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L V Parise

Publications and source records attributed to L V Parise.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modulation of integrin expression in endometrial stromal cells in vitro.

Integrins, a class of cell adhesion molecules found on virtually all cells, display dynamic temporal and spatial patterns of expression in the endometrium during the menstrual cycle and in early pregnancy. To study integrin regulation, we measured the expression of eight different integrin subunits on cultured human endometrial stromal cells obtained from proliferative phase endometrium, using immunofluorescence and flow cytometry. Treatment with estrogen and progesterone induced hormonal changes of decidualization but did not alter the expression of any of the integrins. It is presently unknown whether steroid hormones other than estrogen or progesterone affect integrin expression. In contrast, treatment with several growth factors and cytokines resulted in specific alterations of integrin levels. Epidermal growth factor, transforming growth factor-alpha, and transforming growth factor-beta 1 induced expression of the alpha 1 beta 1 collagen/laminin receptor. There also was a trend towards decreased expression of the alpha 6 subunit in response to interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor-alpha. The expression of alpha 1 beta 1 was accompanied by increased adhesion to collagen but there was no change in the binding to fibronectin and vitronectin. Our findings suggest that some aspects of decidualization may be regulated by steroid hormones, whereas others, such as integrin expression, are regulated by cytokines or growth factors, possibly of trophoblast origin. Integrins are likely to play a role in the interaction between trophoblast and endometrium.

Adult↗
PubMed1993

Integrin alpha 4 beta 1 and glycoprotein IV (CD36) are expressed on circulating reticulocytes in sickle cell anemia.

The abnormal adherence of red blood cells, especially circulating reticulocytes (erythrocyte precursors), to the endothelium is believed to contribute to vascular occlusion observed in patients with sickle cell disease. Although several plasma proteins including von Willebrand factor and fibronectin have been proposed to mediate this adhesion, the mechanism of sickle cell adhesion to the endothelium remains unknown. Using flow cytometry, we screened sickle red blood cells with monoclonal antibodies (MoAbs) against known adhesion receptors and detected integrin subunits alpha 4 and beta 1 and the nonintegrin glycoprotein IV on reticulocytes but not on erythrocytes. No reactivity was detected against integrin subunits alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 2, beta 3, integrin alpha IIb beta 3, or the nonintegrin glycoprotein Ib. Immunoprecipitation of reticulocytes with either alpha 4- or beta 1-specific antibodies identified the alpha 4 beta 1 complex (alpha 4(70) and alpha 4(80) forms), a receptor for fibronectin and vascular cell adhesion molecule-1. An antibody against glycoprotein IV, a receptor reported to bind thrombospondin and collagen, immunoprecipitated an 88-kD protein consistent with its reported M(r). MoAbs against alpha 4 and glycoprotein IV bound to an average of 4,600 and 17,500 sites per reticulocyte, respectively. Identification of alpha 4 beta 1 and glycoprotein IV on reticulocytes suggests both plasma-dependent and independent mechanisms of reticulocyte adhesion to endothelium and exposed extracellular matrix.

Anemia, Sickle Cell↗
PubMed1993

Regulation of ligand binding to glycoprotein IIb-IIIa (integrin alpha IIb beta 3) in isolated platelet membranes.

The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.

Amino Acid Sequence↗
PubMed1993

Evidence for novel binding sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen.

The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen.

Amino Acid Sequence↗
PubMed1992

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor.

Antibodies, Monoclonal↗
PubMed1992

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

Antigens, Differentiation, Myelomonocytic↗
PubMed1991

Ca(2+)-dependent structural transitions of the platelet glycoprotein IIb-IIIa complex. Preparation of stable glycoprotein IIb and IIIa monomers.

The platelet membrane glycoprotein (GP) IIb-IIIa complex is the receptor for adhesive proteins on activated platelets that mediates platelet aggregation. In the present study, factors affecting the structural stability of the purified GP IIb-IIIa complex and the dissociated subunits were investigated. Purified GP IIb-IIIa was incubated in various Ca2+ concentrations, and the percentage of dissociated subunits was quantitated by sucrose gradient sedimentation. Two Ca(2+)-dependent transitions were observed, one at about 60 microM Ca2+, where half of the complexes became dissociated, and the other at 0.1 microM Ca2+, where half of the dissociated subunits became incapable of reforming heterodimer complexes when higher Ca2+ concentrations were readded. This loss in ability to reform heterodimer complexes was caused primarily by a Ca(2+)-dependent transition in GP IIIa, leading to an apparent unfolding of this subunit, followed by the formation of high molecular weight aggregates. The formation of these aggregates was time- and temperature-dependent and could not be reversed by added Ca2+. Although Mg2+ prevented dissociation of GP IIb-IIIa, it failed to promote reassociation of the dissociated subunits. Based on these findings, conditions were developed for the preparation of dissociated GP IIb and GP IIIa such that 70% of the subunits remained functional in that they retained the ability to reform heterodimer complexes.

