological fluids. PCI antigen was assayed by ELISA and PCI activity was measured by its capability to form complexes with APC in the presence of heparin. Seminal plasma from voluntary donors had PCI levels (160 +/- 20 micrograms/mL, mean +/- SD) about 30 or 40 times higher than those found in blood plasma. Patients under a fertilization program had significantly reduced PCI seminal levels (110 +/- 35 micrograms/mL). Seminal plasma PCI retained about 45% of its activity immediately after ejaculation, and the activity rapidly decreased following incubation of seminal plasma at 37 degrees C, in parallel with the appearance of complexes of PCI with prostate-specific antigen (PSA). PCI was present in seminal vesicle secretion, obtained by autopsy, at concentration similar to that observed in semen, was mostly active and was not inactivated by incubation of secretion at 37 degrees C. The mean functional and antigen levels of PCI in urine from normal donors were 0.58 and 0.25 micrograms/mL, respectively, whereas in saliva these levels were 20 and 0.8 ng/mL, respectively. Amniotic fluid contained PCI antigen levels of 2.1 +/- 0.2 microgram/mL. These results show that PCI is secreted in the seminal vesicles in a functional form, and suggest that PSA, a major secretory component of the prostate, is responsible for its inactivation. They also suggest a physiological role of PCI in reproduction, and show that PCI is present in various biological fluids.

PAI-1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects

Impaired fibrinolysis as a result of increased plasminogen activator inhibitor‐1 (PAI‐1) levels in plasma is a common finding in patients with deep vein thrombosis (DVT). A 4G/5G polymorphism in the promoter region of the PAI‐1 gene has been reported to influence the levels of PAI‐1. The 4G allele was found to be associated with higher plasma PAI‐1 activity (act), but contradictory results on the incidence of the 4G allele in DVT patients have been reported. The aim of this study was to analyse whether the PAI‐1 promoter 4G/5G genotype increases the risk of venous thrombosis in subjects with thrombophilic defects, and to determine the distribution of the PAI‐1 4G/5G genotype and its relation to plasma PAI‐1 levels in 190 unrelated patients with DVT in comparison with a control group of 152 healthy subjects. No differences between the 4G/5G allele distribution in the DVT group (0·43/0·57) and in the control group (0·42/0·58) were observed. However, the presence of the 4G allele significantly increased the risk of thrombosis in patients with other thrombophilic defects. Significantly higher PAI‐1 levels were observed in DVT patients than in the controls. Our results also showed significant differences in the plasma levels of PAI‐1 antigen (ag) and PAI‐1 act among the 4G/5G genotypes in DVT patients. A multivariate analysis revealed that, in the DVT group, PAI‐1 ag levels were influenced by the 4G allele dosage, triglyceride levels and body mass index (BMI). The influence of the 4G allele dosage on PAI‐1 levels was independent of the triglyceride levels and BMI. In the control group, no significant correlation between PAI‐1 levels and 4G allele dosage was observed. In conclusion, the PAI‐1 promoter polymorphism was found to have an influence on PAI‐1 levels in DVT patients and on the risk of venous thrombosis in subjects with other genetic thrombophilic defects.

Hyperlipidaemia and venous thromboembolism in patients lacking thrombophilic risk factors

Summary. To ascertain the potential contribution of serum lipids to the development of deep vein thrombosis (DVT), a case–control study was conducted in 143 DVT patients lacking thrombophilic risk factors and in 194 age‐ and sex‐matched controls. DVT patients showed significantly higher body mass indices (BMI), and triglyceride levels than did controls (P < 0·001 and P = 0·045 respectively). Using multivariate analysis, BMI was the only variable which remained statistically different, thus the risk of DVT was associated with obesity (odds ratio = 2·49). These results were confirmed when additional control for fibrinogen and plasminogen activator inhibitor type 1 (PAI‐1) was carried out in a subgroup of cases and controls. When idiopathic (n = 39) and secondary (n = 104) patients with DVT were compared, the former showed a higher mean age, a higher proportion of men, and higher cholesterol levels. Age, sex and total cholesterol were statistically different by multivariate analysis. After age was dichotomized as ≥ 50 years and cholesterol ≥ 5·69 mmol/l, all three variables constituted independent risk factors for idiopathic DVT, with odds ratios of 2·73 for ages ≥ 50 years; 3·72 for men and 2·67 for cholesterolaemia ≥ 5·69 mmol/l. Obesity thus constitutes an independent risk factor for DVT, possibly in part mediated through triglyceride, fibrinogen and PAI‐1 effects on haemostasis. In addition, cholesterolaemia levels of ≥ 5·69 mmol/l constitute an independent risk factor for idiopathic DVT.

