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At least 955 records · Page 53Linked to original sources
PubMed2006

Wiskott-Aldrich syndrome protein is involved in alphaIIb beta3-mediated cell adhesion.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.

Blood Platelets↗
PubMed2004

Platelet factor XIII and calpain negatively regulate integrin alphaIIbbeta3 adhesive function and thrombus growth.

Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb-IIIa (integrin alpha(IIb)beta3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin alpha(IIb)beta3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin alpha(IIb)beta3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin alpha(IIb)beta3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.

Animals↗
PubMed2006

TA205, an anti-talin monoclonal antibody, inhibits integrin-talin interaction.

Talin mediates integrin signaling by binding to integrin cytoplasmic tails through its FERM domain which consists of F1, F2 and F3 subdomains. TA205, an anti-talin monoclonal antibody, disrupts actin stress fibers and focal adhesion when microinjected into fibroblasts. Here, we showed that TA205 caused an allosteric inhibition of integrin alphaIIb beta3 binding to the talin FERM domain and mapped the TA205 epitope to residues 131-150 in talin F1. Furthermore, binding of a talin rod fragment to talin head was partially inhibited by TA205. These findings suggest that talin F1 may be important in regulation of integrin binding and talin head-rod interaction.

Allosteric Regulation↗
PubMed2001

Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors.

Liposome-encapsulated hemoglobin (LEH), a candidate for red blood cell substitute, is cleared from circulation primarily by the phagocytic system, most likely after opsonization of the vesicles by immunoproteins, particularly complement components. Although modification of LEH by polyethylene glycol (PEG) derivatives prolongs its half-life by blocking the opsonization, the half-life is still short as compared with that of red blood cell components. Therefore, this study was performed to elucidate the opsonin-independent mechanisms that regulate phagocytosis of Neo Red Cell (NRC), a PEG-modified LEH, in culture. PKH67 was used as a fluorescence marker, allowing the quantitation of the phagocytosis of NRC by peripheral blood monocytes plus macrophages. The phagocytosis of PKH67-labeled NRC was inhibited by the addition of an excess of unlabeled NRC, indicating that the phagocytosis of PKH67-labeled NRC is specific to NRC, but not to PKH67. The phagocytosis of NRC was blocked about 70% by anti-CD14, 60% by anti-CD36 and 30% by anti-CD51/61 (vitronectin receptor, alpha(v)beta3). These results provided evidence of an opsonin-independent pathway for the phagocytosis of PEG-modified LEH.

Antigens, CD↗
PubMed2002

Glanzmann's thrombasthenia: identification of 19 new mutations in 30 patients.

Glanzmann's thrombasthenia (GT) is a genetically heterogeneous autosomal recessive syndrome associated with a bleeding tendency. To elucidate molecular basis of GT we have screened for mutations 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the alphaIIb and 4 in the beta3 gene have been found. Only two (alphaIIb Pro145Ala and IVS3(-3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in alphaIIb and beta3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of alphaIIb and beta3 genes.

Adolescent↗
PubMed2003

A mutation in the integrin alphaIIb subunit that selectively inhibits alphaIIb beta3 receptor function.

The alpha(IIb) and alpha(v) integrins have been shown to play a significant role in a variety of disease processes. alpha(IIb)beta(3) is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by alpha(IIb)beta(3) is essential for modulation of platelet function. To identify alpha(IIb) residues involved in alpha(IIb)beta(3) ligand binding function, cells expressing a constitutively active variant of alpha(IIb)beta(3) were randomly mutagenized and selected for loss of alpha(IIb)beta(3) ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in alpha(IIb) (Ser9-->Leu). Cells expressing this alpha(IIb) mutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in beta strand A of all seven repeats of alpha(IIb) similarly inhibited ligand binding to alpha(IIb)beta(3). alpha(IIb)S96 maps to the central cavity of the beta-propeller fold of the alpha(IIb) subunit immediately adjacent to a structurally important sequence at the center of the alphaand beta subunit interface. In contrast, substitution of the analogous residues in alpha(v) or alpha(4) did not disrupt the ligand binding function of alpha(v)beta(3) or alpha(4)beta(1). These data support a potential unique structural or mechanistic role for this residue in alpha(IIb)beta(3) receptor function.

