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Biomedical subjects

L A Miles

Publications and source records attributed to L A Miles.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.

Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.

Amino Acid Sequence↗
PubMed1996

Regulation of plasminogen receptor expression on monocytoid cells by beta1-integrin-dependent cellular adherence to extracellular matrix proteins.

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

Carboxypeptidase B↗
PubMed1996

Characterization of cellular binding sites and interactive regions within reactants required for enhancement of plasminogen activation by tPA on the surface of leukocytic cells.

Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and THP-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and with peripheral blood monocytes and neutrophils. The interactions of plasminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturating amounts of U937 monocytoid cells (1.25 x 10(5)/ml) the rate of plasmin generation was 0.39 nM.s-1 versus 0.07 and 0.09 nM.s-1 without cells or without tPA, respectively. The catalytic efficiency of Glu- or Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, respectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 microM. These values are below the plasminogen concentration in plasma (1-2 microM). Moreover, we provide new data indicating that 1) only a specific subset of plasminogen binding sites, i.e. molecules exposing carboxyl terminal lysines on the cell surface, promotes plasminogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfaces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.

Cell Count↗
PubMed1996

Distinct patterns of urokinase receptor (uPAR) expression by leukemic cells and peripheral blood cells.

The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelocytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uPAR-mediated pericellular proteolysis.

Cell Differentiation↗
PubMed1995

The role of an enolase-related molecule in plasminogen binding to cells.

The alpha isoform of enolase is a candidate plasminogen receptor on U937 monocytoid cells [Miles, L. A., Dahlberg, C. L., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991) Biochemistry 30, 1682-1691]. In the present study, an enolase-related molecule was detected on the surfaces of peripheral blood monocytes and neutrophils by fluorescence-activated cell sorting. A mRNA transcript encoding a unique membrane form of an enolase-related molecule was not detected by Northern-blotting and primer-extension analyses, consistent with the cell-surface protein being authentic alpha-enolase. Both the alpha and beta isoforms of purified enolase, bound plasminogen with an affinity similar to that of the cell surface. Moreover, immunopurified alpha-enolase enhanced plasminogen activation by tissue plasminogen activator and blocked the binding of plasminogen to alpha 2-antiplasmin, mimicking functions arising from the association of plasminogen with cells. The interaction of the enolase isoforms with plasminogen was dependent upon recognition of the C-terminal lysyl residue of the enolases by the lysine-binding sites of plasminogen, as the interaction was blocked by (a) peptides with C-terminal lysine residues and (b) an antibody to the C-terminal aspect of enolase. A monoclonal antibody was developed, characterized and utilized to quantify the enolase molecules present on the surface of U937 cells. A substantial number of molecules, 1.8 x 10(6)/cell, was present, accounting for approximately 10% of the plasminogen-binding capacity of these cells. These studies clearly establish the role of enolase as a cell-surface plasminogen-binding site with profibrinolytic functions.

Amino Acid Sequence↗
PubMed1995

The cell biology of the plasminogen system.

The plasminogen system plays a pivotal role in maintaining vascular patency and in cell migration. Binding of plasminogen to surfaces (i.e., fibrin or cells) is of crucial importance in regulating the function of this system. Plasmin(ogen) binds to cells with low affinity and high capacity via its lysine binding sites, which are associated with its kringle domains and recognize carboxy-terminal lysines of cell surface proteins. Upon binding to cellular receptors, plasminogen is more readily activated; bound plasmin has increased enzymatic activity and is protected from inactivation by inhibitors. Plasminogen receptors are modulated by numerous factors, including proteases, steroid hormones, cytokines and the adhesive state of the cells. The apoprotein(a) moiety of lipoprotein(a) is remarkably similar in amino acid sequence to plasminogen. Shared binding sites for lipoprotein(a) and plasmin(ogen) on cell surfaces and in the subendothelial matrix may contribute to the pathogenetic risks associated with elevated levels of lipoprotein(a).

Cell Membrane↗
PubMed1995

Apical topography and modulation of ICAM-1 expression on activated endothelium.

Leukocyte-endothelium interactions and general inflammatory responses are contributed by the regulated expression of intercellular adhesion molecule-1 (ICAM-1) on endothelium. It is now shown by confocal fluorescence microscopy and immunogold transmission electron microscopy that ICAM-1 was exclusively localized on the apical (luminal) membrane of cytokine-activated human umbilical vein endothelial cells. In contrast, other cell adhesion-promoting molecules, including beta 1 integrins, were expressed exclusively on the basolateral endothelial cell membrane, under the same experimental conditions. Kinetic binding studies of a 125I-labeled monoclonal antibody to ICAM-1 revealed that approximately 8% of membrane ICAM-1 on cytokine-activated endothelium was internalized in both coated and non-coated vesicles at 37 degrees C, with a t1/2 of approximately 18 min and a rate of approximately 3200 molecules/minute. This internalization pathway was directly dependent upon the level of ICAM-1 expression on the cell surface. Genetically engineered ICAM-1 transfectants, expressing a 10-fold higher receptor density than activated endothelium, internalized approximately 18% of membrane ICAM-1 at a rate of 75,000 molecules/minute with a t1/2 of approximately 22 min. These findings suggest that a combined pathway of polarized membrane topography and receptor trafficking may regulate ICAM-1-dependent adhesion at the site of vascular injury and endothelial cell activation.

Cell Adhesion↗
PubMed1995

Interaction of Lp(a) with plasminogen binding sites on cells.

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Binding Sites↗
PubMed1994

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Cells, Cultured↗
PubMed1993

Comparison of the lysine binding functions of lipoprotein(a) and plasminogen.

