Plasmin-dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia.

BACKGROUND: The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. OBJECTIVE: To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. METHODS: Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. RESULTS: The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. CONCLUSION: The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

[Experimental model of orthotopic uterus transplantation in the laboratory rat]. / Model experimental de transplant uterin ortotopic la sobolanul de laborator.

This study develops and standardizes an experimental model of uterus transplantation in the laboratory rat. Twelve orthotropic uterus transplantation were done. Animals were randomized in three groups. Postoperative survival was 100% and 75% at 72 hours. Recipients were euthanased at 24 hours, 48 hours and 72 hours and the grafts were harvested. Patency of the microsurgical anastomoses was 100% at 24 hours, 63% at 48 hours and 0% at 72 hours. The explanted uterine grafts were fixed in formalize and analyzed under light microscopy. The acute allograft rejection starts during the second day after transplantation. In additional dissection, anatomy of the pelvic region with regard to the topography of the uterus, tube and ovarian vessels was studied. This model of uterus transplantation in rats proposes a standardized tool for further research regarding cellular mechanisms of the acute allograft rejection and, for future, pregnancy of the transplanted uterus.

Hereditary motor and sensory neuropathy-russe: new autosomal recessive neuropathy in Balkan Gypsies.

A novel peripheral neuropathy of autosomal recessive inheritance has been identified in Balkan Gypsies and termed hereditary motor and sensory neuropathy-Russe (HMSN-R). We investigated 21 affected individuals from 10 families. Distal lower limb weakness began between the ages of 8 and 16 years, upper limb involvement beginning between 10 and 43 years, with an average of 22 years. This progressive disorder led to severe weakness of the lower limbs, generalized in the oldest subject (aged 57 years), and marked distal upper limb weakness. Prominent distal sensory loss involved all modalities, resulting in neuropathic joint degeneration in two instances. All patients showed foot deformity, and most showed hand deformity. Motor nerve conduction velocity was moderately reduced in the upper limbs but unobtainable in the legs. Sensory nerve action potentials were absent. There was loss of larger myelinated nerve fibers and profuse regenerative activity in the sural nerve. HMSN-R is a new form of autosomal recessive inherited HMSN caused by a single founder mutation in a 1 Mb interval on chromosome 10q.

Bemiparin and fluid flow modulate the expression, activity and release of tissue factor pathway inhibitor in human endothelial cells in vitro.

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

In situ analysis of tissue factor-dependent thrombin generation in human atherosclerotic vessels.

Tissue factor (TF) is expressed in human atherosclerotic plaques where it may contribute to the thrombogenicity of the lesions and their progression toward unstable syndromes and acute myocardial infarction. In this study we tested the hypothesis that thrombin generation takes place in the lesion. Localisation of TF, factor VII (FVII), factor X/Xa (FX/Xa), thrombin, thrombin receptor PAR-1 and FXa receptor EPR-1 was done by immunostaining, ligand binding, or immunogold electron microscopy. Quantitation of TF antigen was done using a modified ELISA on fixed tissue sections. The amount of antigen was correlated with the pattern and intensity of immunostaining as detected on consecutive sections using confocal microscopy. TF-dependent generation of FXa on cryosections was used to assess the functional activity of TF. Active thrombin was detected using hirudin as a specific probe, followed by anti-hirudin IgG. Our light microscopy and immunogold electron microscopy results showed that the factors involved in TF-dependent coagulation are localised in atherosclerotic plaques in close proximity and colocalise with active thrombin and fibrin deposits. We have detected 3 to 7-fold increase of TF antigen and TF-dependent FXa generation in atherosclerotic vessels as compared with controls. Hirudin binding proved that active thrombin is present within the lesions. In conclusion, our data show that active coagulation factors are generated within atherosclerotic lesions and co-localise with their cellular receptors. These findings may suggest possible roles of the TF-dependent coagulation pathway in the intramural fibrin deposition and the progression of the atherosclerotic lesions.

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

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

Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels.

