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Y J Legrand

Publications and source records attributed to Y J Legrand.

At least 19 recordsLinked to original sources
PubMed1996

Heterogeneity of microfibrils: role of thrombospondin-microfibrils in the thrombogenicity of the subendothelium.

We report the results of an immunogold electron microscopical analysis on microfibrils from the arterial subendothelium showing that thrombospondin (TSP) is present on 40 nm-diameter structures joining 8-10 nm-diameter microfibrils containing fibrillin. They differ from type VI collagen which forms 3-5 nm-diameter microfibrils. TSP containing microfibrils (TSP-MF) extracted from human umbilical arteries did not contain fibrillin or type VI collagen. Blood platelet interactions with TSP-MF were not modified by anti-fibrillin or anti-type VI collagen antibodies. In situ, vWF was bound to cross-linked microfibrils, at the level of their 40 nm junction, and a double-labeling with the anti-thrombospondin and anti-vWF antibodies was observed. In vitro, vWF binding to TSP-MF was not inhibited by anti-fibrillin or anti-type VI collagen antibodies. These results suggest a structural and functional heterogeneity of microfibrils and emphasize the role of TSP-MF in the thrombogenicity of the subendothelium.

Collagen↗
PubMed1994

Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.

We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.

Animals↗
PubMed1994

Proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha) down regulate synthesis and secretion of thrombospondin by human endothelial cells.

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) b cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin-1 beta (IL-1 beta) or human tumor necrosis factor-alpha (TNF-alpha) caused a time-and dose-dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL-1 beta and TNF-alpha in combination ha a greater effect than either agent alone. Interferon-gamma (IFN-gamma) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with lL-1 beta, either alone or in combination with TNF-alpha, had no significant effect on the steady-state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL-1 beta decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.

Cells, Cultured↗
PubMed1993

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF-GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.

Actin Cytoskeleton↗
PubMed1993

Spectrophotometric evaluation of the adhesion of blood platelets to collagen and microfibrils.

We present an easy method in which the adhesion of platelets to collagen or to MFs was measured in a spectrophotometer, after an incubation of hypercitrated platelet rich plasma (PRP), or of a platelet suspension, with an inducer, followed by the filtration of non adhering platelets through translucent Isopore membrane filters (pore diameter = 5 microns). The adhering platelets, which are retained on the filter, were stained by Coomassie blue to quantify the adhesion by the simple reading of the O.D. 580 nm of the stained platelets which appear as a blue spot on the translucent membranes.

Blood Platelets↗
PubMed1993

Role of thrombospondin in the adhesion of human endothelial cells in primary culture.

The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.

Cell Adhesion↗
PubMed1992

Molecular mechanism of the interaction of subendothelial microfibrils with blood platelets.

This short review summarizes the present state of our knowledge concerning the mechanisms of the interaction between subendothelial microfibrils and blood platelets. Microfibrils associated with elastin and with basement membranes have been shown to be able to promote platelet adhesion and subsequent activation and aggregation. A 128 kDa thrombospondin (TSP)-like glycoprotein of microfibrils (GP 128) is involved in their reactivity towards platelets, as established from specific inhibition by antibodies against GP 128 and against TSP of the microfibril-platelet interaction. This interaction only occurs in the presence of plasma von Willebrand factor (vWF). VWF effectively binds to microfibrils; this binding does not implicate GP 128, but a 97 kDa protein of microfibrils not yet characterized. In addition, from data based on inhibition of platelet adhesion and aggregation by monoclonal antibodies against platelet membrane glycoprotein Ib (GP Ib), it appears that GP Ib, a platelet membrane receptor for vWF, acts in the vWF dependent microfibril-platelet interaction as a receptor for VWF bound to protein 97 kDa in microfibrils. A tentative model is presented to illustrate the complex mechanism of the microfibril-platelet interactions: recognition of vWF bound to microfibrils by GP Ib would be the first step, which would then allow subsequent binding of GP 128 to its receptor on the platelet membrane.

Actin Cytoskeleton↗
PubMed1991

Thrombospondin: a component of microfibrils in various tissues.