Amino Acid Substitution↗
PubMed2005

The regulation of expanded human nasal chondrocyte re-differentiation capacity by substrate composition and gas plasma surface modification.

Optimizing re-differentiation of clinically relevant cell sources on biomaterial substrates in serum containing (S+) and serum-free (SF) media is a key consideration in scaffold-based articular cartilage repair strategies. We investigated whether the adhesion and post-expansion re-differentiation of human chondrocytes could be regulated by controlled changes in substrate surface chemistry and composition in S+ and SF media following gas plasma (GP) treatment. Expanded human nasal chondrocytes were plated on gas plasma treated (GP+) or untreated (GP-) poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer films with two compositions (low or high PEG content). Total cellularity, cell morphology and immunofluorescent staining of vitronectin (VN) and fibronectin (FN) integrin receptors were evaluated, while post-expansion chondrogenic phenotype was assessed by collagen types I and II mRNA expression. We observed a direct relationship between cellularity, cell morphology and re-differentiation potential. Substrates supporting high cell adhesion and a spread morphology (i.e. GP+ and low PEG content films), resulted in a significantly greater number of cells expressing alpha5beta1 FN to alpha(V)beta3 VN integrin receptors, concomitant with reduced collagen type II/ImRNA gene expression. Substrates supporting low cell adhesion and a spherical morphology (GP- and high PEG content films) promoted chondrocyte re-differentiation indicated by high collagen type II/I gene expression and a low percentage of alpha5beta1 FN integrin expressing cells. This study demonstrates that cell-substrate interactions via alpha5beta1 FN integrin mediated receptors negatively impacts expanded human nasal chondrocyte re-differentiation capacity. GP treatment promotes cell adhesion in S+ media but reverses the ability of low PEG content PEGT/PBT substrates to maintain chondrocyte phenotype. We suggest alternative cell immobilization techniques to GP are necessary for clinical application in articular cartilage repair.

Adult↗
PubMed1998

Identification of a novel truncated alphaIIb integrin.

Integrin alphaIIb beta3 requires its cytoplasmic tails to participate in tumor cell adhesion, spreading, and migration. Using 3' rapid amplification of cDNA ends, we have amplified two alphaIIb cDNAs from human leukemia, prostate adenocarcinoma, and melanoma cells. One of these is the predicted wild-type alphaIIb cDNA, and the other is a novel truncated alphaIIb variant. This variant is unique in that it lacks the transmembrane and cytoplasmic portions of the alphaIIb light chain. The truncated alphaIIb integrin protein is expressed by human leukemia, prostate adenocarcinoma, and melanoma cells but not by platelets or normal prostate epithelial or normal breast epithelial cells. Tumor cells secrete this protein and deposit it on the extracellular matrix. To our knowledge, this is the first report of a naturally occurring variant of an alpha integrin that lacks the transmembrane and cytoplasmic tail.

Alternative Splicing↗
PubMed1997

Spatial and temporal analyses of integrin and Muc-1 expression in porcine uterine epithelium and trophectoderm in vitro.

The expression of the glycoproteins Muc-1 and integrin subunits (alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3), all of which may be involved in the control of uterine receptivity, was examined in cultured porcine uterine epithelial (pUE) cells. Integrin subunit expression was also determined in trophoblastic vesicles generated from Day 12 and Day 15 pig conceptuses. Immunocytochemistry was performed on pUE cells cultured on several substrates (glass, serum, fibronectin, or Matrigel) in DME/F12 medium with 5% charcoal/dextran-stripped fetal bovine serum either containing no steroids or supplemented with estrogen, progesterone, or estrogen plus progesterone, or in medium with 5% complete fetal bovine serum. In addition, pUE cells were grown on Matrigel-coated Millicell filter inserts to facilitate development of structurally and functionally distinct apical and basal domains (i.e., polarized pUE cells) and were maintained in the same medium with supplements. The major findings in this investigation are that 1) Muc-1 and alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3 integrin subunits are expressed on pUE cells in vitro; 2) Muc-1 and several integrin subunits are modulated by steroid hormones if culture conditions are provided that permit development of cell polarity; 3) the expression and steroid-induced modulation of Muc-1 and several integrin subunits in polarized pUE cells are similar to those detected in vivo; and 4) the expression of alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3 integrin subunits by trophoblastic vesicles is identical to their expression on trophectoderm of intact conceptuses in vivo. These results suggest that the properties of polarized pUE cells and trophoblastic vesicles are comparable to those of their counterparts in vivo and are, therefore, useful as in vitro model systems to study conceptus-endometrial interactions during the periimplantation period and the control of uterine receptivity to implantation.