Tissue factor (TF) pathway inhibitor (TFPI) is the major downregulator of the procoagulant activity of the TF-factor VIIa (FVIIa) complex (TF. FVII). The active TF present in the atherosclerotic vessel wall is proposed to be responsible for the major complication of primary atherosclerosis, namely, acute thrombosis after plaque rupture, but our knowledge of the sites of TFPI expression in relation to TF remains fragmentary. The aim of this study was to investigate the expression, localization, and activity of TFPI and its relation to the activity and distribution of TF in the normal and atherosclerotic vessel wall. We applied a novel approach in which serial cross sections of human vascular segments were used to perform a complete set of assays: immunolabeling for TFPI and/or TF, in situ hybridization for the expression of TFPI mRNA, ELISA for the determination of TFPI antigen, and functional assay for the activity of TFPI and TF. In healthy vessels, TFPI protein and mRNA are present in luminal and microvascular endothelial cells (ECs) and in the medial smooth muscle cells (SMCs). In atherosclerotic vessels, TFPI protein and mRNA frequently colocalized with TF in ECs overlying the plaque and in microvessels, as well as in the medial and neointimal SMCs, and in macrophages and T cells in areas surrounding the necrotic core. At the ultrastructural level, immunogold electron microscopy confirmed the localization of TFPI in ECs, macrophages/foam cells, and SMCs. In ECs and SMCs, the gold particles decorated the plasmalemma proper and the caveolae. ELISA on cross sections revealed that atherosclerotic tissues contain more TFPI than do the healthy vessels. TFPI was functionally active against TF. FVIIa-induced coagulation, and its activity was higher in those tissues that display less TF. The largest amount of TFPI and TF were detected in complicated arterial plaques. By immunofluorescence, TFPI colocalized with platelet- and fibrin-rich areas within the organized thrombi. Atherosclerotic vessel sections promote activation of factor X, which is dependent on the presence of TF and enhanced by preincubation of the sections with anti-TFPI IgG. Taken altogether, our results suggest that TFPI is largely expressed in the normal vessel wall and enhanced in the atherosclerotic vessel, in a manner suggesting a significant role of TFPI in the regulation of TF activity.

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons.

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.

Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor-factor VIIa complex (TF. FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF. FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2. 4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue (TF)-dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be physiological importance in controlling TF-mediated thrombin generation.

Altered distribution of some surface glycosaminoglycans and glycoconjugates on human blood platelets in diabetes mellitus.

The distribution of some proteoglycan anionic sites and carbohydrate moieties on the diabetic platelet surface has been investigated by cytochemical methods. Human blood platelets obtained from either diabetic patients (serum glycaemia 9.6-19.2 mmol/L) or normal donors were labeled with specific stains (Ruthenium Red, Safranin O) or with biotinylated lectins (WGA, RCA(120))/Straptavidin-gold. All the probes were processed in a standardised manner for transmission electron microscopy. Results showed a modified pattern of diabetic platelet surface labeling as compared to normals. The anionic sites in the glycocalyx (particularly proteoglycans), evenly distributed all over the normal platelet plasma membrane, suffered a marked redistribution, with cluster formation, on the surface of diabetic platelets. The sugar moieties were also differently exposed: as compared to normal, diabetic platelets expressed a 1.5-fold lower number of WGA-binding sites (sialic acid) and a 2.5-fold higher number of RCA(120)-binding sites (galactose residues). We conclude that the distribution of some platelet surface components, potentially implicated in the adhesion and aggregation processes, is significantly modified on platelets from diabetic patients.

Enhanced prothrombin and intrinsic factor X activation on blood platelets from diabetic patients.

The present work was conducted to bring information about the enhanced procoagulant activity of blood platelets in diabetes mellitus, as compared to normal control platelets. The inquiry was performed on platelets from either human patients or hamsters with streptozotocin-induced diabetes. The experiments comprised functional assays of factor Xa and thrombin generation in the presence of normal or diabetic platelets, using saturating amounts of coagulation factors. The prothrombinasic and tenasic complexes activation was measured by using discontinuous amidolytic tests with chromogenic substrates (S-2238 for FXa and S-2222 for thrombin). The results showed that, in contrast to the low prothrombin and factor X converting activity of normal platelets, the thrombin and FXa generation on diabetic platelets was increased by 3-7 times for either humans or hamsters. These findings may be relevant for the pathogenesis of thrombotic complications known to occur in diabetes mellitus.

Some Major Plasmalemma Proteins of Human Diabetic Platelets are Involved in the Enhanced Platelet Adhesion to Cultured Valvular Endothelial Cells.

