A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

[Implication of hyaluronic acid in normal and pathological angiogenesis. Application for cellular engineering]. / Implication de l'acide hyaluronique dans l'angiogenèse normale et pathologique, application à l'ingénierie cellulaire.

Angiogenesis is a physiological process that allows the formation of new blood vessels, either from the local vascular structures, or from circulating endothelial progenitor cells, mobilized from the bone marrow, and attracted to the neovascularization site. This mechanism is controlled by pro-angiogenic molecules. It is crucial to supply oxygen and nutrients to tissues during growth, embryonic development or tissue regeneration in response to injuries. Thus, the dermis part of the skin is highly vascularized by a dense network of small and medium arteries and of capillaries and venules. In case of injury, rapid tissue repair is possible through this vascular network. However, once the vascularization is restored in tissue repair, the process of angiogenesis is negatively regulated by anti-angiogenic molecules. Controling the balance between pro-and anti-angiogenic agents is crucial and its deregulation leads to serious disease. The extracellular matrix plays an important role in controlling angiogenesis, allowing at least, the distribution of growth factors and the regulation of endothelial cell migration. Among these matrix components, hyaluronic acid plays a major role in the mechanical properties of connective tissues in ensuring their hydration. This glycosaminoglycan is a large size polymer, whose breakdown products strongly act on angiogenesis, especially in pathological situations (cancer, inflammation). Regarding its biological and mechanical properties, hyaluronic acid is used as matrix in tissue engineering, for improving the revascularization of tissues like skin.

Successful treatment of intractable epilepsy with ketogenic diet therapy in twins with ALG3-CDG.

BACKGROUND: Congenital disorders of glycosylation (CDG) is a heterogeneous group of congenital metabolic diseases with multisystem clinical involvement. ALG3-CDG is a very rare subtype with only 24 cases reported so far. CASE: Here, we report two siblings with dysmorphic features, growth retardation, microcephaly, intractable epilepsy, and hemangioma in the frontal, occipital and lumbosacral regions. RESULTS: We studied two siblings by whole exome sequencing. A pathogenic variant in ALG3 (NM_005787.6: c.165C > T; p.Gly55=) that had been previously associated with congenital glycolysis defect type 1d was identified. Their intractable seizures were controlled by ketogenic diet. CONCLUSION: Although prominent findings of growth retardation and microcephaly seen in our patients have been extensively reported before, presence of hemangioma is a novel finding that may be used as an indication for ALG3-CDG diagnosis. Our patients are the first reported cases whose intractable seizures were controlled with ketogenic diet. This report adds ketogenic diet as an option for treatment of intractable epilepsy in ALG3-CDG.

Circulating endothelial progenitor cells in systemic sclerosis: association with disease severity.

BACKGROUND: Heterogeneous data have been reported regarding the detection and number of circulating endothelial progenitor cells (EPCs) in systemic sclerosis (SSc). OBJECTIVE: We investigated the number of circulating EPCs using recent recommendations and we quantified their late outgrowth in patients with SSc and healthy controls. PATIENTS AND METHODS: EPCs, defined as Lin-/7AAD-/CD34+/CD133+/VEGFR-2+ cells, were quantified in 50 patients with SSc (mean age: 55 (16) years, disease duration: 9 (9) years) and 26 controls (mean age: 53 (19) years) by cell sorting/flow cytometry and by counting late outgrowth colony-forming units (CFU). RESULTS: Patients with SSc displayed higher circulating EPC counts than controls (median 86 (5-282) vs 49 (5-275)) EPCs for 1 million Lin- mononuclear cells; p = 0.01). Lower EPC counts were associated with the higher Medsger's severity score (p = 0.01) and with the presence of past and/or current digital ulcers (p = 0.026). There was no difference for the number of late outgrowth EPC-CFUs between patients with SSc and controls in cell culture evaluation. The formation of colonies was associated with higher levels of circulating EPCs (p = 0.02) and the number of colonies correlated with levels of EPCs (R = 0.73, p = 0.0004), validating our combination of fluorescence-activated cell sorter surface markers. CONCLUSIONS: We quantified circulating EPCs with an accurate combination of markers herein validated. Our data demonstrate increased circulating EPC levels in SSc, supporting their mobilisation from bone marrow. Furthermore, the subset of patients with digital vascular lesions and high severity score displayed low EPC counts, suggesting increased homing at this stage. The predictive value of this biomarker now warrants further evaluation.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma.

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Circulating endothelial progenitors in vascular repair.

