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Biomedical subjects

S Menashi

Publications and source records attributed to S Menashi.

At least 19 recordsLinked to original sources
PubMed1995

The extracellular matrix produced by bovine corneal endothelial cells contains progelatinase A.

Progelatinase A is a matrix metalloproteinase involved in the turnover of extracellular matrix (ECM). We report that the ECM produced by bovine corneal endothelial (BCE) cells contains progelatinase A free of tissue inhibitor of metalloproteinase (TIMP2). The matrix-bound progelatinase A can be activated by APMA to generate a 62 kDa and a 45 kDa species with enzymatic activity and is inhibited by TIMP2. The bound progelatinase can be released after treatment of the ECM with gelatinase B. These studies suggest that the ECM can function as a reservoir for progelatinase A which may be readily available for cells in processes such as metastasis, angiogenesis, inflammation and wound healing.

Animals↗
PubMed1995

The effect of platelets on invasiveness and protease production of human mammary tumor cells.

Interaction of tumor cells with platelets facilitates metastasis of tumor cells. It has been proposed that platelets protect tumor cells against the host's immune defense and enhance tumor-cell extravasation. In the present work we show that platelets increase the invasiveness of 3 mammalian cell lines (MCF-7, ZR-51 and MDA-MB231) through extracellular matrix, and propose this as an additional mechanism by which platelets facilitate metastasis. Since gelatinase and urokinase have both been implicated in degradation of the extracellular matrix and cell migration, and therefore in tumor invasion, we have also analyzed whether the interaction of platelets with tumor cells can modify the secretion of these proteases by tumor cells. MDA-MB231, which was the most invasive cell line among the 3 tested and was the most potent in inducing platelet aggregation, secreted the highest level of urokinase and was the only one in which gelatinase was detected. While platelets had no significant effect on the urokinase activity expressed by these cells, they induced in MDA-MB231 an important increase in the secretion of gelatinase, which can be reproduced by both platelet membrane and platelet releasate of activated platelets. This increase in gelatinase could be responsible, at least in part, for the increased invasiveness of these cells, since added TIMP-1 significantly reduced the number of cells which traversed matrigel.

Blood Platelets↗
PubMed1994

Fibrinolysis potentiating activity following endothelial cells-platelet interaction.

When porcine endothelial cells in culture are incubated in the presence of human platelets, a 90kDa neutral proteinase activity is generated on casein gel (PECAP-Platelet Endothelial Cell Activated Protease). This activity was undetected when platelet extract or serum free EC conditioned medium were analysed under similar conditions. The optimum pH, isoelectric point, molecular weight and inhibitory profile of this activity were similar to Glu-plasmin. However, the low plasminogen content (less than 50ng/ml) in the conditioned medium of endothelial cells incubated with platelet could not contribute alone to this activity and the presence of a plasmin potentiating factor was suggested. This factor was separated from plasminogen by lysine-Sepharose chromatography.

Animals↗
PubMed1993

Comparative study of fibrinolytic activity on 937 line after stimulation by interferon gamma, 1,25 dihydroxyvitamin D3 and their combination.

Previous study showed that the secretion of urokinase (UK) by monoblastic cell line U 937 and the number of binding sites for urokinase and for plasminogen (Plg) on the cell surfaces were augmented by interferon gamma (INF tau). This induction led to an increase in fibrinolytic activity on cell surfaces. A similar increase was also observed when treating the U 937 cells with 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3. Here we report that the combination of these two agents induced a 2.7 fold increase in the plasminogen activator activity on U 937 cell surfaces in comparison with 1 fold increase induced by INF tau and 1.3 fold increase by 1,25(OH)2D3. As evaluated by a flow cytometer, the increased fibrinolytic activity induced by the combination of INF tau and 1,25(OH)2D3 could be attributed to the increase of the number of binding sites both for UK (3.7 x 10(4) vs 1.2 x 10(4) per cell) and for Plg (16.2 x 10(4) vs 3.6 x 10(4) per cell), accompanied by an increased expression of CD 14, which is an antigen of differentiation on cell surfaces. These results suggest that the expression of urokinase receptors and plasminogen receptors may be coupled together by unknown intracellular mechanisms during cell differentiation, and support the idea that the concomitant regulation of these two receptors for UK and Plg is an important aspect in cell associated-fibrinolytic activity.

Calcitriol↗
PubMed1993

Endothelial cell proteases: physiological role and regulation.

