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PubMed2001

Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line.

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.

Adhesiveness↗
PubMed1996

Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways.

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.

Amino Acid Sequence↗
PubMed1998

Role of beta1 and beta3 integrins in human smooth muscle cell adhesion to and contraction of fibrin clots in vitro.

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alphavbeta3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha5, beta1, and alpha5/beta1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alphavbeta3, anti-alphav, anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha5beta1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.

Antibodies↗
PubMed2000

Integrin expression and IgA nephropathy: in vitro modulation by IgA with altered glycosylation and macromolecular IgA.

BACKGROUND: Signal transduction by mesangial cell (MC) integrins regulates cell growth and survival, extracellular matrix production, and organization. The aim of the study was to investigate human MC integrin modulation by differently glycosylated IgA and macromolecular IgA, which are thought to play a pathogenetic role in IgA nephropathy (IgAN). METHODS: MCs were incubated with purified human polymeric IgA, heat-aggregated IgA, IgA glycoforms generated by enzymatic hydrolysis of saccharide residues and serum fractions from IgAN patients, and controls isolated by lectin affinity and containing IgA with peculiar glycan patterns. Integrins were quantitated by flow cytometry. RESULTS: Cultured MCs highly expressed alphavbeta3 and some alpha3beta1; alphavbeta3 was up-regulated by matrix components (P < 0.02). In vitro desialylated and degalactosylated polymeric human IgA enhanced alphavbeta3 expression on cultured MCs (P < 0.001). Serum IgA glycoforms isolated from IgAN patients with high exposure of internal sugars, GalNAc, Neu5Ac2,6GalNAc, and Man enhanced alphav expression on cultured MCs more than healthy controls. CONCLUSIONS.: These data support the hypothesis that IgA glycation plays a role in modulating the cell-matrix interaction, and that this mechanism can be operating in IgAN.

Antibodies, Monoclonal↗
PubMed2005

Membrane-proximal {alpha}/{beta} stalk interactions differentially regulate integrin activation.

The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alphaIIbbeta3 and alphaVbeta3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn(2+). Although alphaVbeta3 showed robust ligand binding in the presence of Mn(2+), alphaIIbbeta3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alphaIIb and alphaV revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alphaIIb/alphaV domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alphaIIbbeta3 susceptible to Mn(2+) activation was the alphaV calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn(2+)-induced alphaVbeta3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alphaIIbbeta3 when alphaIIbbeta3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.

Animals↗
PubMed2003

Analysis of retinal pigment epithelium integrin expression and adhesion to aged submacular human Bruch's membrane.

PURPOSE: Uncultured aged retinal pigment epithelium (RPE) does not resurface aged Bruch's membrane after 24 hours in organ culture. These experiments assess whether culturing alters RPE integrin expression and resurfacing of Bruch's membrane. METHODS: RNA was isolated from uncultured and cultured RPE of aged adult donor and fetal eyes. Integrin subunit messenger RNA (mRNA) expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and semiquantitative analysis of the amplified products. Cell surface integrin expression was assessed using flow cytometry. Passaged cultured fetal RPE and primary cultured aged RPE were seeded onto Bruch's membrane, and resurfacing was assessed with scanning electron microscopy. RESULTS: Uncultured fetal RPE had low levels of alpha3 and beta5 mRNA compared to passaged cultured fetal RPE. Uncultured aged RPE had decreased alpha1-5 mRNA compared to primary cultured aged RPE. Cultured aged RPE had decreased beta4 and beta5 mRNA compared to passaged cultured fetal RPE. Flow cytometry confirmed the expression of alpha1-5, alphav, and beta1 protein on cultured fetal RPE and alpha1-3 and beta1 protein on cultured aged RPE. Twenty-four hours after seeding, cultured fetal and aged RPE resurfaced 99% +/- 1.3% and 76% +/- 22%, respectively, of aged submacular Bruch's membrane specimens from which native RPE had been debrided, exposing the native RPE basement membrane. Cultured fetal and aged RPE resurfaced 97% +/- 3.1% and 39% +/- 35%, respectively, of specimens in which the inner collagenous layer was exposed. CONCLUSIONS: Uncultured aged RPE has low amounts of integrin subunits that form receptors for laminin, fibronectin, and collagens. Culturing up-regulates integrins and promotes more efficient aged RPE attachment to and survival on aged Bruch's membrane.

Aged↗
PubMed2004

Matrix-bound sixth Ig-like domain of cell adhesion molecule L1 acts as an angiogenic factor by ligating alphavbeta3-integrin and activating VEGF-R2.

