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PubMed1999

Selective Sp1 and Sp3 binding is crucial for activity of the integrin alphaV promoter in cultured endothelial cells.

Human integrin alphavbeta receptors are expressed in a number of cells and their expression is regulated at the level of transcription and by post-transcriptional mechanisms. A substantial body of research exists on the structure, function, molecular biology and physiological significance of alphav integrin receptors. However, the importance of particular cis-acting DNA elements or trans-acting nuclear factors in the regulation of the alphav gene promoter is still not adequately understood. Previous functional analysis of the alphav gene 5' flanking region in transfected cultured cells identified cis elements critical for alphav transcription within a 222-bp region. To define further the location of this enhancing element, we performed DNase I footprinting of the human alphav gene promoter between -522 and the translation initiation site. For this purpose, nuclear extracts of alphavbeta3-positive cells, human umbilical vein endothelial cells, were used. Nuclear proteins of endothelial cells strongly protected essentially one region corresponding to the sequence between -194 and -172 of the alphav promoter region. Electrophoretic mobility shift assays with different oligonucleotides, and competition analysis identified a CTCCTCCTC sequence that is directly involved in the transcriptional activity of the alphav promoter. Purified Sp1 alone produced an identical footprint, and DNA binding assays using anti-Sp1 and anti-Sp3 antibodies showed that transcription factors Sp1 and Sp3 were the major nuclear proteins bound to this region.

Antigens, CD↗

PubMed2001

Extracellular signal-regulated kinase (ERK) activation is required for GP Ibalpha-dependent endothelial cell migration.

The GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibalpha, and echistatin inhibited alphavbeta3 binding. Albo A, B and echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibalpha monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to alphavbeta3, EC GP Ibalpha initiates ERK activation, and regulates ERK-induced EC migration on vWF.

Antibodies, Monoclonal↗

PubMed1999

A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.

Antibodies, Monoclonal↗

PubMed2000

Structure and function of murine alphaIIbbeta3 (GPIIb/IIIa): studies using monoclonal antibodies and beta3-null mice,.

The alphaIIbeta3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet alphaIIbeta3, utilizing both beta3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine alphaIIbbeta3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of alphaIIbbeta3 (60-80,000 copies/platelet). Like their human counterparts, murine alphaIIb and beta3 exhibit different electrophoretic motilities under nonreducing (aIIb 135k Da; beta3 92k Da) and reducing (aIIb 120k Da; beta3 108k Da) conditions, and the alphaIIbbeta3 complex is dissociated by EDTA at pH 8 and 37 degrees C. Murine beta3 is less susceptible to proteolysis by plasmin than is human beta3. In addition to defective platelet aggregation, mouse platelets lacking alphaIIbbeta3 and alphaVbeta3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, beta3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, beta3-null platelets have altered tyrosine phosphorylation patterns following thrombin- and collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.

Animals↗

PubMedDate not supplied

Insights into phenylalanine derivatives recognition of VLA-4 integrin: from a pharmacophoric study to 3D-QSAR and molecular docking analyses.

The very late antigen-4 (VLA-4), also known as integrin alpha4beta1, is expressed on monocytes, T- and B-lympohocytes, basophils, and eosinophils and is involved in the massive recruitment of granulocytes in different pathological conditions such as multiple sclerosis and asthma. VLA-4 interacts with its endogenous ligand VCAM-1 during chronic inflammation, and blockade of VLA-4 /VCAM-1 interaction is a potential target for immunosuppression. Two classes of VLA-4 antagonists have so far been reported: beta-amino acid derivatives containing a diaryl urea moiety (BIO-1211) and phenylalanine derivatives (TR-14035). With the aim of clarifying the structural basis responsible for VLA-4 recognition by phenylalanine derivatives, we developed a combined computational study on a set of 128 antagonists available through the literature. Our computational approach is composed of three parts. (i) A VCAM-1 based pharmacophore was constructed with a restricted number of phenylalanine derivatives to identify the region of the protein that resembles synthetic antagonists. The pharmacophore was instrumental in constructing an alignment of a set of 128 compounds. This alignment was exploited to build a pseudoreceptor model with the RECEPTOR program. (ii) 3D-QSAR analysis was carried out on the computed electrostatic and steric interaction energies with the pseudoreceptor surface. The 3D-QSAR analysis yielded a predictive model able to explain much of the variance of the 128 antagonists. (iii) A homology modeling study of the headpiece of VLA-4 based on the crystal structure of alphavbeta3 was performed. Docking experiments of TR-14035 into the binding site of VLA-4 aided the interpretation of the 3D-QSAR model. The obtained results will be fruitful for the design of new potent and selective antagonists of VLA-4.