We used antisera directed against human platelet thrombospondin (TSP) and microfibril-associated GP 128 to localize the presence of these glycoproteins in fixed sections of human placenta or porcine arteries and skin by immunogold labeling, using electron microscopy. These two antibodies reacted with both human and porcine tissues and always recognized the same structures. In all three tissues the antibodies were associated with the basement membranes and, more precisely, with the microfibrillar structures present at the junction between the basement membrane and the adjacent connective tissue. This localization indicates that GP 128 and TSP are associated with the microfibrils, and suggests their possible role in the attachment of basement membrane to the connective tissue meshwork. Their presence in microfibrils associated with the subendothelial basement membrane in arteries may be important in regard to the thrombogenicity of the subendothelium since, after an endothelial lesion, they may be directly accessible to blood platelets.

Actin Cytoskeleton↗
PubMed1990

Binding of plasma von Willebrand factor by arterial microfibrils.

We developed an ELISA test to measure the binding of plasma von Willebrand factor (vWF) to arterial microfibrils and compared this interaction to the binding of vWF to collagen under the same conditions. We found that vWF binds to microfibrils in a similar manner as it binds to collagen: the binding was independent of the presence of cations, and temperature of incubation and was displaced by 1 M NaCl. Using purified 125I-vWF we showed that the binding was saturable and could be displaced by cold vWF in excess. Using immunoblotting we showed that vWF binds to a 97 kDa protein present in the microfibrils different from the 128 kDa thrombospondin-like structure (GP 128) which in microfibrils is known to interact with blood platelets. These results indicate that in the subendothelium, microfibrils bind plasma vWF and this reinforces the thrombogenic role of these structures.

Cations, Divalent↗
PubMed1990

Collagen-induced platelet activation mainly involves the protein kinase C pathway.

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.

Blood Platelets↗
PubMed1988

Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils.

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.

Blood Coagulation↗
PubMed1988

Immunochemical identification of a thrombospondin-like structure in an arterial microfibrillar extract.

Arterial microfibrils contain a 128 Kd collagenase and pepsin resistant glycoprotein (GP 128) essential for their ability to induce platelet aggregation. A previous report (Fauvel F. et al, (1984) Biochem. Biophys. Res. Comm., 123, 114-120) showed that GP 128 and thrombospondin (TSP) synthetized by endothelial cells each inhibited the aggregation of platelets by microfibrils and not by collagen. We used a monospecific antiplatelet TSP IgG in an immunoblotting assay for the identification of a TSP-like structure in untreated, collagenase-treated and pepsin-treated arterial microfibrils. The only constituent recognized in the three samples of microfibrils was GP 128. Fab fragments of this IgG provoked a dose dependent inhibition of the microfibril induced platelet aggregation (50% inhibition with 0.25 mg, 100% inhibition with 1 mg); in contrast, they did not affect collagen induced aggregation. The results indicate that a glycoprotein constituent with a thrombospondin-like antigenicity is involved in the thrombogenic properties of arterial microfibrils.

Actin Cytoskeleton↗
PubMed1987

Effect of a collagen-derived octapeptide on phosphoinositide turnover and 43K protein phosphorylation in collagen-activated platelets.

A collagen-derived octapeptide KPGEPGPK which specifically inhibits the activation of platelets by collagen has been tested for its ability to affect the collagen-induced phosphoinositide breakdown and protein phosphorylations. Collagen produced a transient decrease followed by a rapid resynthesis of [32P]-phosphatidyl 4-5 bisphosphate (PIP2) and 4-mono phosphate (PIP). Octapeptide, at a concentration preventing aggregation but allowing shape change, did not impair the phosphoinositide breakdown, whereas the P43 phosphorylation was strongly inhibited. Higher concentrations of peptide which did not permit any shape change were needed to hinder the PIP2 and PIP decrease. Therefore, the octapeptide appears to affect early events of the collagen-induced platelet activation involving the P43 phosphorylation, independently of its effect on the receptor-stimulated phosphoinositide hydrolysis.

Blood Platelets↗
PubMed1986

Activation of platelets by microfibrils and collagen. A comparative study.

