Biomedical subjects

Y Legrand

Publications and source records attributed to Y Legrand.

At least 19 recordsLinked to original sources

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↗

PubMed1995

Thrombospondin modulates human breast adenocarcinoma cell adhesion to human vascular endothelial cells.

Thrombospondin (TSP), a M(r) 450,000 cytoadhesive glycoprotein, has been shown to potentiate tumor cell metastasis in mice by a mechanism that involves the hemostatic system of the host. In this study, the potential involvement of TSP in the interaction of human mammary adenocarcinoma MCF-7 cells with human umbilical vein endothelial cells (HUVECs) in culture was investigated. Using an ELISA, preconfluent HUVECs synthesized 100-fold more TSP than did MCF-7 cells during 24 h of culture (20 versus 0.2 microgram/10(6) cells). Confocal microscopy localized TSP within intercellular junctions between aggregated MCF-7 cells in suspension. On adherent cells, TSP exhibited a patchy distribution both on the cell surface and in the cytosol. In HUVECs, TSP strongly stained the perinuclear space and was also found in association with cytoskeletal microfibrils. Flow cytometric analysis indicated the presence of a large number of unoccupied receptors for TSP on MCF-7 cells. Binding studies using [125I]TSP demonstrated the presence of 1.6 x 10(6) sites/cell with an apparent Kd of 28 nM. Attachment of radiolabeled MCF-7 cells to a TSP-coated substrate and to HUVEC monolayers was inhibited in the presence of a polyclonal antibody to TSP (10 micrograms/ml) or increasing concentrations (1-10 micrograms/ml) of soluble TSP. Neither nonimmune IgG nor the cell adhesion peptide Gly-Arg-Gly-Asp-Ser (100 micrograms/ml) inhibited these interactions. Inhibition was also observed with heparin (10 micrograms/ml), suggesting the participation of TSP heparin-binding domain(s) and heparin-like molecules. In the presence of an excess of soluble TSP or anti-TSP antibody, MCF-7 cells did not form aggregates in suspension and preformed aggregates were readily dissociated by the addition of soluble TSP. These results indicate that mammary adenocarcinoma cells use TSP to form aggregates and to attach to human endothelial cells. These interactions may have physiological implications during the hematogenous spread of tumor cells.

Adenocarcinoma↗

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

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↗

PubMed1991

Hemostasis in patients undergoing extracorporeal circulation: the effect of aprotinin (Trasylol).

The administration of aprotinin during extracorporeal circulation (ECC) reduces blood loss. To explore the mechanism of this effect, a placebo-controlled double-blind study was performed in 20 patients (10 were administered with a high dose of aprotinin, 10 with placebo) undergoing a primary, elective operation of coronary artery bypass grafting (CABG) with ECC. Biological tests were performed at 4 different time points during the operation. A marked reduction in the placebo group of ristocetin-induced platelet agglutination (binding of von Willebrand factor [vWF] to platelet glycoprotein [GP] Ib) was shown during ECC and at the end of surgery, but not in the aprotinin group. This abnormality is not related to the hydrolysis of vWF or GP Ib, since washed platelets were resuspended in pooled normal plasma which provided a constant amount of vWF in this test and since the plasma concentration of the fragment of GP Ib (glycocalicin) did not correlate with this abnormality. Despite a high concentration of heparin (5-7 IU/ml) in patient's plasma during bypass, activation of blood coagulation in both groups was evidenced by an increase in ATm (thrombin-modified antithrombin III) level. The level of ATm in the placebo group reached a maximum at the end of ECC during rewarming, while in the aprotinin group, ATm level at this time point was significantly lower than in the control group. In comparison to the placebo group, the generation of the fibrin degradation products (DDE complexes) was inhibited by aprotinin during ECC, but the level of DDE complexes in the aprotinin group was slightly elevated after ECC, although much less than in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗

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↗

PubMed1988

Molecular weight and shape of angiotensin-I converting enzyme. A neutron scattering study.

