αIIb Gene Research Articles

Analysis of platelet membrane glycoprotein polymorphisms in Glanzmann thrombasthenia showed the French gypsy mutation in the αIIb gene to be strongly linked to the HPA-1b polymorphism in β3

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.

Identification of distal regulatory regions in the human αIIb gene locus necessary for consistent, high-level megakaryocyte expression

The alphaIIb/beta3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of alphaIIb, and studies of the alphaIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the alphaIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) alphaIIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the alphaIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the h(alpha)IIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the h(alpha)IIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of h(alpha)IIb mRNA that were about 30% of the native m(alpha)IIb mRNA level. These constructs also resulted in detectable h(alpha)IIb/m(beta)3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the alphaIIb gene for tissue specificity, but also characterizes the distal organization of the alphaIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the alphaIIb gene at high levels during megakaryopoiesis.

Analysis of the Amino Acid Requirement for a Normal αIIbβ3 Maturation at αIIbGlu324 Commonly Mutated in Glanzmann Thrombasthenia

SummaryGlanzmann thrombasthenia is an inherited bleeding disorder arising from quantitative or qualitative defects of the αIIbβ3 integrin of platelets. Here, we report that PCR-SSCP analysis and DNA sequencing revealed a homozygous single base pair substitution in exon 12 of the αIIb gene leading to a Glu324 (E) to Lys (K) substitution in the αIIb subunit in a patient with Type I disease. As this mutation is found on at least 3 continents, the codon for Glu324 may be a mutational hotspot of the disease. To better understand this mutation, we analyzed the effect of substituting E324 with A324, L324, D324, Q324, N324, S324, as well as K324, looking at both αIIbβ3 maturation and cell surface expression in transiently transfected Cos-7 cells. The maturation state of the receptor clearly correlated with the level of cell membrane expression. Maturation efficiency was dependent on the electric charge as well as the size of the side chain of the amino acid present in what is a highly conserved N-terminal position in the third β-strand of blade 5 of the αIIbβ-propeller.

Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment

To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment

AbstractTo analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

Characterization of the Murine Platelet αIIb Gene and Encoded cDNA

AbstractThe αIIb/β3 receptor is central to platelet aggregation. Biological studies of this receptor have been limited by the inability to reproduce αIIb/β3 function in a cell system. Increasingly, efforts are being directed at studies of this receptor in mice models. The structure of murine (m) β3 has been reported. We now have sequenced the mαIIb gene and found that it has the same size and organization as the human gene. The exon/intron borders are reported here, as are the distances between exons. mαIIb protein is 1,033 amino acids (aa), 7 and 5 aa shorter than human (h) and rodent (r) αIIb, respectively, with 79% and 90% homology, respectively. As part of the comparative analysis of the 3 known αIIb chains included in this report, we found that a particular region of the αIIb N-terminal β-propeller is highly conserved and speculate that it directly participates in ligand binding.

The Human Platelet αIIb Gene Is Not Closely Linked to Its Integrin Partner β3

AbstractαIIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation. Both genes are on chromosome 17q21.32. Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis. In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either αIIb or β3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and β3 as follows: cen-THRA1-BRCA1-D17S579/αIIb-β3-qter, with a distance of 1.3 centiMorgans (cM) between αIIb and β3 and the two genes being oriented in the same direction. PFGE genomic and YAC clone analysis showed that the β3 gene is distal and ≥365 kb upstream of αIIb. Additional restriction mapping shows αIIb is linked to the erythrocyte band 3 (EPB3) gene, and β3 to the homeobox HOX2b gene. Analysis of αIIb+-BAC and P1 clones confirm that the EPB3 gene is ∼110 kb downstream of the αIIb gene. Sequencing the region surrounding the human αIIb locus showed the Granulin gene ∼18 kb downstream to αIIb, and the KIAA0553 gene ∼5.7 kb upstream. This organization is conserved in the murine sequence. These studies show that αIIb and β3 are not closely linked, with αIIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.

Insights and Questions Arising from Studies of a Mouse Model of Glanzmann Thrombasthenia

In summary, studies of beta 3-deficient mice demonstrate that they provide an excellent model of the human bleeding disorder, Glanzmann thrombasthenia (GT), and replicate essentially all features of the human disease. Furthermore, these mice reveal additional defects consequent upon the absence of integrins alpha v beta 3 and alpha IIB beta 3 that had not become evident from studies of the human patients. These include defects in placental development and in bone resorption. In contrast, despite published evidence suggesting a role for alpha v beta 3 in angiogenesis, beta 3-null mice have not yet shown any defects in this process. The availability of these mice will allow extensive further studies of GT pathogenesis and therapy and of the roles of beta 3 integrins in different forms of angiogenesis, including the pathologically important angiogenic processes in the retina and in response to tumors. Furthermore, they open the way to studies of a variety of other processes in which roles for beta 3 integrins have been suggested.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin αIIb Subunit in a Patient With a Glanzmann's Thrombasthenia-Like Syndrome

AbstractThe integrin αIIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of αIIb and β3. We now report the molecular analysis of αIIbβ3 from a patient with a Glanzmann's thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of αIIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of αIIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the αIIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated αIIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type αIIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the αIIb cytoplasmic domain.

Ultrastructural Analysis of Bone Marrow Hematopoiesis in Mice Transgenic for the Thymidine Kinase Gene Driven by the αIIb Promoter

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the αIIb murine promoter of the gene encoding the αIIb-subunit of the platelet integrin αIIbβ3 (Tropel et al, Blood90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1+ and CD34+ cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the αIIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic αIIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.© 1998 by The American Society of Hematology.

A 2.7-kb Portion of the 5′ Flanking Region of the Murine Glycoprotein αIIb Gene Is Transcriptionally Active in Primitive Hematopoietic Progenitor Cells

AbstractThe continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the α subunit of the platelet integrin αIIbβ3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the αIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5′-flanking region of the murine αIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the αIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.

An Ets/Sp1 Interaction in the 5′-Flanking Region of the Megakaryocyte-Specific αIIb Gene Appears to Stabilize Sp1 Binding and Is Essential for Expression of This TATA-Less Gene

The megakaryocyte-specific integrin, alpha IIb, is the alpha-subunit of the alpha IIb/beta 3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alpha IIb gene is specifically expressed in megakaryocytes, the 5'-flanking region was studied as a potential model for megakaryocyte-specific gene expression. Previous studies have defined some of the important regulatory elements in the 5'-flanking region of this gene. The present studies focus on the issue of the molecular basis by which this TATA-less gene is properly transcribed. A GA-rich region centered 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site. Binding to this site is of low affinity, but is markedly improved by interaction with protein(s) binding at an Ets-consensus site approximately 20 bp further upstream. Mutation of the Ets site greatly reduces the ability of Sp1 to bind to its site. Trans-acting nuclear factors binding to and interaction of the proteins at these two sites have direct effects on the observed promoter activity in primary megakaryocyte transient expression studies. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alpha IIb gene.