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R L Nachman

Publications and source records attributed to R L Nachman.

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

Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.

Amino Acid Sequence↗
PubMed1991

Lipoprotein (a) regulates plasminogen activator inhibitor-1 expression in endothelial cells. A potential mechanism in thrombogenesis.

Lipoprotein (a) (Lp(a)) is a low density lipoprotein-like particle which contains the plasminogen-like apolipoprotein a. Lp(a) levels are elevated in patients with atherosclerotic coronary artery disease. Recent studies suggest that Lp(a) competitively inhibits plasminogen binding to the endothelial cell and interferes with surface-associated plasmin generation. In this study, we present evidence for the presence of Lp(a) in the microvasculature of inflamed tissue. In addition, we demonstrate that Lp(a) regulates endothelial cell synthesis of a major fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). In cultured human endothelial cells, Lp(a) enhanced PAI-1 antigen, activity, and steady-state mRNA levels without altering tissue plasminogen activator activity or mRNA transcript levels. This effect was cell-specific. Although other lipoproteins did not coordinately raise PAI-1 mRNA levels in endothelial cells, low density lipoprotein treatment selectively raised the level of the 3.4-kilobase mRNA species of PAI-1 without a concomitant increase in PAI-1 activity or antigen. Endothelial cell exposure to Lp(a) did not cause generalized endothelial cell activation since the functional activity and mRNA levels for tissue factor, platelet-derived growth factor and interleukin-6 were not elevated following Lp(a) exposure. These data suggest a molecular mechanism whereby Lp(a) may support a specific prothrombotic endothelial cell phenotype, namely by increasing PAI-1 expression.

Cell Line↗
PubMed1991

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.

Antigens, Differentiation↗
PubMed1991

Endothelial cell fibrinolytic assembly.

Endothelial cells play a critical role in thromboregulation by controlling the assembly of fibrinolytic constituents on the membrane. The assembly system illustrated in FIGURE 6 is characterized by the binding of circulating glu-plasminogen to a membrane receptor (Pathway 1). A membrane-associated protease (possibly plasmin) converts the inactive zymogen into a catalytically more efficient zymogen lys-plasminogen (Pathway 2). T-PA binds to a specific receptor, retains its catalytic activity, and is protected from its natural inhibitor PAI-1. The membrane provides a favorable environment for plasmin generation (Pathway 3) at the vessel surface and contributes to the maintenance of a physiological nonthrombogenic state. The immobilization and surface activation of plasminogen provides an important mechanism for localizing proteolytic activity at the surface of other cells such as macrophages and tumor cells. Lp(a), a plasminogen-like lipoprotein, by competing at the endothelial surface for plasminogen binding down-regulates endothelial cell plasmin generation and may thus promote localized thrombogenesis that over a period of time contributes to progressive atherosclerosis.

Animals↗
PubMed1990

Cytokine-enhanced expression of glycoprotein Ib alpha in human endothelium.

Platelet glycoprotein Ib is a major platelet membrane protein composed of two disulfide-linked chains, termed the alpha and beta chains. The larger alpha chain (GpIb alpha), a platelet receptor for von Willebrand factor, plays a major role in mediating platelet adhesion to the subendothelium. Our laboratories have previously reported synthesis of a protein in human endothelial cells that is immunoprecipitated with polyclonal and monoclonal antibodies to platelet GpIb alpha. Lopez et al. (Lopez, J. A., Chung, D. W., Fujikawa, K., Hagan, F. S., Papayannopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5615-5619) have reported the cloning of GpIb alpha from a human erythroleukemia (HEL) cell cDNA library. Using this clone as probe, we have isolated two partial GpIb alpha clones from a human umbilical vein endothelial cell lambda gt11 cDNA library. These clones were localized within HEL-derived GpIb alpha cDNA by sequence and restriction enzyme analysis. Additionally, they detected the same message species in HEL and tonsilar RNA that was detected with the HEL GpIb alpha cDNA. Low level GpIb alpha mRNA expression was detected in cultured human umbilical vein endothelial cells, which was increased by treatment of the cells with tumor necrosis factor-alpha. This effect was enhanced by pretreatment with interferon-gamma. Additionally, localization of GpIb alpha in endothelium of fresh tonsilar tissue was demonstrated by immunohistochemistry and in situ hybridization. GpIb alpha may play a role in mediating platelet or other effector cell adhesion to activated endothelium.

Cloning, Molecular↗
PubMed1990

Thrombospondin forms complexes with single-chain and two-chain forms of urokinase.

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.

Amino Acid Sequence↗
PubMed1989

Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis.