Endothelial Colony Forming Cells (ECFCs) are obtained in culture from Circulating Endothelial Progenitor Cells. They display all characteristics of endothelial cells and they display stem cells features. Cord blood-derived ECFCs (CB-ECFCs) have a high clonogenic and proliferative potentials, and exhibit vascular repair capabilities useful for the treatment of ischemic diseases. However, the link between immaturity and functional properties of CB-ECFCs is still poorly defined. We showed that these cells have a high clonogenic potential and are capable to be efficiently reprogrammed into induced pluripotent stem cells. Moreover, we analyzed the expression of a broad panel of genes involved in embryonic stem cell properties. We define a novel stem cell transcriptional signature for CB-ECFCs fora better characterization and stratification according to their stem cell profile. We then improved the yield of CB-ECFC production for obtaining cells more functional in fewer passages. We used Glycosaminoglycans (GAG), components from the extracellular matrix which potentiate heparin binding growth factor activities. GAG mimetics were designed, having the capacity to increase the yield of ECFC during the isolation process, to increase the number of colonies, improve adhesion, proliferation, migration and self-renewal. GAG mimetics have thus great interest for vascular regeneration in combination with ECFC. Our results show that CB-ECFC are immature cells harboring specific functions such as formation of colonies, proliferation and formation of vascular structures in vitro and in vivo.

Transcriptional regulation of the human CD9 gene: characterization of the 5'-flanking region.

The CD9 antigen, initially discovered on B lineage leukemic cells, belongs to the tetraspan superfamily of surface molecules. If no precise function has been assigned to any of these molecules, there are some indications that they could be involved in cell adhesion and cell migration, as well as malignant progression. The CD9 antigen is associated with surface proteins such as VLA integrins or HB-EGF precursor. Transfection of CD9 in melanoma cells reduces tumor growth and metastasis. The heterogenous distribution of the CD9 antigen suggests a complex regulation of its expression. We have previously characterized the CD9 gene and shown that transcription could be initiated at several sites in the TATA-less 5'-flanking region. We show here, using as a model two human leukemic cell lines with erythromegakaryocytic potential, HEL and K562, that the [-205, -154] region supports a promoter activity when cloned ahead of a CAT reporter gene. Mutagenesis analysis suggested the presence of a positive element located within the [-170, -154] region. Gel shift experiments using HEL extracts were compatible with the binding of the transcriptional factor Sp1 to the [-237, -205] region and indicated that a non-identified protein binds to the 3' end of the [-205, -154] region.

The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.

Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Regulation of gene transcription during the differentiation of megakaryocytes.

Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. Thus studies on the transcriptional regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation of this lineage. In previous experiments, the promoter of GPIIb gene was isolated and we have shown that a fragment extending 643 bp upstream the transcription start site was able to control the cell specificity of a reporter gene in transfection experiments of different permanent cell lines. Most of the transcriptional activity is contained in an enhancer containing binding sites for members of the GATA and ets transcription factors families. The transcription factor GATA1 is not only a major regulator of the transcription of erythroid genes, but it also regulates the expression of GPIIb and other megakaryocytic genes. We suggest that the lineage specificity and the temporal activation of GPIIb gene during hematopoiesis rely on the activity of a repressor that has been identified on the promoter. To test this hypothesis, we have developed a cell model allowing the study of the megakaryocytes differentiation from very immature progenitors to fully differentiated cells. This model is based on the differentiation of mouse embryonic stem cells. We have obtained megakaryocytes together with erythrocytic and granulo-macrophagic cells. The transfection in these ES cells of GPIIb promoter constructs mutated or not on different regions, including the repressor element will provide important information on the mechanisms controlling gene activation or repression during megakaryocyte differentiation.

Expression of coagulation factor IX in a haematopoietic cell line.

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.

Cord blood-circulating endothelial progenitors for treatment of vascular diseases.

Adult peripheral blood (PB) endothelial progenitor cells (EPC) are produced in the bone marrow and are able to integrate vascular structures in sites of neoangiogenesis. EPCs thus represent a potential therapeutic tool for ischaemic diseases. However, use of autologous EPCs in cell therapy is limited by their rarity in adult PB. Cord blood (CB) contains more EPCs than PB, and they are functional after expansion. They form primary colonies that give rise to secondary colonies, each yielding more than 10(7) cells after few passages. The number of endothelial cells obtained from one unit of CB is compatible with potential clinical application. EPC colonies can be securely produced, expanded and cryopreserved in close culture devices and endothelial cells produced in these conditions are functional as shown in different in vitro and in vivo assays. As CB EPC-derived endothelial cells would be allogeneic to patients, it would be of interest to prepare them from ready-existing CB banks. We show that not all frozen CB units from a CB bank are able to generate EPC colonies in culture, and when they do so, number of colonies is lower than that obtained with fresh CB units. However, endothelial cells derived from frozen CB have the same phenotypical and functional properties than those derived from fresh CB. This indicates that CB cryopreservation should be improved to preserve integrity of stem cells other than haematopoietic ones. Feasibility of using CB for clinical applications will be validated in porcine models of ischaemia.