Amino Acid Sequence↗

PubMed2006

The specificity and function of the metal-binding sites in the integrin beta3 A-domain.

The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta3 A-domain, beta3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to alphavbeta3 but not alphaIIbbeta3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta3 A-domain.

Binding Sites↗

PubMed1998

Glycoprotein IIb Leu214Pro mutation produces glanzmann thrombasthenia with both quantitative and qualitative abnormalities in GPIIb/IIIa.

Glanzmann thrombasthenia is an inherited bleeding disorder due to a functional reduction or absence of platelet GPIIb/IIIa (alphaIIbbeta3) integrin receptors. Based on a prolonged bleeding time and absence of platelet aggregation in response to physiologic agonists, a 55-year-old white man was diagnosed as having Glanzmann thrombasthenia. The patient's platelet fibrinogen level was approximately 5% of normal. As judged by complex-dependent monoclonal antibody (MoAb) binding, surface expression of platelet GPIIb/IIIa receptors was less than 5.5% of normal, whereas the binding of an anti-GPIIIa specific MoAb (7H2) was approximately 12% of normal. Immunoblot analysis of the patient's platelet lysates showed approximately 35% of normal levels of GPIIIa, approximately 30% of normal levels of GPIIb, and an abnormally migrating fragment of GPIIb. Biotinylation of the surface proteins on the patient's platelets followed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed only GPIIb and GPIIIa subunits of normal size. Surface expression of platelet alphavbeta3 receptors was 192% of normal, suggesting that the patient's' defect was in GPIIb. Sequence analysis of the patient's GPIIb cDNA identified a T to C transition at nucleotide 643, predicting a Leu214Pro substitution. Direct sequencing of GPIIb exon 6 indicated that the patient is homozygous for the mutation. The nature of the Leu214Pro mutation was analyzed by expression in Chinese hamster ovary (CHO) cells. As judged by subunit-specific MoAb binding, surface expression of mutant receptors was approximately 60% of normal, but these receptors were not recognized by the complex-dependent monoclonal antibodies, 10E5 and 7E3. In addition, mutant receptors pretreated with the ligand-induced binding site MoAb AP5 were not recognized by the activation-dependent MoAb PAC-1 and mutant expressing CHO cells did not adhere to immobilized fibrinogen. These data suggest that the Leu214Pro mutation in GPIIb disrupts the structural conformation, and either directly or indirectly, the ligand binding properties of the heterodimeric complex. This is in accord with studies from other integrins that have implicated a beta-turn in a homologous region as important in ligand binding. Thus, the Leu214Pro mutation appears to produce the Glanzmann thrombasthenia phenotype by both qualitative and quantitative abnormalities. In addition, the mutation appears to confer susceptibility of the GPIIb subunit to proteolysis.

Animals↗

PubMed1999

Effect of exogenous gonadotropins on endometrial maturation in oocyte donors.

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.

Adult↗

PubMed2004

Targeted mutagenesis of the angiogenic protein CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-heparan sulfate proteoglycan coreceptor-mediated cellular functions.

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin alpha6beta1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha6beta1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support alpha6beta1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha6beta1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished alpha6beta1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alphavbeta3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha6beta1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha6beta1-HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha6beta1-HSPG-specific activities in vivo.

Amino Acid Sequence↗

PubMed1998

Vitronectin expression and interaction with receptors in smooth muscle cells from human atheromatous plaque.

Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, alphavbeta3 and alphavbeta5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, alphavbeta3 and alphavbeta5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors.