The molecular weight of angiotensin-I converting enzyme from pig lung has been determined to be 112000 (+/- 6000) by neutron scattering. This is somewhat lower than the value determined by SDS-PAGE and than previous estimates which, however, show a very wide range of values. A quantitative analysis of the amino acid and carbohydrate content was made in order to determine the enzyme concentration. The small angle neutron scattering technique also gives information on the molecular shape, yielding a radius of gyration of 44.5 +/- 1.5 A which, for the observed molecular weight, indicates that the angiotensin converting enzyme molecule is clearly elongated in aqueous solution. Dynamic light scattering experiments confirm this conclusion.

Amino Acids↗

PubMed1985

Inhibition of some activities of thrombin by a collagen-derived octapeptide.

The effect of a collagen-derived octapeptide on some properties of thrombin is presented. This peptide provoked a dose- and time-dependent prolongation of the thrombin-induced plasma and fibrinogen clotting time and inhibited the polymerization of fibrin generated from fibrinogen by thrombin. It did not affect the polymerization of fibrin monomers; it was also without effect on the coagulation of plasma or fibrinogen by reptilase. The prolongation of the fibrinogen clotting time depended on the duration of the incubation of thrombin and the octapeptide and not on the duration of the incubation of fibrinogen and the octapeptide. The inhibition was therefore ascribed to an interference with thrombin, rather than with fibrinogen. A preincubation of the octapeptide with thrombin resulted in an inhibition of the thrombin-induced platelet aggregation. The effect of the octapeptide on thrombin has been related to the presence of positively and negatively charged groups, because uncharged analogue sequences were without effect on these activities of thrombin.

Adult↗

PubMed1983

Non enzymatic glycosylation increases platelet aggregating potency of collagen from placenta of diabetic human beings.

Pepsin extracted collagen and an acid soluble glycoprotein were purified from placentas of normal and diabetic human beings. Diabetic samples exhibit a significant increase in ketoamine-linked glucose whereas both amino acid and carbohydrate composition were unaffected. This excess non enzymatic condensation of glucose on free amino-groups was found to increase platelet aggregating potency of these proteins independently of any modification in fiber morphology.

Amino Acids↗

PubMed1983

Nonenzymatic glycosylation of collagen in diabetes: incidence on increased normal platelet aggregation.

The effect of normal and nonenzymatically glycosylated rat type I acid-soluble collagen on normal human platelets was investigated. Glycosylated collagen was obtained either after in vitro incubation with glucose or from rats made diabetic by streptozotocin. The amount of nonenzymatically bound glucose was as follows: normal 2.3, diabetic 7.6 and in vitro glycosylated collagen 9.0 nmol/mg. When investigated under conditions leading to identical diameters and molecular packings for all the samples, the aggregation potency was markedly stronger for diabetic and glycosylated collagens than for normal ones. These results show the potential role of this posttranslational modification of collagen which can be considered as a risk factor in the thrombotic pathogeny of diabetes.

Animals↗

PubMed1982

[Thrombogenicity of the vessel: role of microfibrils and of collagen].

Thrombogenicity of the vessel wall: role of the microfibrils and collagen. The study of platelet adhesion to rabbit aortic subendothelium preincubated with highly specific collagenase has revealed that platelets adhere to the microfibrils of the elastic lamina. To certify that an interaction between microfibrils and platelets can occur, microfibrils from two different origins were isolated: placental microfibrils extracted from the villi of human placenta, and aortic microfibrils extracted from adult bovine aorta. Both preparations were histologically homogeneous, and differed in their amino acid composition with an acidic character more pronounced for placental than for aortic microfibrils. Both preparations were able to induce platelet aggregation in plasma, but not after platelet isolation and resuspension in buffer. An interesting feature was the fact that when normal platelets were isolated, washed and resuspended in plasma from severe VWD patients, they were not aggregated by placental or aortic microfibrils. This defect was corrected after perfusion of cryoprecipitate to one patient. Moreover, monoclonal antibody directed against platelet glycoprotein Ib inhibited the aggregation of platelets to microfibrils, not to collagen; this suggested that an axis platelet GPI-FVIII/VWF-microfibrils could represent a pathway for platelet/subendothelium interaction. The adhesion of platelets to collagen seems to involve the staggering of a short amino acid sequence along a collagen fibre. This possibility arises from the requirement for the preservation of the quaternary structure of collagen in the induction of platelet adhesion/aggregation in vitro, and also from the identification and synthesis of a nonapeptide derived from type III collagen, which is also to specifically inhibit the aggregation of platelets by collagen, following its binding to platelet membrane.