Regions of apoprotein(a) of lipoprotein(a) [Lp(a)] exhibit striking primary sequence homology to the kringles of plasminogen. The kringles of plasminogen are lysine binding structures and mediate interactions of plasmin(ogen) with substrates and inhibitors. In the current study, the lysine binding properties of Lp(a) have been compared to those of plasminogen and isolated kringle 4 of plasminogen (K4). An analytical assay was implemented to quantitate the interaction of kringle-containing molecules with lysine-Sepharose beads. Radioiodinated ligands, Lp(a), plasminogen, and K4, bound to the beads, and their interactions were inhibited by lysine analogues in a dose-dependent fashion. A series of omega-aminocarboxylic acids inhibited Lp(a), plasminogen, and K4 binding to the lysine-Sepharose beads, but marked differences in the effectiveness of these compounds were observed with each ligand. In this series of compounds, 6-aminohexanoic acid was the most potent inhibitor of binding to lysine-Sepharose for all three ligands. The pH had little effect on the inhibition of plasminogen binding by these compounds. For Lp(a), a low pH caused a marked decrease in inhibition by the 5-carbon and 4-carbon omega-amino acids. In addition, tranexamic acid was 750-fold more potent than lysine in inhibiting plasminogen and 55-fold more potent for K4 binding to the beads. In contrast, the differential potency of these compounds on Lp(a) binding was only 3-fold. These results suggest that the kringles of Lp(a) possess lysine binding functions which are similar, but not identical, to those of plasminogen and its K4.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1993

Competition between plasminogen and tissue plasminogen activator for cellular binding sites.

Cellular receptors for plasminogen and tissue plasminogen activator (t-PA) regulate plasminogen activation and cell-associated proteolytic activity. The characteristics of the interactions of both ligands with monocytes and monocytoid cell lines bear certain similarities, including affinity (kd approximately 1 mumol/L) capacity and susceptibility to carboxypeptidase treatment. Therefore, we have undertaken the present study to determine directly whether t-PA and plasminogen share common binding sites on cells. We found that recombinant human single-chain t-PA (rt-PA) could inhibit the binding of 125I-plasminogen to the cells and, conversely, plasminogen could inhibit 125I-rt-PA binding. This relationship was observed with 9 cell types, including both adherent cells and cells in suspension. In addition, under several conditions of cell treatment, plasminogen and t-PA receptor expression was modulated in parallel. Furthermore, molecules that have been implicated as candidate plasminogen receptors, gangliosides, and an alpha-enolase--related molecule, also interacted with t-PA. These results suggest that at least a component of the binding sites for plasminogen is shared with t-PA. Occupancy of these sites by either or both ligand(s) should result in arming the cells with the proteolytic activity of plasmin.

Binding Sites↗
PubMed1993

Processing of chromaffin granule proteins: a profusion of proteases?

Evidence suggests that proenkephalin and members of the chromogranin/secretogranin family of proteins are prohormone precursors, giving rise to a variety of peptides with biologic activity. However, the specific proteases responsible for cleaving these proteins in vivo have not been fully established. Several candidate proteases have been described, some of which have been shown to cleave these proteins in vitro. Proteolytic processing of the chromogranins may be particularly complex, occurring in specific tissue-dependent patterns. To account for this level of complexity several protease systems may be operative, either alone or in concert, both within the neurosecretory granule and in the extracellular space. Specific proteases which are available within neurosecretory cells or in the local extracellular environment, and which may cleave these prohormones include PC1 and PC2 (recently described members of the Kex2/furin family of endoproteases), as well as kallikrein, acetylcholinesterase, and, more recently, the plasminogen/plasmin protease system. The potential role of these specific proteases in the processing of proenkephalin and the chromogranins is discussed, in particular, in the context of possible processing clues available from recent analysis of cDNA and genomic intron/exon structure.

Animals↗
PubMed1993

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a).

Affinity Labels↗
PubMed1991

Binding of tissue plasminogen activator to human monocytes and monocytoid cells.

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.

Arginine↗
PubMed1991

Cellular regulation of fibrinolysis.

By virtue of their capacity to bind plasminogen activators and plasminogen, to accelerate plasminogen activation and to protect bound plasmin from inactivation by alpha 2 antiplasmin, cells can harness the broad proteolytic activity of plasmin to their surface. Most cells bind plasminogen with a very high capacity, a relatively low affinity (Kd approximately 1 microM) and recognize the lysine binding sites of the molecule. Gangliosides serve as non-protein plasminogen binding sites, and a subset of membrane proteins with carboxy-terminal lysine residues also serve as receptors. The alpha isoform of enolase possesses a carboxy-terminal lysine and is a prominent plasminogen binding protein of cells. Cells of the monocytoid lineage, including peripheral blood monocytes, can markedly upregulate their expression of plasminogen receptors. The capacity to modulate expression of receptors for fibrinolytic components establishes an additional mechanism by which the cell-surface regulates the function of the plasminogen system.

Animals↗
PubMed1991

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.

Amino Acid Sequence↗
PubMed1990

Plasminogen receptors in the mediation of pericellular proteolysis.

A wide variety of cells bind plasminogen with very high capacity, with similar affinity and recognize the same structural features within the plasminogen molecule. As a consequence of binding to cell surfaces, plasminogen is more readily activated to plasmin. Plasmin remains cell-bound where it can degrade matrix constituents and is protected from inactivation by alpha 2-antiplasmin. Thus, the functional consequence of plasminogen binding to cells is pericellular proteolysis, permitting cell migration. Both proteins and nonprotein cell-surface constituents function as plasminogen binding sites. Gangliosides exhibit the appropriate properties of the non-protein plasminogen receptors.

Binding Sites↗