Angiogenic signals can be matrix attached or freely diffusible. Here, the sixth Ig-like domain of L1 (L1Ig6), a ligand for alphavbeta3-integrin, was investigated. This domain was expressed as a fusion protein having a substrate sequence for factor XIII to enable covalent binding into three-dimensional fibrin matrices. Matrix-bound L1Ig6 induced endothelial cell (EC) process extension in vitro, which was associated with ligation and phosphorylation of alphavbeta3-integrin. VEGF-R2 and alphavbeta3 were observed to co-associate after stimulation with either L1Ig6 or VEGF-A165, whereas no co-association with bFGF-R was observed. Furthermore, VEGF-R2 was tyrosine phosphorylated after stimulation with L1Ig6, even in the absence of exogenous VEGF-A165, indicating close cooperation between VEGF-R2 and alphavbeta3. Angiogenesis was investigated in vivo by stimulating chicken chorioallantoic membranes (CAMs) with L1Ig6-modified matrices with or without co-incorporation of VEGF-A165 or bFGF. Matrix-immobilized L1Ig6 induced angiogenesis to a similar degree as VEGF-A165; co-stimulation with bFGF increased vascular branching, whereas VEGF-A165 did not. Matrix-immobilized L1Ig6 induced up-regulation of alphav in CAMs by a similar degree as stimulation with VEGF-A165, and this up-regulation was increased further by co-stimulation with matrix-bound L1Ig6 and VEGF-A165. alpha5 and beta1 levels were not increased. The similarity of action of matrix-bound L1Ig6 and soluble VEGF-A165 indicate a close link between specific ligation of alphavbeta3-integrin and VEGF-R2 and suggest the possible use of matrix-bound L1Ig6 in local therapeutic angiogenesis.

Angiogenesis Inducing Agents↗
PubMed2004

Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells.

Helicobacter pylori colonizes the human stomach and causes gastric disease. The resulting gastric damage is a multi-step process involving several molecular factors and different target cells. Th1 cytokines released by neutrophils and lymphoid cells that infiltrate gastric mucosa, nitric oxide production and inducible nitric oxide synthase (iNOS) are associated with immune activation and tissue injury. Many other molecular processes such as apoptosis, as well as angiogenic factors and integrins, are involved in H. pylori pathogenesis. We used cancer gastric cells AGS and MKN as experimental models to evaluate apoptotic rates, iNOS gene expression with and without the presence of interferon-gamma (IFN-gamma), placenta growth factor gene expression and alphav modulation. Our results show that AGS cells stimulated with H. pylori underwent apoptosis. Moreover, the addition of IFN-gamma caused a further increase in iNOS gene expression and in the apoptotic rates. We also found early modulation in PlGF and alphav expression, and noted that p53 and bax gene expression was involved in the apoptotic process. Taken together, these findings demonstrate that H. pylori employs a series of mechanisms to avoid the host defense and cause gastric mucosa damage. One H. pylori pathogenic mechanism for causing gastric damage is the induction of iNOS-dependent apoptosis that is strongly enhanced by IFN-gamma. Thus, data obtained indicate that Th1 cytokines such as IFN-gamma, via modulation of iNOS gene expression, may contribute to an increase in the pathogenicity of H. pylori infections.

Apoptosis↗
PubMed1997

Cloning and characterization of a novel integrin beta3 subunit.

We have identified a novel integrin beta3 subunit, termed beta3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3'-untranslated region of the beta3C subunit differs from the previously reported beta3A (platelet) and beta3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The beta3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the beta3A gene. HEK 293 cells were stably co-transfected with alphaV and either beta3C (HEKbeta3C) or beta3A (HEKbeta3A). The viability of HEKbeta3C cells was lower than that of HEKbeta3A cells, and HEKbeta3C cells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both alphaVbeta3A and alphaVbeta3C isoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKbeta3A, HEKbeta3C cells failed to adhere to osteopontin, an alphaVbeta3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the beta3 integrin in cell adhesion and suggest a potential role for the beta3C integrin subunit in modulating cell-matrix interactions.

Amino Acid Sequence↗
PubMed2005

Three-dimensional EM structure of the ectodomain of integrin {alpha}V{beta}3 in a complex with fibronectin.

Integrins are alphabeta heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin alphaVbeta3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn(2+)-bound alphaVbeta3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most alphaVbeta3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound alphaVbeta3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of beta3, with FN9 just adjacent to the synergy site binding region of alphaV. We conclude that the ectodomain of alphaVbeta3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

Binding Sites↗
PubMed2004

Synthesis and biological evaluation of a (99m)Tc-labelled cyclic RGD peptide for imaging the alphavbeta3 expression.