Animals↗

PubMed1981

Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function.

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

Antibodies, Monoclonal↗

PubMed1981

Gray platelet syndrome: alpha-granule deficiency. Its influence on platelet function.

The ultrastructure, cytochemistry, biochemistry, and functions of platelets from two patients with the familial gray platelet syndrome are described. Ultrastructural studies showed a lack of alpha-granules in the megakaryocytes in the vicinity of a marrow myelofibrosis and/or in platelets. A normal number of mitochondria and a slight increase of dense bodies was confirmed by labeling the whole patient with mepacrine. Platelet peroxidase (in the dense tubular system) and catalase-positive granules (revealed by the cytochemistry) were present. Platelets from both patients had severe deficiency of beta TG either after tritonization (less than 4% of normal) or thrombin treatment (less than 15% of normal), and the plasma beta TG levels were slightly increased. Functional studies of these platelets showed an uptake of [14C]5HT inhibited by reserpine similar to that in the control platelets. Thromboxane formation was within normal limits in the presence of arachidonic acid, ADP, collagen, or ionophore A23187, indicating that (1) the cyclooxygenase/thromboxane synthetase systems were not altered and (2) the phospholipase activities were not impaired. Although the platelet adhesion to prepolymerized fibrillar type III collagen and the ADP-, arachidonic acid-, and ionophore A23187-mediated aggregations were apparently normal, in every case the release of [14C]5HT was either at the low side of the normal range or decreased. This abnormality was increased when collagen or thrombin was added to either PRP or washed platelets from both patients, where at the same time, the aggregation and the release were markedly reduced. The results suggest that alpha-granules or their content has an influence on not only the platelet aggregation but also the dense bodies involved in the [14C]5HT release reaction.

Adolescent↗

PubMed1981

[The adhesion of blood platelets to collagen: molecular features of collagen (author's transl)].

This review deals with some structural features of the collagen molecules involved in the adhesion of platelets representing the initial step of hemostasis, thrombosis, and (partly) atherosclerosis. The adhesion occurs at the level of a vascular lesion or deendothelialized area, whatever the genetic type of collagen. In vitro experiments with purified collagens have shown that vascular interstitial collagens (types I and III, the latter present in subendothelium) as well as basement membrane-derived collagens (types IV and V) induce an adhesion of platelets, provided that an ordered arrangement linked to the quaternary and tertiary structures of their molecule is preserved. Whatever the quaternary structure, the important point seems to be the size of the fibers and more precisely the availability of an optimal number of adhesion sites on multimerized fibers. Various direct or indirect proofs (for example, the occurrence of the impairment of collagen multimerization on platelet adhesion/aggregation) are reviewed. Our recent studies on interstitial collagens have shown the involvement of certain specific amino-acid sequences obtained after cyanogen bromide cleavage of collagen. These are the C-terminal alpha1 (I) CB6 peptide of the alpha 1 chains of type I collagen (216 amino acids) and the central alpha1 (III) CB4 peptide from type III collagen (149 amino acids) Cleavage of this last peptide by chymotrypsin, hydroxylamine, and trypsin has suggested the possibility that a nonapeptide (sequence gly-lys-hyp-gly-glu-hyp-gly-pro-lys) is a minimum site of adhesion for platelets. This assumption has been reinforced by the fact that a synthetic nonapeptide with this sequence specifically inhibits the aggregation of platelets to collagen in vitro. The adhesion of platelets may consequently be due to the repetitive staggering of short amino acid sequences (such as this nonapeptide from type III collagen) along the rigid structure formed by a multimerized collagen fiber.

Basement Membrane↗