Endothelial cell-derived proteases can be classified according to their physiological role. The proteases involved in extracellular matrix degradation are important in endothelial cell migration and thereby in angiogenesis. They include the urokinase-type plasminogen activator (uPA) and the metalloproteases, collagenases, gelatinases and stromelysin. uPA secreted from endothelial cells remains associated with the cell membrane, on specific receptors localized in the vicinity of the receptors for plasminogen. This favours the local activation of plasminogen into plasmin. Plasmin, generated on the cell surface, is fully active as it is not inhibited by alpha 2-antiplasmin. Plasmin acts directly by degrading some components of the extracellular matrix and indirectly by activating the prometalloproteases. Secretion of PAI by migrating cells is generally stimulated by the same factors that induce uPA secretion, limiting the degradation of the matrix to the pericellular path. The degradation of the fibrin clot involves the tissue-type plasminogen activator tPA, which like the uPA activates plasminogen to plasmin. This system is also regulated by two different mechanisms. On the one hand, fibrin itself favours its own degradation by formation of a ternary complex, fibrin-plasminogen-tPA, in which the affinity of tPA for plasminogen is markedly increased, as compared to the affinity of unbound tPA. In addition, plasmin generated on the clot is protected from inhibition by alpha 2-antiplasmin. On the other hand, as for uPA, tPA is inhibited by PAI-1. The importance of the regulation of this system is illustrated by the thrombotic risk observed when there is either a decrease in tPA or an increase in PAI-1, and inversely by haemorrhages in the case of increase in tPA.

Animals↗
PubMed1993

Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function.

Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01-0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with < 0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min, indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31 +/- 0.90 for resting platelets, down-regulated to 0.84 +/- 0.13 (P < 0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63 +/- 2.21 (P > 0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.

Antithrombins↗
PubMed1991

Modulation of endothelial cells fibrinolytic activity by platelets.

Interaction between endothelial cells (EC) and platelets in culture was shown to regulate the fibrinolytic system of the aortic EC. Untreated porcine EC from aorta exhibited almost no net fibrinolytic activity and zymographic assay have shown a single fibrin lysis band of 105 kDa corresponding to a tPA-PAI complex. Incubation of aortic EC with intact platelets stimulated a cell-associated fibrinolytic activity of the urokinase type as evidenced by a plasminogen-dependent fibrin independent amidolytic activity, and the appearance of a new 48 kDa lysis band on zymography. However, in the culture medium of platelet-treated aortic EC, a new lysis band of 92 kDa appeared with no associated amidolytic activity suggesting that the 48 kDa plasminogen activator secreted by the aortic EC after treatment with platelets is complexed to the inhibitor PAI1. This modulation of fibrinolytic activity depends on the EC origin since it is not observed with pulmonary artery EC, and represents a new concept in fibrinolysis regulation by cell-cell interaction.

Animals↗
PubMed1990

Endothelial proteases stimulated by blood platelets.

Endothelial cells secrete a protease which is activated in the extracellular medium by platelets. This protease PECAP, degrades casein and fibrinogen, and its characteristics differentiate it from the other known blood and vascular proteases.

Blood Platelets↗
PubMed1990

[Endothelial cell proteases and their modulation by platelets].

Endothelial cells produce and secrete a large number of proteases which are implicated in various disease states. These proteases fall into two classes: serine proteases include plasminogen activators (t-PA) and urokinase (u-PA) and play a major role in fibrinolysis, tissue repair and carcinogenesis; and metalloproteases include collagenases and stromelysine, two enzymes involved in the tissue remodelling that occurs during angiogenesis and tumor growth. The authors have recently identified two other proteases in porcine aortic endothelial cell culture medium. One is an elastase-like enzyme of the metalloprotease group, whereas the other is a new protease whose molecular weight is 85 Kd and whose activity becomes apparent only after exposure of the endothelial cells to platelets. The term Platelet Endothelial Cell Activated Protease accurately describes this enzyme. PECAP degrades casein and fibrinogen. Because PECAP is not inhibited by the usual inhibitors of the various classes of proteases, it remains at present unclassified.

Blood Platelets↗
PubMed1989

Elastase-like activity in cultured aortic endothelial cells.

Cultured porcine aortic endothelial cells were studied for cellular and secreted elastase activity. We describe an activity hydrolyzing the synthetic elastase substrate, succinyl(alanine)3 nitroanilide, but not elastin, which was shown to be membrane located and was not secreted to the culture medium. A different neutral proteinase activity degrading insoluble elastin was demonstrated in the culture medium following its fractionation by gel filtration high performance liquid chromatography (HPLC). Since no elastinolytic activity could be directly detected in the conditioned medium, it is likely that the chromatographic separation removed an endogenous inhibitor.

Animals↗
PubMed1989

Interaction of platelets with endothelial cells: activation of a novel neutral protease.