Purification and characterization of plasma protein C inhibitor

Plasma protein C inhibitor (PCI) was purified to homogeneity (greater than 95%) with good recovery (greater than 25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr = 57,000 into two products with Mr = 54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr = 54,000. PCI did not detectably inhibit alpha-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 x 10(4), 0.22 x 10(4), 0.08 x 10(4), 0.61 x 10(4), 2.01 x 10(4), 6.50 x 10(4), and 9.03 x 10(4) M-1s-1 in the absence of heparin and 1.58 x 10(6), 0.43 x 10(6), 0.03 x 10(6), 0.52 x 10(6), 0.09 x 10(6), 0.18 x 10(6) and 0.74 x 10(6) M-1s-1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 x 10(4) and 0.26 x 10(4) M-1s-1 in the absence of heparin and 0.54 x 10(6) and 0.71 x 10(6) M-1s-1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PGI and is not essential for the heparin stimulation of this reaction.

Thrombin‐activatable fibrinolysis inhibitor in young patients with myocardial infarction and its relationship with the fibrinolytic function and the protein C system

Summary. Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a fibrinolytic inhibitor. Studies in coronary artery disease have reported increased TAFI activity (TAFI Act) and low TAFI antigen (TAFI Ag) levels. This controversy might be explained by the polymorphisms of its gene. Only the Thr325Ile polymorphism modulates both TAFI Ag and Act levels in vitro. This study assessed TAFI Ag and Act levels, TAFI Thr325Ile polymorphism, the fibrinolytic and protein C systems and some prothrombotic mutations in a young patient group (n = 127, aged < 51 years, with myocardial infarction) and a control group (n = 99) with similar characteristics. Patients exhibited hypofibrinolysis and higher plasminogen activator inhibitor‐1 (PAI‐1) levels. Although TAFI Ag was lower, TAFI Act level was significantly higher in patients and positively correlated with PAI‐1, protein C inhibitor and the euglobulin lysis time. No differences between groups were found according to the Thr325Ile polymorphism. Irrespective of the genotype, patients had higher TAFI Act levels. The Ile‐325 variant exhibited lower TAFI Ag levels. We suggest that the hypofibrinolysis observed in these patients results from an increase in both PAI‐1 and TAFI Act, which is not related to the Thr325Ile polymorphism. Patients have high TAFI Act with low TAFI Ag levels, probably because of an increased stability of TAFI related to a fibrinolytic hypofunction.

The effect of estrogen replacement therapy with or without progestogen on the fibrinolytic system and coagulation inhibitors in postmenopausal status.

OBJECTIVE The aim of this study was to analyze several fibrinolytic components and coagulation inhibitors in postmenopausal women and had to evaluate the effect of hormone replacement therapy. STUDY DESIGN Several hemostatic parameters were evaluated in 75 postmenopausal women before and after 3 to 4 and 12 months of hormone therapy. RESULTS An increase in plasma fibrinolytic activity primarily related to a significant increase in tissue-type plasminogen activator and a decrease in plasminogen activator inhibitor type 1 was observed in women receiving hormone replacement therapy. A significant decrease in protein S and lipoprotein(a) was detected under therapy. No modifications in tissue-type plasminogen activator/plasminogen activator inhibitor-1 and activated protein C/alpha 1-antitrypsin complexes, urokinase activity, plasminogen, and antithrombin III were detected. CONCLUSIONS The increase in fibrinolytic activity and the decrease in lipoprotein(a) levels observed in women receiving hormone replacement therapy could help decrease the risk of coronary disease associated with the postmenopausal state.