AIM: The alphavbeta3 integrin is involved in tumour induced angiogenesis and tumour metastasis. We describe the synthesis and evaluation of a (99m)Tc-labelled RGD analogue for the visualisation of alphavbeta3 integrin expression. METHODS: The linear peptides were assembled on a solid support. Cyclisation was performed under high dilution conditions. For conjugation with the chelator peptide, a water soluble carbodiimide was used. Radiolabelling was carried out due to standard procedures with high radiochemical yield and radiochemical purity. For in vivo evaluation, nude mice bearing alphavbeta3-positive human melanoma M21 and alphav-negative human melanoma M21-L or Balb/c mice bearing alphav-positive murine osteosarcoma were used. RESULTS: Activity accumulation of (99m)Tc-DKCK-RGD 240 min p. i. was 1.1% ID/g in the alphavbeta3-positive melanoma and 0.3% ID/g in the negative control tumour. In the osteosarcoma model 2.2% ID/g was found 240 min p. i. Planar gamma camera images allowed contrasting visualisation of alphavbeta3-positive tumours 240 min p. i. Blocking of the tumour using the alphavbeta3-selective pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) reduces activity accumulation in the tumour to background level. However, 240 min p. i. highest activity concentration was found in kidneys resulting in low tumour/kidney ratios. Metabolite analysis 240 min p. i. showed approximately 60% intact tracer in kidneys and 80% in the tumour. Only 24% intact tracer was found in blood 30 min p. i. CONCLUSION: (99m)Tc-DKCK-RGD allows imaging of alphavbeta3-positive tumours in mice. However, pharmacokinetics as well as metabolic stability of the tracer have to be improved for potential clinical application.

Amino Acid Sequence↗
PubMed1999

Overexpression of protein kinase C-alpha in MCF-7 breast cancer cells results in differential regulation and expression of alphavbeta3 and alphavbeta5.

MCF-7 breast cancer cells stably transfected with protein kinase C-alpha (MCF-7-PKC-alpha cells) show anchorage-independent growth and exhibit increased tumorigenicity in nude mice. Since integrins are involved in tumor growth and metastatic spread, we investigated whether integrin expression is differentially regulated in MCF-7-PKC-alpha cells. Fluorescence-activated cell sorting revealed that alphavbeta3 is highly expressed on MCF-7-PKC-alpha cells, but is undetectable on MCF-7V cells (MCF-7 cells transfected with vector only). In contrast, MCF-7-PKC-alpha cells have reduced expression of alphavbeta5. Blocking experiments with antibodies to alphavbeta3 and alphavbeta5 revealed that these receptors are used by MCF-7-PKC-alpha cells to adhere primarily to vitronectin and osteopontin. Consistent with heterodimer expression, MCF-7-PKC-alpha cells express increased beta3 and decreased beta5 on their surface. Surface expression of alphav on MCF-7-PKC-alpha cells is unchanged. Western blotting, Northern analysis, and nuclear run-on assays indicated that post-translational mechanisms increase the surface expression of beta3 on MCF-7-PKC-alpha cells. In contrast, reduced beta5 transcription diminishes beta5 surface expression on MCF-7-PKC-alpha cells. These results indicate that overexpression of PKC-alpha in MCF-7 cells alters beta5 and beta3 expression by transcriptional and post-translational mechanisms, respectively, resulting in altered heterodimer expression. These findings suggest that the increased metastatic capacity of tumor cells with elevated protein kinase C levels may result, in part, from modulation of integrin expression.

Animals↗
PubMed1996

One-step affinity purification of recombinant alphavbeta3 integrin from transfected cells.

Vitronectin receptor (alphavbeta3 integrin) is present on the surface of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alphavbeta3 from Chinese hamster ovary (CHO) cells transfected with alphavbeta3 (VNRC3 cells). The method consists of two steps: lysis of the cells and affinity chromatography of the lysate on a GRGDSPK-Sepharose column. The yield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding to the alphav and beta3 subunits, and was recognized by monoclonal antibodies directed against alphav and the alphavbeta3 complex, but not by monoclonal antibody specific for the alphaIIbbeta3 complex. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen was not observed. Purified recombinant alphavbeta3 demonstrated greater immunoreactivity with LM 609, an alphavbeta3 complex-specific monoclonal antibody, than alphavbeta3 purified from placenta. As visualized by SDS-polyacrylamide gel electrophoresis, preparations of placenta-derived alphavbeta3 contained several contaminating proteins that were not present in preparations of recombinant alphavbeta3 purified from the transfected CHO cells.

Animals↗
PubMed2004

Increased expression levels of integrin alphavbeta5 on scleroderma fibroblasts.