Calcium↗
PubMed1991

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.

Amino Acids↗
PubMed1990

Glycoprotein IIIa is phosphorylated in intact human platelets.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.

Alkaloids↗
PubMed1987

Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a platelet cell-surface receptor for fibrinogen and fibronectin. A carboxyl-terminal decapeptide of the fibrinogen gamma-chain (Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val LGGAKQAGDV] and a tetrapeptide (Arg-Gly-Asp-Ser (RGDS] from the fibrinogen alpha-chain and the fibronectin cell-binding domain appear to mediate the binding of these ligands to GP IIb-IIIa. The present study was designed to examine the effects of these and related peptides on the structure of purified platelet GP IIb-IIIa. Treatment of GP IIb-IIIa with various synthetic peptides affected the glycoprotein so that GP IIb alpha became a substrate for hydrolysis by thrombin. The order of potency of these peptides was as follows: RGDS greater than LGGAKQAGDV greater than KGDS greater than RGES. This is the same order of potency in which these peptides inhibit fibrinogen binding to platelets. This effect was time-, temperature-, and concentration-dependent; RGDS induced a half-maximal effect at approximately 60 microM. In addition, RGDS, but not RGES, decreased the intensity of the intrinsic protein fluorescence of GP IIb-IIIa. Finally, the decapeptide or RGDS decreased the sedimentation coefficient of GP IIb-IIIa from 8.5 to 7.7 or 7.4 S, respectively, whereas RGES had a minimal effect. This decrease was accompanied by an increase in the Stoke's radius from 74 to 82 A with RGDS or 85 A with the decapeptide, indicating a peptide-induced unfolding of the GP IIb-IIIa complex. This change in conformation may be related to changes in the distribution and function of GP IIb-IIIa on the platelet surface that occur when adhesive proteins or peptides from the GP IIb-IIIa binding domains of these proteins bind to GP IIb-IIIa.

Binding Sites↗
PubMed1987

The platelet membrane glycoprotein IIb/IIIa complex. Structure, function, and relationship to adhesive protein receptors in nucleated cells.

The GPIIb/IIIa complex functions as the aggregation site on the platelet membrane surface. This complex has been purified, characterized biochemically and morphologically, and reconstituted into phospholipid vesicles. Fibrinogen and fibronectin bind to reconstituted GPIIb/IIIa with many of the properties that characterize their binding to intact platelets. The GPIIb/IIIa complex appears to be a member of a widely distributed family of cell-surface glycoproteins that mediate cellular interactions. The terms cytoadhesins30 and integrins39 have been suggested for the members of this family of two-subunit molecules. The aminotermini of the alpha subunits of these molecules have been sequenced and appear to be homologous. Three beta subunits have been identified for this family of receptors, indicating that many alpha subunits have a common beta subunit. The three beta subunits have been sequenced, and there is about a 40 to 50% identity among their amino acid sequences. It thus appears that the receptors mediating cellular interactions have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1986

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex.

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.

Amino Acid Sequence↗
PubMed1985

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex.

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.

Amino Acids↗
PubMed1985

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology.

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.

Blood Platelets↗
PubMed1984

Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A2/prostaglandin H2 receptor interaction.

The mechanism by which the active metabolites of arachidonic acid (AA), i.e., thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2) induce platelet aggregation is not understood. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP, whereas other studies have proposed that this response is ADP-independent. In the present report, we used the specific TXA2/PGH2 receptor antagonist, 13-azaprostanoic acid (13-APA), and the ADP antagonist, ATP, to examine the contribution of TXA2/PGH2 or secreted ADP to aggregation. We found that 13-APA, but not ATP, deaggregates platelets stimulated by AA or U46619 (a TXA2/PGH2 mimetic). In contrast, ADP-induced aggregation was reversed in response to ATP but not to 13-APA. These results suggest that TXA2/PGH2-stimulated aggregation is mediated through TXA2/PGH2 receptor occupation. Furthermore, secreted ADP does not appear to be required for maintenance of the AA-aggregation response.

Adenosine Diphosphate↗