Determination of plasma protein C inhibitor and of two activated protein C-inhibitor complexes in normals and in patients with intravascular coagulation and thrombotic disease

We developed an ELISA to quantitate complexes of activated protein C (APC) with a major plasma APC inhibitor, alpha 1-antitrypsin (alpha 1AT) in human plasma based on the sandwich principle using two different antibodies directed towards protein C and alpha 1AT, respectively. This ELISA test was specific for APC:alpha 1AT complexes and sensitive to greater than or equal to 150 pg complex. Fifty-one of 56 healthy donors had APC:alpha 1AT complex levels above the detection limit (3 ng/ml) ranging from 4 to 14 ng/ml (mean value +/- SD: 7.6 +/- 2.5 ng/ml). Patients (n = 10) with disseminated intravascular coagulation (DIC) had detectable levels of APC:alpha 1AT complex ranging from 21 to 125 ng/ml (median: 69 ng/ml). Complexes of APC with plasma protein C inhibitor (PCI) were also measured using an ELISA sandwich assay. None of the 30 healthy donors had detectable levels (greater than or equal to 5 ng/ml) of APC:PCI complex, and plasma samples from 9 of 10 DIC patients had detectable concentrations of APC:PCI complex ranging from 10 to 63 ng/ml (median: 22 ng/ml). APC:alpha 1AT complex was detected in 25 of 26 patients with deep venous thrombosis (DVT), with levels ranging from 5 to 136 ng/ml (median: 23 ng/ml), whereas APC:PCI was detected in only 6 DVT patients, with levels between 11 and 105 ng/ml. PCI antigen levels in 70 normals ranged from 56 to 175% (mean +/- SD: 99.1% +/- 24.2%). PCI antigen levels were decreased in DIC patients, in patients with cerebral arterial thrombosis, and in DVT patients undergoing heparin therapy, but not in patients with myocardial infarction. PCI antigen levels were decreased much further in DVT patients receiving heparin compared to those not receiving heparin, showing that heparin therapy is associated with a decrease in PCI levels. The detection in normal subjects and in thrombotic patients of circulating APC:inhibitor complexes supports the view that the protein C pathway is activated during DIC and DVT. Moreover, it emphasizes that both PCI and alpha 1AT are physiologic inhibitors of APC. Thus, measurement of APC complexes may provide sensitive parameters for specific detection of activation of the clotting and protein C pathways.

MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors

STUDY QUESTION Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? SUMMARY ANSWER This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. WHAT IS ALREADY KNOWN?: An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. STUDY DESIGN, SIZE, DURATION Case-control study that involved 51 women with endometriosis and 32 women without the disease (controls). PARTICIPANTS/MATERIALS, SETTING, METHODS The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. LIMITATIONS, REASONS FOR CAUTION Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. WIDER IMPLICATIONS OF THE FINDINGS Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

microRNAs expression in endometriosis and their relation to angiogenic factors

BACKGROUND Endometriosis is a common, multifactorial disease in which angiogenesis may be involved in the growth of endometrium outside the uterus. microRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. The objective of this study was to analyze several miRNAs related to angiogenesis and the angiogenic factors, vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), in endometriotic lesions (ovarian endometrioma, peritoneal lesion and rectovaginal nodule) and eutopic endometrium from women with endometriosis. METHODS TaqMan real-time PCR was used to assess the expression of the miRNAs (miR-15b, -16, -17-5p, -20a, -21, -125a, -221 and -222), while VEGF-A and TSP-1 mRNA were assessed by real-time PCR, with SYBR Green I and VEGF-A and TSP-1 protein levels were quantified by ELISA. Included in the study were 58 women with endometriosis and 38 control women. RESULTS In paired samples, ovarian endometrioma showed significantly lower VEGF-A mRNA (P = 0.02) and protein (P = 0.002) expression than eutopic endometrium and higher expression of miR-125a (P = 0.003) and miR-222 (P <0.001). However, ovarian endometrioma had significantly higher expression of the angiogenic inhibitor TSP-1 and lower expression of miR-17-5p than eutopic endometrium (P < 0.001). Moreover, a significant inverse correlations between miR-222 and VEGF-A protein levels (-0.267, P = 0.018) and between miR-17-5p and TSP-1 protein levels (-0.260, P=0.022) were observed. Peritoneal lesions showed a significant increase in VEGF-A in comparison with ovarian endometrioma (P < 0.01). CONCLUSIONS Expression levels of miRNAs related to angiogenesis were different in eutopic endometrium from that observed in ovarian endometrioma. This could influence the expression of angiogenic factors and play a role in the pathogenesis of endometriosis.