Arteriosclerosis↗

PubMed2003

Different effects of various anti-GPIIb-IIIa agents on shear-induced platelet activation and expression of procoagulant activity.

Inhibitors of the platelet glycoprotein (GP)IIb-IIIa receptor (integrin alphaIIbbeta3) reduce acute thrombotic events in patients with coronary artery disease. To characterize the mechanism of action of these drugs, we evaluated the effects of different GPIIb-IIIa antagonists on shear-induced platelet aggregation, activation, and the expression of procoagulant activity. Samples of platelet-rich plasma from 16 volunteers were exposed to the shear rate of 10 800 s-1 for 6 min in an optically modified cone-plate viscometer. Abciximab, tirofiban and eptifibatide inhibited aggregation to a similar extent (mean +/- SD: 74.1 +/- 8.5%, 69.5 +/- 13.6%, 65.6 +/- 17.0%, respectively), but only abciximab inhibited significantly microparticle release associated with shear-induced platelet activation (64.4 +/- 13.6%, P = 2.2 x 10-7; tirofiban = 20.0 +/- 23.4%; eptifibatide = 23.9 +/- 17.4%). P-selectin platelet surface translocation was also strongly inhibited by abciximab, weakly by eptifibatide, but not by tirofiban. The addition of anti-alphavbeta3 to tirofiban enhanced the inhibiting effects on shear-induced P-selectin translocation and microparticle release. Shearing of platelet-rich plasma shortened the re-calcification clotting time after addition of kaolin from 106.9 +/- 14.3 to 94.2 +/- 10.7 s (mean +/- SD; P = 0.0013). This effect, which is mediated by the appearance of procoagulant phospholipids on the surface of sheared platelets and microparticles, was prevented by abciximab and by the combination of tirofiban and anti-alphavbeta3, but not by tirofiban alone or eptifibatide. The ability to inhibit shear-induced platelet activation, as evidenced by microparticle release and P-selectin surface translocation as well as the expression of procoagulant activity, differentiates the effects of anti-GPIIb-IIIa agents, which may explain the distinct antithrombotic efficacy of the agents.

Abciximab↗

PubMed1996

Cell-type specific recognition of RGD- and non-RGD-containing cell binding domains in fibrillin-1.

The fibrillins are large glycoprotein components of 10-nm microfibrils found in the extracellular matrix of most tissues. Microfibrils play a role in elastic fiber assembly and serve to link cells to elastic fibers in the extracellular matrix. To determine whether fibrillin-1 specifically interacts with receptors on cells from fibrillin-rich tissues, we evaluated whether two cell types that produce different types of fibrillin can adhere to purified fibrillin-1 in cell adhesion assays. Our results indicate that both cell types attach and spread on fibrillin-1 and that the RGD sequence in the fourth 8-cysteine motif mediates this interaction. Fibroblast attachment to fibrillin-1 was sensitive to inhibition by antibodies to the alphavbeta3 receptor and by peptides encoding the RGD sequence in fibrillin-1 and the second RGD sequence in fibrillin-2. In contrast, adhesion of auricular chondroblasts to fibrillin-1 was only partially inhibited by these reagents, suggesting that some cell types recognize a second, non-RGD binding site within the fibrillin molecule. These findings confirm and extend ultrastructural studies that suggest a direct interaction between microfibrils and the cell surface and provide a functional explanation for how this association occurs.

Amino Acid Sequence↗

PubMed1999

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

Amino Acid Substitution↗

PubMed2004

Nonpeptide alphavbeta3 antagonists. Part 11: discovery and preclinical evaluation of potent alphavbeta3 antagonists for the prevention and treatment of osteoporosis.

3-(S)-Pyrimidin-5-yl-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5e) and 3-(S)-(methylpyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5f) were identified as potent and selective antagonists of the alpha(v)beta(3) receptor. These compounds have excellent in vitro profiles (IC(50) = 0.07 and 0.08 nM, respectively), significant unbound fractions in human plasma (6 and 4%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in an in vivo model of bone turnover following once-daily oral administration, these two compounds were selected for clinical development for the treatment of osteoporosis.

Administration, Oral↗

PubMed2002

Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis.

The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.

Binding Sites↗