Animals↗
PubMed2006

Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2006

Molecular mediators of alphavbeta3-induced endothelial cell survival.

The alphavbeta3 integrin interaction with the extracellular matrix (ECM) plays an essential role in inhibiting apoptosis in endothelial cells. We have recently shown that alphavbeta3 ligation on rat aortic endothelial cells (RAECs) specifically activates the transcription factor nuclear factor kappaB (NF-kappaB) and promotes cell survival. Inhibiting NF-kappaB nuclear translocation abolished the protective effect of alphavbeta3 ligands. Here, we report that ligation of alphavbeta3 by its ligand, osteopontin (OPN), induces the phosphorylation and activation of inhibitory kappa B kinase beta IKKbeta and promotes the specific degradation of inhibitory kappa Balpha (IkappaBalpha) in RAECs. Overexpression of a dominant negative (DN) IKKbeta protein prevents IkappaBalpha phosphorylation, NF-kappaB activation, and inhibits the protective effects of OPN. The NF-kappaB-inducing kinase (NIK) has been shown to be one of the upstream kinases involved in IKK activation. OPN-mediated NF-kappaB activity is increased upon NIK wild-type (WT) overexpression and blocked following NIK DN overexpression. In addition, NIK-/-mouse embryonic fibroblasts (MEFs) plated on OPN display reduced NF-kappaB activity and decreased IkappaBalpha phosphorylation compared to NIK+/+MEFs. Finally, functional inhibition of integrin beta3-dependent NF-kappaB signaling decreases OPN-induced IkappaBalpha, IKKbeta and NIK phosphorylation. These studies for the first time show that the alphavbeta3-NF-kappaB-dependent endothelial survival pathway is dependent on IkappaBalpha, IKKbeta, and NIK.

Animals↗
PubMed2005

A 13-bp deletion in alpha(IIb) gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of alpha(IIb)beta3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the alpha(IIb) gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes alpha(IIb)beta3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300-600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective alpha(IIb)beta3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in alpha(IIb) gene, TT1616-7 deletion in beta3 gene, and IVS14: -3C --> G in beta3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of beta3 and was expressed as dysfunctional alpha(IIb)beta(3). None of 15 unrelated Jordanian patients carried any of the described mutations.

Amino Acid Substitution↗
PubMed1997

Characterization of the interaction between the Staphylococcus aureus clumping factor (ClfA) and fibrinogen.

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of biomaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen gamma chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the gamma chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen gamma chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221-550) recognized intact recombinant fibrinogen gamma chains, but failed to recognize recombinant fibrinogen gamma chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen gamma chains contains a binding site for the integrin alphaIIb beta3 (glycoprotein gpIIb/IIIa) receptor on platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Hawiger, J. (1989) Biochemistry 28, 2915-1919; Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl. Acad. Sci. USA 89, 10729-10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701-706]. We now show that Clf33 inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 +/- 0.19 microM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal gamma-chain peptide effectively inhibited this interaction.

Adenosine Diphosphate↗
PubMed2004

Reduced beta3-endonexin levels are associated with enhanced urokinase-type plasminogen activator receptor expression in ApoE-/- mice.