To extend our investigations on the interaction between diabetic platelets and endothelium, we tried to identify the molecular components involved in the increased adhesiveness of diabetic platelets to cultured valvular endothelial cells (VEC). Platelets from diabetic patients were radiolabeled with ((3)H]-adenine, incubated for 30 min at 37°C with confluent VEC grown in medium containing 4.5 g/L glucose, and the monolayer-associated radioactivity was used to calculate the adhesion index. To identify the plasmalemma proteins involved in the adhesion process, platelets were incubated for 30 min prior to (the) adhesion assay with one of the following monoclonal antibodies: AP-2 (anti GP IIb-IIIa), AP-5 (anti GP IIIa), TM 83 (recognizes an epitope other than the fibrinogen binding site in GP IIIa), PECAM 1.2 (anti PECAM-1) or a polyclonal anti-fibronectin receptor (anti FnR). In addition, two synthetic peptides, RGDS and GPRP, applied alone or together, were used. The effect of paraformaldehyde fixation of diabetic platelets on their adhesion was also tested. The results showed that except for TM 83, all antibodies reduced significantly (∼45%) the adhesion index of diabetic platelets to VEC. The synthetic peptides also decreased the adhesion by ∼30%. Paraformaldehyde-fixed diabetic platelets fail to adhere to VEC. Taken together these observations suggest that: (1) platelet GP IIb-IIIa complex, PECAM-1 and FnR may be instrumental in the increased adhesion of diabetic platelets to VEC; (2) fibrinogen binding sites in the GP IIb-IIIa complex and fibrinogen/fibrin are important contributors to the adhesion process and (3) impairment of diabetic platelets adhesion by chemical fixation, supports the role of cytoskeletal proteins reorganization and redistribution of some plasmalemma components during adhesion.

Increased adhesion of human diabetic platelets to cultured valvular endothelial cells.

Diabetes is accompanied by impaired platelet function and accelerated vascular disease. To find out whether a correlation exists between these two complications, and if modifications occurring in diabetic platelets influence their relationship with endothelium, we have studied the interaction between platelets isolated from plasma of diabetic patients and bovine valvular endothelial cells (VEC), in culture. For quantitative analysis, normal and diabetic [3H]-adenine-labeled platelets were incubated with confluent VEC grown in Dulbecco's modified Eagle medium, containing 4.5 g/l glucose, for 30 min at 37 degrees C. After extensive washing and solubilization of the monolayer, the calculated adhesion index showed a two-fold increased adherence of diabetic platelets to VEC as compared to normal platelets. Statistical analysis (by Pitman randomization test) indicated that the adhesion was significantly higher (p = 0.0003) than that of normal platelets to VEC. To partially identify the membrane components implicated in the adhesion process, either platelets or VEC were treated with neuraminidase, trypsin or heparinase prior to the adhesion assay. Trypsin or neuraminidase treatment of platelets significantly diminished their adherence to VEC, suggesting a role of platelets sialylated glycoproteins in the adhesion process. Neuraminidase or heparinase treatment of VEC increased the adhesion of both normal and diabetic platelets, indicating that the cell membrane sialyl residues and heparan sulfate participate in the normal thromboresistant properties of VEC. Transmission and scanning electron microscopy revealed a close apposition between platelets and VEC with the formation of an adhesion plaque, characterized by fine fibrillar bridges between the plasma membranes of the two cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the platelet surface charge in rabbits with experimental hypercholesterolemia.

The effect of hypercholesterolemia on the platelet surface charge was examined in rabbits fed a lipid-rich diet (0.5% cholesterol and 5% butter). The strong anionic sites were detected with cationized ferritin (CF) pI 8.4, and the sialic acid concentration was evaluated by biochemical assays. In normal rabbits (average plasma cholesterol 0.36 +/- 0.05 mg/ml, and total platelet sialic acid 30.03 +/- 6 micrograms/mg protein) the platelet surface displayed a homogeneous distribution of CF, which also labeled the open canalicular system. Beginning with the third week of diet, at a plasma cholesterol level of 4.6 +/- 0.3 mg/ml, a reduction in the overall platelet negative charge was observed. As the diet progressed and the plasma cholesterol level increased, the CF binding to platelet surface diminished up to an almost total disappearance when the plasma cholesterol reached 18 mg/ml (the 20th week of diet). At the same time a progressive decrease in the sialic acid content up to 5.1 micrograms/mg protein was detected. These results suggest that diet-induced hyperlipidemia causes significant alterations in the platelet surface negative charge, especially in the sialic acid content.