PubMed Health⌕ Search

Biomedical subjects

J Lahav

Publications and source records attributed to J Lahav.

At least 37 records · Page 2Linked to original sources
PubMed1992

Lupus anticoagulant antibodies inhibit collagen-induced adhesion and aggregation of human platelets in vitro.

The effect of circulating lupus anticoagulant on platelet interaction with collagen and other proteins was tested, with the aim of understanding the role of membrane phospholipids in platelet function. Plasma samples from 26 systemic lupus erythematosus (SLE) patients, containing circulating lupus anticoagulant (LAC), were examined for their effect on adhesion and aggregation of normal human platelets. We find that SLE plasma, but not normal plasma, inhibits platelet adhesion to collagen in a concentration-dependent manner. At a plasma concentration of 1% the inhibition was 73 +/- 9% (mean +/- SD). In sharp contrast, there was no effect on platelet adhesion to fibronectin. Purified IgG from the same plasma samples also had an inhibitory effect. At 15 micrograms/ml (comparable in IgG concentration to 0.1% plasma) it inhibited adhesion to collagen by 33 +/- 11%. Inhibition could be abolished by preincubation of the LAC-containing plasma with cardiolipin (CL), phosphatidylinositol (PI), and, to a lesser extent, phosphatidylserine (PS) but not with phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Inhibition could also be abolished by preincubation of the LAC-containing plasma with a 10-fold excess of washed normal platelets. The effect of 1% LAC plasma on platelet aggregation was as striking, showing 79 +/- 26% inhibition of collagen-induced aggregation, and it could also be abolished by preincubation of the LAC plasma with cardiolipin. In contrast, the effect of LAC plasma on thrombin-induced aggregation was rather modest. Our results indicate that antiphospholipid antibodies interfere with platelet adhesion and stimulation by collagen in vitro and point to an important role of external plasma membrane phospholipids, particularly PI, in collagen-induced platelet activation.

Adult↗
PubMed1992

Differentiation-controlled synthesis and binding of thrombospondin to granulosa cells.

Thrombospondin (TSP) is a large glycoprotein, synthesized by several matrix-forming cells and incorporated into their extracellular matrix. In several cell types its presence supports cell growth and proliferation. To investigate the role of this protein in cell differentiation, we studied the hormonal effect of TSP production and receptor-mediated binding to primary granulosa cells prepared from diethylstilbestrol-treated immature female rats. These cells can be induced to differentiate by FSH, 8-bromo-cAMP (8-Br-cAMP), or forskolin. Progesterone production is induced during differentiation, and its level of synthesis is an important manifestation of the differentiated phenotype. We find that undifferentiated granulosa cells synthesize and secrete TSP. The protein comprises about 0.5% of the total cell protein, and it is the major protein secreted in culture. Treatment of the cells with FSH or 8-Br-cAMP reduces TSP production dramatically, and forskolin completely inhibits it. In parallel, we observed that the undifferentiated cells bind TSP specifically with a Kd of 1.8 nM, and the number of binding sites per cell is 1.7 x 10(5). This binding can be prevented by excess TSP or an anti-TSP monoclonal antibody (B7-3). This ability to bind TSP is completely lost after induction of differentiation by FSH or 8-Br-cAMP. Our findings show that both the production and binding of TSP to granulosa cells are tightly controlled by normal cell differentiation and indicate that changes in TSP are correlated with the passage of the cell through the stages of maturation, a passage that also involves changes in cell shape and extracellular interactions and in the steroidogenic capacity of these cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin.

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.

Affinity Labels↗
PubMed1988

Gray platelet syndrome in the elderly.

A 68-year-old male who suffered from thrombocytopenia and mild splenomegaly for 18 years was found to present agranular gray platelets on peripheral blood smear. Bone biopsy revealed a mild, diffuse, reticular fibrosis with no collagen, and electron microscopy of the platelets showed an absence of almost all the alpha-granules. Platelet thrombospondin and fibronectin analysed by SDS-polyacrylamide gel electrophoresis and Rocket immunoelectrophoresis were absent. Follow-up of 4 years showed the same parameters with no evidence of active myeloproliferative or dysmyelopoietic disorders. Hemorrhagic diathesis was limited to ecchymoses and postprostatectomy bleeding, necessitating platelet transfusion. This led us to conclude that our patient probably had a constitutional primary alpha-granule deficiency or gray platelet syndrome. This extremely rare defect has been described in less than 10 patients, all of them very young. Our observation shows that these patients may have a long, uneventful survival.