Endothelial cells play a critical role in thromboregulation by virtue of a surface-connected fibrinolytic system. Cultured endothelial cells synthesize and secrete tissue-type plasminogen activator (t-PA) which can bind to at least two discrete sites on the cell surface. These binding sites preserve the catalytic activity of t-PA and protect it from its physiological inhibitor (PAI-1). N-terminal glutamic acid plasminogen (Glu-PLG), the main circulating fibrinolytic zymogen, also interacts specifically with the endothelial cell surface. Binding is associated with a 12-fold increase in catalytic efficiency of plasmin generation by t-PA which may reflect conversion of Glu-PLG to its plasmin-modified form, N-terminal lysine plasminogen (Lys-PLG). Lipoprotein(a) is an atherogenic lipoprotein particle which contains the plasminogen-like apolipoprotein(a) bound to low density lipoprotein. We report here that lipoprotein(a) interferes with endothelial cell fibrinolysis by inhibiting plasminogen binding and hence plasmin generation. In addition, we demonstrate lipoprotein(a) accumulation in atherosclerotic lesions. These findings may provide a link between impaired cell surface fibrinolysis and progressive atherosclerosis.

Apolipoproteins↗
PubMed1989

Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion.

An adhesive interaction between activated platelets and mononuclear phagocytes may contribute to the role these cells play in regulating inflammation, thrombosis, and atherosclerosis. We have previously shown that this adhesive interaction is mediated by the expression of the glycoprotein thrombospondin (TSP) on the surface of activated platelets. We now show that TSP-dependent platelet-monocyte interactions are mediated by glycoprotein IV (GPIV), an intrinsic membrane protein recently identified as a cell surface TSP receptor. Monoclonal antibodies to GPIV bound to cells of the human monocytoid line U937 as assessed by flow cytometry and inhibited the binding of 125I-TSP to the cell surface by 83%. U937 cells preincubated with anti-GPIV were not rosetted by thrombin-stimulated platelets (72% inhibition compared with control anti-monocyte antibodies). In addition, when platelets were stimulated in the presence of saturating concentrations of monoclonal antibodies to GPIV, only 18% of U937 cells were rosetted (78% inhibition). Control antibodies including anti-GPIb did not inhibit rosette formation. These data suggest that TSP can cross-link platelets and monocytes via an interaction with GPIV on the surface of both cells. This molecular bridge may mediate platelet-macrophage communication in various pathophysiologic settings.

Antibodies, Monoclonal↗
PubMed1989

A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.

Animals↗
PubMed1989

Thrombospondin: phenomenology to function.

Since the initial description of thrombospondin in 1971, our knowledge of the protein has reached a point where some understanding of its physiologic and pathologic roles has accrued. Thrombospondin is a multifunctional glycoprotein whose demonstrated affinities for multiple ligands make it a versatile molecule that can participate in mediating or modulating cell adhesion, fibrin polymerization, fibrinolysis, and development. The recently defined primary structure will make an understanding of the functional aspects of this molecule more accessible, and the next several years promises to define the role of thrombospondin in human biology.

Amino Acid Sequence↗
PubMed1988

Case 11-1988.

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Diagnosis, Differential↗
PubMed1988

Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment.

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.

Cell Line↗
PubMed1988

Identification and isolation of a platelet GPIb-like protein in human umbilical vein endothelial cells and bovine aortic smooth muscle cells.

Glycoprotein Ib (GPIb) is an intrinsic platelet membrane protein that plays a major role in platelet adherence and mediates ristocetin-dependent platelet von Willebrand factor binding. Recent reports that the platelet membrane glycoprotein complex IIb/IIIa is expressed in several cell types prompted us to look for GPIb expression in other vascular cells. Immunoperoxidase staining of human stomach and skin histologic sections with polyclonal as well as monoclonal anti-GPIb antibody revealed the presence of GPIb in the endothelial cell and smooth muscle cell layers. Western blotting using monospecific polyclonal anti-GPIb antibodies confirmed the presence of immunoreactive GPIb in human umbilical vein endothelial and bovine aortic smooth muscle cell cultures. Fab fragments of a monoclonal anti-GPIb antibody were used to immunoprecipitate [3H]leucine labeled GPIb from metabolically labeled cells. The GPIb in these cells was functional as measured by ristocetin-dependent cell agglutination and by vWF binding. Endothelial cells as well as smooth muscle cells bound 125I-labeled vWF in a ristocetin-dependent manner, with a Kd of 7.9 nM.

Agglutination↗
PubMed1988

Endothelial cell-mediated conversion of Glu-plasminogen to Lys-plasminogen. Further evidence for assembly of the fibrinolytic system on the endothelial cell surface.