Proteolysis of extracellular matrix components is required for cell migration occurring in atherosclerotic lesion formation. In the present study, gene expression of the urokinase plasmingen activator receptor (uPAR) and underlying mechanisms were analyzed during the development of atherosclerosis in the aorta of apolipoprotein E-deficient mice (apoE(-/-)). A significant increase of uPAR expression was detected in the atherosclerotic tissue with advancing plaque-dimension. As uPAR gene transcription involves the transcription factor nuclear factor-kappaB (NF-kappaB), we analyzed nuclear NF-kappaB activity in vascular tissue of apoE-deficient mice. Congruent to uPAR, we could detect an increase in NF-kappaB activity, which underlines the chronic inflammatory component of the disease. Recently we reported that beta(3)-endonexin, a protein that modulates beta(3)-integrins, regulates uPAR expression through direct interaction with subunits of the NF-kappaB-complex. Herein we could show that beta(3)-endonexin protein is expressed in aortic tissue of mice. Moreover, in contrast to uPAR or NF-kappaB, the expression of beta(3)-endonexin was reduced in extracts of advanced atherosclerotic aortic tissue. The cytoplasmic protein beta(3)-endonexin regulates function of beta(3)-integrins. We revealed that integrin stimulation of endothelial cells led to an enhanced NF-kappaB activity and secretion of the NF-kappaB dependent chemokine monocyte chemoattractant protein-1 (MCP-1). The beta(3)-integrin dependent increase in MCP-1 was notedly reduced in cells that overexpressed beta(3)-endonexin. These results provide strong evidence that beta(3)-endonexin acts as a regulating factor in the integrin-mediated signal transduction and the present findings imply a pathophysiological role of beta(3)-endonexin in atherogenesis.

Animals↗
PubMed2005

The unique N-terminus of R-ras is required for Rac activation and precise regulation of cell migration.

The Ras family GTPase, R-Ras, elicits important integrin-dependent cellular behaviors such as adhesion, spreading and migration. While oncogenic Ras GTPases and R-Ras share extensive sequence homology, R-Ras induces a distinct set of cellular behaviors. To explore the structural basis for these differences, we asked whether the unique N-terminal 26 amino acid extension of R-Ras was responsible for R-Ras-specific signaling events. Using a 32D mouse myeloid cell line, we show that full-length R-Ras activates Rac and induces Rac-dependent cell spreading. In contrast, truncated R-Ras lacking its first 26 amino acids fails to activate Rac, resulting in reduced cell spreading. Truncated R-Ras also stimulates more beta3 integrin-dependent cell migration than full-length R-Ras, suggesting that the N-terminus may negatively regulate cell movement. However, neither the subcellular localization of R-Ras nor its effects on cell adhesion are affected by the presence or absence of the N-terminus. These results indicate that the N-terminus of R-Ras positively regulates specific R-Ras functions such as Rac activation and cell spreading but negatively regulates R-Ras-mediated cell migration.

Actins↗
PubMed1999

Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibody of c7E3 Fab (abciximab; ReoPro).

Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers. The expression of endothelial cell integrin alpha(v)beta3 has been shown to increase during vascular proliferation associated with human tumors. Selective antagonists of alpha(v)beta3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells. Monoclonal antibody 7E3, an antagonist of the human, but not murine, integrins alpha(v)beta3 and alphaIIbbeta3 (GPIIb/IIIa), inhibits platelet aggregation. It is the parent antibody of a mouse/human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications (c7E3Fab; abciximab; ReoPro). To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects, we established integrin alpha(v)beta3-negative human melanoma tumors in full-thickness human skin grafted onto SCID mice. The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31. Administration of 7E3 prevented or significantly inhibited the growth of tumors, and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors. These results support the previous findings that blockade of integrin alpha(v)beta3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of alpha(v)beta3 and alphaIIbbeta3 are effective in blocking tumor growth and angiogenesis.

Journal Article↗
PubMed2005

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Alleles↗
PubMed2003

Analysis of platelet membrane glycoprotein polymorphisms in Glanzmann thrombasthenia showed the French gypsy mutation in the alphaIIb gene to be strongly linked to the HPA-1b polymorphism in beta3.

We have tested the DNA of a large series of Glanzmann thrombasthenia patients for polymorphisms in platelet membrane glycoproteins. To our surprise, we noted a high prevalence of the HPA-1b allele of beta3, the minority allele in a normal population. This proved to be due to the presence of nine patients homozygous for the so-called French gypsy mutation (IVS15[ + 1]G-->A) in alphaIIb. Seven of these patients were homozygous for the HPA-1b alloantigen and the other two heterozygous HPA-1a/1b. As the alphaIIb and beta3 genes are both on chromosome 17, it is highly probable that the French gypsy mutation first arose on a chromosome encoding HPA-1b. For other adhesion receptors, no major differences were seen in the distribution of the A1, A2 and A3 alleles in the alpha2 gene, or in the Kozak or HPA-2 polymorphisms of GPIbalpha, suggesting that none of these alleles result in increased survival in Glanzmann thrombasthenia.

Amino Acid Substitution↗