Aged↗
PubMed1988

Thrombospondin inhibits adhesion of endothelial cells.

Adsorption of thrombospondin to a substratum inhibits adhesion of endothelial cells to that substratum. Four hours after plating of cells on glass covered with thrombospondin, the number of cells bound per unit area was only 8% of that bound to fibronectin, and 20% of that which could bind to albumin. While on fibronectin cells assumed a well-spread configuration with time in culture, on thrombospondin they stayed completely round. On surfaces constructed by sequential incubation of glass with thrombospondin and fibronectin or other proteins, thrombospondin retained its inhibitory effect on cell adhesion. Fibronectin surfaces treated with thrombospondin lost 50% of their capacity to adhere endothelial cells. Cell spreading was also greatly impaired. These observations indicate that thrombospondin, which is a component of the extracellular matrix, can modulate adhesion of endothelial cells to the matrix.

Animals↗
PubMed1988

The build-up of the thrombospondin extracellular matrix. An apparent dependence on synthesis and on preformed fibrillar matrix.

Thrombospondin (TSP) is a multifunctional protein synthesized by several cell types in culture, among them endothelial cells, and incorporated into the extracellular matrix (ECM) of these cells. In vitro it has been detected in most interstitial spaces, and its presence has been suggested to be developmentally controlled. With the aim of elucidating the role of TSP in the extracellular matrix, we studied the build-up of this protein in the matrix as a function of cell type density and age in culture. The development of the TSP matrix was compared to that of fibronectin (FN) and of von Willebrand Factor (VWF) in the same cultures using our monoclonal anti-TSP antibody B7-3 and monospecific anti-FN or anti-VWF antibodies. In cultures of bovine aortic endothelial cells (BAEC) we observed that both the pattern and distribution of TSP in the matrix changed with cell density and time in culture; it started as a completely amorphous dense layer of protein when the cells were sparse and changed to well organized fibrils when the cells had been confluent for a while. The first appearance of the fibrillar arrays of TSP coincides perfectly with that of the FN fibers; extracellular VWF also was first detected at this point. From this time in culture, there was an increasing coincidence of the TSP with the FN and the VWF matrices suggesting the formation of a complex multicomponent structure of the fibrillar network.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1988

Thrombospondin inhibits adhesion of platelets to glass and protein-covered substrata.

Glass and protein-covered surfaces when treated with the platelet-secreted glycoprotein thrombospondin lose their capacity to bind unstimulated platelets. In comparison to the number that bind to fibronectin-covered glass surfaces, less than 3% bind to thrombospondin-covered glass surfaces. When the fibronectin-covered surface is incubated with thrombospondin, it loses 87% of its binding capacity for platelets. The inhibitory effect of thrombospondin on platelet binding increases with increasing amounts of the adsorbed protein and reaches maximal values at 65% saturation of the adsorption of thrombospondin to the surface. Platelet spreading on the surface is also completely inhibited by thrombospondin. These data suggest that thrombospondin is nonthrombogenic and can modulate platelet adhesion to the subendothelium.

Adsorption↗
PubMed1987

The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains.

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen.

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.

Animals↗
PubMed1984

Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding.

Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix.

Binding, Competitive↗
PubMed1982

Analysis of platelet adhesion with a radioactive chemical crosslinking reagent: interaction of thrombospondin with fibronectin and collagen.

We have analyzed the interactions of platelets and platelet-secreted proteins with proteins bound to glass substrata. Using a newly developed radioactive crosslinking reagent, N-succinimidyl-3-[(2-nitro-4-azidophenyl)-2-aminoethyldithio] propionate, we observed crosslinking between thrombospondin released from the platelets and surface-bound fibronectin or collagen. The crosslinking was selective and specific, since thrombospondin showed little crosslinking to substratum-bound bovine serum albumin or ovalbumin, even though it bound to these surfaces. Furthermore, although albumin and fibrinogen released by platelets bound to fibronectin-coated substrata as well as did thrombospondin, these two proteins crosslinked at much lower levels than seen for thrombospondin. Interaction between fibronectin and thrombospondin was confirmed by affinity chromatography. These results suggest that thrombospondin and fibronectin may interact during platelet-substratum adhesion or during platelet-platelet aggregation, or both.