Lysine-plasminogen (Lys-PLG), the plasmin-modified form of native glutamic acid-plasminogen (Glu-PLG), displays enhanced binding affinity for fibrin and also enhanced activation by urokinase and tissue plasminogen activator. We previously demonstrated high-affinity, specific, and functional binding of Glu-PLG as well as tissue plasminogen activator to cultured human umbilical vein endothelial cells (HUVEC). In the present study, we demonstrate binding of Lys-PLG to HUVEC, as well as conversion of Glu-PLG to Lys-PLG at the cell surface. Binding of Lys-PLG to HUVEC was saturable, reversible, epsilon-aminocaproic acid-sensitive, and involved two saturable sites with Kd's of 142 pM and 120 nM, respectively. Upon incubation with Glu-PLG, HUVEC, as well as endothelium in situ, partially converted the ligand to a Lys-PLG-like species. Conversion by HUVEC was blocked by diisopropyl-fluorophosphate, but not by other serine protease inhibitors, including alpha 2-plasmin inhibitor. Eluates of intact umbilical cord vessels contained Lys-PLG by immunoblot analysis. Lys-PLG was also identified immunohistochemically on the endothelial surface of vessels from a variety of normal and inflamed tissues. Thus, endothelial cells appear to actively modify circulating Glu-PLG, bind Lys-PLG to their surface, and thus enhance the fibrinolytic potential of the blood vessel wall.

Binding Sites↗
PubMed1988

Binding of Lys-plasminogen to monocytes/macrophages.

The ability of mononuclear phagocytes to assemble and activate components of the fibrinolytic system on their surfaces may be crucial in effecting an efficient inflammatory response. Lys-plasminogen, the plasmin modified form of this zymogen, was found to bind specifically and with high affinity to murine peritoneal macrophages and to cells of the human monocytoid line U937. This modified plasminogen has been shown to be a more efficient substrate for plasminogen activators than native Glu-plasminogen. Binding was lysine binding site dependent, rapid and reversible. In contrast, although native Glu-plasminogen bound specifically to these cells, affinity was low. Lys-plasminogen inhibited the binding of Glu-plasminogen but the opposite was not true. Molecular analysis of the bound ligands indicated that Glu-plasminogen was converted to Lys-plasminogen and Lys-plasminogen to plasmin on the cell surface but not in the supernatant. Peritoneal macrophages from patients with indwelling catheters and tissue macrophages in chronic inflammatory lesions were shown to express immunologically identified Lys-plasminogen on their surfaces. Therefore binding and surface activation of kinetically favored Lys-plasminogen may provide an important physiological mechanism for localizing proteolytic activity on the surface of inflammatory cells.

Animals↗
PubMed1987

Tumor necrosis factor-mediated release of platelet-derived growth factor from cultured endothelial cells.

Platelet-derived growth factor (PDGF) is a 30,000-Mr glycoprotein that is chemotactic and mitogenic for vascular smooth muscle cells (SMC). It is also a potent vasoconstrictor. In the present study, we found that the macrophage-derived polypeptide, tumor necrosis factor (TNF), releases a factor from human umbilical vein endothelial cells (EC) that is mitogenic for SMC. Postculture medium from TNF-stimulated EC induced a 90% increase in mitogenesis is compared with controls. This effect was half-maximal at a TNF dose of 114 pM, reflected a 2.5-fold increase in PDGF-specific mRNA synthesis, and peaked at 15 h of TNF stimulation. Mitogenic activity was completely abrogated by preincubation of postculture medium with antibody to platelet PDGF. Stimulation of EC with IL-1 (60-240 pM) led to the release of similar mitogenic activity. Thus, in addition to its effects on the hemostatic and adhesive properties of EC, TNF also promotes release of PDGF, which may serve to modulate proliferation of vascular SMC during wound healing, inflammation, and atherogenesis.

Cell Division↗
PubMed1987

Histidine-rich glycoprotein inhibits the antiproliferative effect of heparin on smooth muscle cells.

Histidine-rich glycoprotein (HRGP), an alpha-glycoprotein in human plasma that is also present in platelets and macrophages, binds heparin with high affinity and neutralizes its anticoagulant activity. We now report that HRGP specifically inhibits the antiproliferative effect of heparin on arterial smooth muscle cells while other heparinoid-binding proteins do not influence mitogenesis. The multicellular inflammatory response to endothelial injury characterized, in part, by the influx of platelets and macrophages, may be associated with HRGP release into the arterial microenvironment. This release of HRGP may allow smooth muscle cell proliferation and atherogenesis by inhibiting the action of endothelial cell-derived heparinoid substances.

Animals↗