The activation of a new neutral protease of MW 85,000 was demonstrated on interaction between porcine aortic endothelial cells and human and porcine platelets in culture. The activity of this enzyme, PECAP (platelet endothelial cell activated protease), was detected by electrophoresis on polyacrylamide gels impregnated with the substrate casein. Our results showed that the platelet-endothelial cell interaction did not involve induction of synthesis of de novo enzyme, but rather an activation of a latent enzyme. PECAP cleaved casein and fibrinogen, but had no activity against gelatin or elastin. It was not inhibited by inhibitors of metalloproteases (EDTA, 1.10 phenanthroline), serine proteases (phenylmethylsulfonyl fluoride, elastatinal), or cysteine proteases (iodoacetate, N-ethylmaleimide) and it seems to be unrelated to the previously known proteases of mesenchymal and hematopoietic cells.

Animals↗
PubMed1987

Collagen in abdominal aortic aneurysm: typing, content, and degradation.

The collagen content of the media of infrarenal aorta has been compared in age- and sex-matched normal aorta and dilated and nondilated atherosclerotic aorta. The proportion of collagen was increased in aneurysmal aorta from 62% to 84% and appears to be the result of preferential elastin degradation. The ratio of type I to type III collagen, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of specific cyanogenbromide peptides, did not vary significantly from 2:1 in any of the three groups of aortas. There was no evidence of increased collagenase activity in unruptured aneurysmal aorta. Collagenase activity was increased in ruptured aneurysmal aorta but could only be satisfactorily measured after resolution from the endogenous tissue inhibitor of metalloproteinases. We suggest that only limited collagen turnover occurs in the media of abdominal aortic aneurysms before rupture. A subgroup of three patients with a significant family history of aneurysm had lower amounts of type III collagen in the aortic media, suggesting that abnormalities in type III collagen may be one of the genetic factors contributing to familial clustering of aneurysms.

Aorta, Abdominal↗
PubMed1987

Whole blood clotting tests: an evaluation of five methods.

Five different methods of measuring the clotting time of fresh whole blood have been compared. The Biobridge was the best instrument, and was able to measure both anticoagulation and hypercoagulation accurately and reproducibly.

Blood Coagulation Tests↗
PubMed1986

Pepsinogen--an immunoglobulin binding artefact in 'collagen' preparations.

It has previously been shown that extracts of human articular cartilage, many many of which contain type II collagen, react with heat-aggregated immunoglobulin and artificially prepared immune complexes. Sera from patients with rheumatoid arthritis and psoriatic arthritis, but not from patients with inflammatory bowel disease, react with these extracts. There are two distinct patterns of binding, either as low molecular weight immune complexes or as free antibody directed against collagen. Aggregate-binding activity identified in extracts of human articular cartilage following pepsin digestion was found to be distinct from collagen in its salt solubility. Further purification of this aggregate-binding factor by SDS gel electrophoresis has shown it to be an artefact resulting from the binding of small immune complexes to pepsinogen present in the pepsin preparation used to digest the cartilage.

Antigen-Antibody Complex↗
PubMed1984

Characterization of the calcium-sequestering process associated with human platelet intracellular membranes isolated by free-flow electrophoresis.

By using density-gradient fractionation and high-voltage free-flow electrophoresis, human platelet membranes were separated into highly purified subfractions of surface (SM) and intracellular (IM) origin. Associated exclusively with the IM fraction is an ATP-dependent Ca2+ uptake that, in the absence of oxalate, reaches steady-state levels in 5-10 min. When Ca2+-EGTA buffers were used to control the external Ca2+ concentrations (range 0.1-50 microM) there was an increase in the intravesicle steady-state level of Ca2+ up to 10 microM external Ca2+ concentration. Above this level the intravesicle space becomes saturated at a concentration between 10 and 20 nmol of Ca2+ X (mg of protein)-1. The ionophore A23187 promotes a rapid and almost total release of the sequestered Ca2+ (greater than 90%, t1/2 1-2 min). The presence of oxalate in the external medium greatly enhances the Ca2+ accumulation to levels as high as 200 nmol X (mg of protein)-1, but the uptake process is more variable and rarely reaches steady-state level even after 2 h incubation. Moreover, accumulation in the presence of oxalate effects ionophore release with less than 80% depletion in 45-60 min. These findings, taken together with the known presence in the platelet of a wide variety of functional and metabolic processes triggered by this cation, suggest that the platelet IM has a key role in controlling cytosolic Ca2+ concentrations.

Adenosine Triphosphate↗
PubMed1982

Localization of cyclo-oxygenase and thromboxane synthetase in human platelet intracellular membranes.

Platelet mixed membrane fractions can be separated into discrete vesicle subpopulations of surface and intracellular origin. Intracellular membrane vesicles are the predominant site of phospholipid-modifying enzymes that liberate arachidonic acid. We report the selective enrichment in intracellular membranes of cyclo-oxygenase and thromboxane synthetase activities. Surface membrane fractions show no such enrichment. These results suggest that a sequence of activities leading to the biosynthesis of thromboxane from arachidonate is associated with the intracellular membrane elements known as dense tubular membranes.

Arachidonic Acid↗