Azides↗
PubMed1982

Fibronectin binds to the C1q component of complement.

Fibronectin immobilized to plastic tubes binds soluble C1q with a Kd of 82 +/- 2.6 nM. The binding of fibronectin to C1q is relatively insensitive to pH but is sensitive to ionic conditions. C1q covalently bound to Sepharose selectively binds cellular fibronectin produced by a hamster fibroblast cell line. The globular head regions of C1q have no effect on the binding of C1q to fibronectin but the collagenous tails of C1q interfere competitively with a Ki of 59 nM. We conclude that fibronectin binds C1q via its collagen-like tail region and thus the process resembles the binding of fibronectin to gelatin. This is further emphasized by our observation that gelatin binds to fibronectin immobilized on plastic tubes with a Kd of 131 nM. Because fibronectin stimulates endocytosis in several systems and promotes the clearance of particulate material from the circulation, these results suggest the possibility that fibronectin could function in the clearance of C1q-coated material such as immune complexes or cellular debris.

Animals↗
PubMed1981

Involvement of fibronectin, Von Willebrand factor, and fibrinogen in platelet interaction with solid substrata.

The proteins fibronectin (FN), Von Willebrand factor (VWF), and fibrinogen are believed to play a role in platelet function. They are distributed between the plasma and the platelet pool in the resting state and undergo redistribution upon platelet activation. We have studied their expression on the surface of the platelet and their mobilization following platelet binding to substrata. For the purpose of studying protein expression on the surface of intact platelets either adherent to a substratum or in suspension, the enzyme-linked immunosorbent assay (ELISA) was elaborated and modified. Using this technique as well as immunofluorescence, we found that antiserum raised against carefully washed human platelets recognized FN, VWF, and fibrinogen as well as platelet surfaces. However, specific antisera against these three proteins failed to bind to the surface of unactivated gel-filtered platelets. When gel-filtered platelets were exposed to plastic or fibrillar collagen, they adhered and spread. Such platelets did bind antibodies against FN, VWF, and fibrinogen, Moreover, when the adherent platelets were incubated with FN or with VWF in the absence of ristocetin, they bound these proteins in a concentration-dependent fashion. The patterns of the bound proteins were not similar, suggesting a different spatial distribution of binding sites. These findings indicate that platelet activation by adhesion to substrata mobilize both endogenous and exogenous pools of these proteins, thereby making them surface associated and probable participants in further binding properties of the activated platelet.

Adult↗
PubMed1980

Effect of ouabain on electrical conductance of frog skins. Evidence against recycling of sodium.

Ouabain (10(-4) M) added to the serosal solution of isolated frog skin not only stops the active transport of Na+ but causes a dramatic increase in the electric conductance of the skin. The effect also appears when ouabain is added to amiloride-pretreated skins, ruling out the possibility of a cellular effect. The similarity between the effect of ouabain on normal and amiloride-pretreated skins indicates that no appreciable recycling of Na+ across the basolateral membrane of frog skin takes place. The change of Na+ efflux after ouabain is added to amiloride-pretreated skins parallels the change of electric conductance, indicating that besides blocking the Na+ pump, ouabain affects the paracellular shunt pathway between the cells.

Amiloride↗
PubMed1978

A thermodynamic analysis of the correlation between active Na+ transport and the rate of oxygen consumption in epithelia.

Active transport in epithelia is discussed in terms of the relationships between oxygen consumption and sodium flux as affected by each of the two corresponding thermodynamic forces. Analysis is presented of the use of nonequilibrium thermodynamics as a total in elucidating coupling and stoichiometry, and in evaluating drug action in the system. The analysis leads to the quantitative characterization of active transport in "two-flow" systems in terms of two plots: oxygen consumption nad sodium flow, each as a function of electrical potential difference, at constant affinity and constant concentrations. The relevant characteristic parameters are then shown to be represented by the slopes and intercepts of the two plots, the ratios of the slopes and of the intercepts, and by the difference--as well as the ratio--of the ratios. Distinction is made between experimental conditions in which the phenomenological coefficients remain constant and those in which these coefficients undergo appreciable changes. In terms of the above analysis, and examination is made of the effect of commonly used drugs. It is shown that while drugs may effect both the affinity and the phenomenological coefficients, they invariably affect the latter--at least in the cases hitherto reported.

Animals↗