Previous works demonstrated that microfibrils stimulate blood platelets to aggregate. The present study compares the activation of platelets by human placental and bovine aortic microfibrils and by type III collagen. We studied the morphological changes occurring in in platelets during their activation and aggregation, as well as the kinetics of the release reaction and thromboxane B2 formation. As for collagen, the microfibrils-induced platelet aggregation followed a lag phase, during which progressive emission of pseudopodes and centralization of organelles occurred. Aggregation was associated with secretion of beta-thromboglobulin and adenylic adenylic nucleotides, and with formation of thromboxane B2; it was established that the kinetics of secretion and the aggregation curve were parallel. Microfibrils-induced aggregation was also inhibited by ethylenediamine tetraacetic acid, creatine phosphate-creatine phosphokinase, and aspirin, showing that it was calcium-dependent and required a secretion of ADP and formation of endoperoxide and thromboxane. The response to microfibrils was much more rapid than to collagen; placental microfibrils reacted faster than aortic microfibrils. The requirement of plasma in the microfibrils platelets interaction was confirmed: 10 microliters is the minimal amount of plasma necessary for an aggregation of platelets in 400 microliters of buffer. This fact supports the idea of the existence of two different pathways in the interaction between platelet and the subendothelium, depending on the vascular structure (microfibrils or collagen) involved, even though the sequence of events leading to the formation of an aggregate is similar.

Adenosine Diphosphate↗
PubMed1985

Evidence that a collagen derived octapeptide inhibits fibrinogen binding to platelets stimulated by collagen and not by ADP.

A synthetic octapeptide derived from type III collagen which specifically inhibits the activation and aggregation of platelets by collagen without affecting their adhesion was assayed on the collagen and ADP dependent fibrinogen binding to platelets. With 20 micrograms/ml collagen, the octapeptide (6 mM) inhibited by 68% the fibrinogen binding: this inhibition was correlated (p less than 0.01) to a decrease in the velocity of aggregation, suggesting that the fibrinogen binding might influence this parameter. The octapeptide did not affect the ADP-induced platelet aggregation and fibrinogen binding. This indicates that the octapeptide does not inhibit the binding of fibrinogen to its receptor directly, but interferes with some step(s) preceding the collagen-induced expression of the fibrinogen receptor.

Adenosine Diphosphate↗
PubMed1985

Collagen derived octapeptide inhibits platelet procoagulant activity induced by the combined action of collagen and thrombin.

Platelet prothrombin converting activity was measured in a system using washed human platelets and purified coagulation factors Xa, Va and prothrombin. Exposure of platelet prothrombin converting activity evoked by collagen or the combined action of collagen and thrombin was effectively inhibited when a collagen derived octapeptide was added prior to platelet activation. Half maximal inhibition of prothrombinase activity of platelets stimulated by collagen plus thrombin- or collagen alone was obtained at 0.9 mM and 0.5 mM octapeptide, respectively. This suggests a modifying effect of thrombin on the platelet-collagen interaction. Octapeptide either alone or in combination with thrombin was unable to enhance platelet procoagulant activity. The increased prothrombin converting activity seen upon treatment of platelets with ionophore A23187 was not affected by octapeptide, added either before or after treatment with ionophore. It is concluded that octapeptide specifically interferes with the platelet-collagen interaction required to generate a procoagulant surface which enhances the rate of thrombin formation.

Blood Platelets↗
PubMed1984

Aortic endothelial cells in culture secrete glycoproteins reacting with blood platelets.

The culture medium of bovine aortic endothelial cells contains proteins which inhibit the aggregation of platelets induced by aortic microfibrils but not by type III collagen. From this medium, fibronectin, thrombospondin and a glycoprotein with MW of 128 Kd (GP 128), similar to a glycoprotein described in a microfibrillar extract from bovine aorta were separated by affinity and ion exchange chromatography. GP 128 was further purified by molecular sieve chromatography on SW 3000 column. GP 128 inhibited the aggregation of platelets by microfibrils. This suggests a role of GP 128 in the platelet/subendothelium interaction.

Amino Acids↗