Integrin alphavbeta5 is a receptor for vitronectin, a plasma glycoprotein that is also distributed in extracellular matrix of various tissues. Matrix-bound vitronectin has the potential to stabilize the active form of plasminogen activator inhibitor-1, resulting in the inhibition of the plasmin-mediated pericellular proteolytic cascade. In this study, we compared the levels of alphavbeta5 and matrix-bound vitronectin between normal and scleroderma fibroblasts and investigated the association with fibrosis. We demonstrated that alphavbeta5 was up-regulated on scleroderma fibroblasts. The up-regulated alphavbeta5 contributed to the increase in vitronectin-binding ability in scleroderma fibroblasts, which led to the vitronectin-dependent activation of plasminogen activator inhibitor-1. In immunohistochemistry, the alphav and beta5 subunits were stained strongly on scleroderma fibroblasts and the amount of vitronectin was increased in the pericellular matrix of those cells. The transient overexpression of alphavbeta5 on normal fibroblasts enhanced the human alpha2(I) collagen promoter activity through Sp-1 and Smad3 as well as the vitronectin-dependent plasminogen activator inhibitor-1 activity. This effect on the promoter activity was also observed in the absence of vitronectin and completely disappeared in the presence of anti-alphavbeta5 antibody. These results indicate that the up-regulated alphavbeta5 may contribute to the phenotypical alteration of scleroderma fibroblasts, while at the same time suppressing the plasmin-mediated pericellular proteolytic cascade.

Blotting, Northern↗
PubMed1995

Human melanoma integrins contribute to arrest and stabilization potential while flowing over extracellular matrix.

To form distant metastases, tumour cells must stabilize adhesive interactions that prevent detachment at secondary sites. Primary receptor-ligand interactions alone may not maintain prolonged adhesive contacts without secondary events that lead to adhesion stabilization. Computerized imaging methods enable us to examine various substrates for: (i) the wall shear adhesion threshold (WSAT), a measure of the dynamic adhesive potential of tumour cells; (ii) the number of tumour cells that adhered; and (iii) the adhesion stabilization lag time (ASLT) or length of time required for tumour cells to stabilize adhesive contacts capable of withstanding high wall shear force (up to 100 dynes/cm2). The relative WSAT ratios found were: wheat germ agglutinin (WGA) > laminin > fibronectin > vitronectin > collagen I > collagen IV > von Willebrand factor (vWF) (the greater the shear rate the higher the adhesive potential). The relative stabilization ratios found were as follows: laminin < fibronectin < vitronectin < collagen IV < collagen I < vWF < WGA (shorter times correlate with greater stabilization potential). Stabilization data using fibronectin as a substrate correlated the best with metastatic potential. Using three melanoma lines of different metastatic potential semiquantitative reverse transcriptase-polymerase chain reaction (PCR) showed a two- to four-fold increase in alpha1, alpha3, alpha4, alpha5, alpha6, and ICAM-1 in the highly metastatic 70W cells compared to the MeWo and non-metastatic 3S5 melanoma cells. There were no differences in alphav, beta1 and beta3 levels among the three melanoma lines, and PCR products for alphaIIb, alpha2, CD36, or ICAM-2 were not detected. The 70W cells also had higher levels of alphax and beta2 (CD11/CD18 and p150 leukocyte antigen) than either the MeWo or 3S5 cells. The data indicate that melanoma cells exhibit differences in the adhesion properties under fluid shear and differences in the expression of adhesion components that correlate with their metastatic potential.

Base 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↗
PubMed1998

Role of the alpha-subunit 326GRV sequence in the surface expression of fibrinogen and vitronectin receptors.

The platelet GPIIb-GPIIIa heterodimer (integrin alphaIIbbeta3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The 326GRV motif in GPIIb is highly conserved in the alpha-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327-->His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327 or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327 or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327 in the alpha-subunit of the vitronectin receptor, to His did not prevent the surface expression of alphav-GPIIIa complexes. Thus the conformational constraint imposed by His327 seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

Amino Acid Sequence↗
PubMed2003

Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts.

Osteoclasts from osteopontin-deficient mice exhibit decreased CD44 surface expression [corrected]. Osteopontin (OPN)/alphavbeta3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-alpha), to be a potent activator of CD44 surface expression. ROK-alpha activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-alpha promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44.ERM.actin complex formation. Osteoclasts from OPN-/- mice exhibited an approximately 55-60% decrease in basal level ROK-alpha phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-alpha/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-alpha by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-alpha and CD44 surface expression. Neutralizing antibodies to alphav, beta3, or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-alphav or -beta3 antibodies blocked OPN-induced phosphorylation of ROK-alpha, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-alpha in alphavbeta3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between alphavbeta3 and CD44 for osteoclast motility and bone resorption.

Animals↗