Angiogenesis in systemic sclerosis: impaired expression of vascular endothelial growth factor receptor 1 in endothelial progenitor-derived cells under hypoxic conditions.

OBJECTIVE: To assess angiogenesis and explore the expression and regulation of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), and VEGFR-2, the leading mediators of angiogenesis, in SSc patients and controls. METHODS: Late-outgrowth endothelial progenitor cells (EPCs), isolated from the peripheral blood of systemic sclerosis (SSc) patients and controls, and human umbilical vein endothelial cells (HUVECs) were assessed under normal and hypoxic conditions. Genomic background was evaluated in a large case-control study (including 659 patients with SSc and 511 controls) using tag single-nucleotide polymorphisms on VEGFR1 and VEGFR2 genes. RESULTS: EPCs from SSc patients had the phenotype of genuine endothelial cells and displayed in vitro angiogenic properties similar to those of HUVECs and control EPCs under basal conditions, as determined by flow cytometry, tube formation, and migration assay. However, after 6 hours of hypoxic exposure, EPCs from SSc patients exhibited lower induced expression of VEGFR-1 at the messenger RNA and protein levels, but similar VEGF and VEGFR-2 expression, compared with HUVECs or EPCs from healthy controls. There was no evidence of defective expression of hypoxia-inducible factor 1alpha. These results were supported by the lower serum levels of soluble VEGFR-1 found in SSc patients (n = 187) compared with healthy controls (n = 48) (mean +/- SD 163.7 +/- 98.5 versus 210.4 +/- 109.5 pg/ml; P = 0.0042). These abnormalities did not seem to be related to genomic background. CONCLUSION: Our findings shed new light on the possible role of VEGFR-1 in the main vascular disturbances that occur in SSc and lead to more severe disease.

Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells.

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)

The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements.

Much information on regulation of the transcription of megakaryocytic genes stems from studies on the glycoprotein IIb (GPIIb) gene, an early and specific marker of this lineage. Transcriptional activity is controlled by the association of positive promoter elements corresponding to binding sites for the transcription factor GATA-1 and a member of the Ets family. In the present study, we show that these elements are not directly involved in the control of cell specificity. In contrast, we identified a sequence located between -170 and -73 that exhibited a repressor activity based on an analysis of the transcriptional activity of 5'-deleted GPIIb promoter fragments transfected in the nonhematopoietic HeLa cells. Further analysis of this repressor by substitution mutagenesis of the -139/-63 region showed that bases -120/-116 and -102/-93 were required for full repressor activity. The repressor is able to interact differentially with GPIIb promoter elements active in the megakaryocytic HEL, the erythroid K562, the monocytic U937, or the nonhematopoietic HeLa cell lines, indicating that it controls GPIIb gene tissue specificity. In addition, direct evidence for tissue-specific interaction between this repressor and the GPIIb -598/ -406 enhancer was obtained when these elements were set in the context of a heterologous SV40 promoter. Interestingly, the same repressor element controlling tissue specificity of the GPIIb gene may also control its temporal expression during megakaryocyte differentiation, based on recent evidence obtained by Fong and Santoro (J Biol Chem 269:18441, 1994). Finally, we found that the -120/-116 GPIIb sequence was part of a consensus motif shared by promoters of other megakaryocyte-specific genes, suggesting a common repressor mechanism.

Organization of the human CD9 gene.

The CD9 antigen was originally described as a 24-kDa molecule present on B-lineage-derived acute lymphoblastic leukemia cells and developing B lymphocytes. Platelets also express a large amount of CD9 antigen and can be activated by CD9 antibodies. We report here the structure of the CD9 gene, which is composed of 8 exons spanning more than 20 kb. There is no TATA or CAAT box in the 5'-flanking domain of the CD9 gene, but a 120-bp region extremely rich in C and G (88%) contains several Sp 1 binding sites and a consensus site for the binding of zinc-finger proteins of the Krox/EGR family. The CD9 antigen belongs to a new cell surface protein family. The organization of its gene closely resembles the organization of the genes for two other members of this protein family, TAPA1 and CD63, which share with CD9 respectively 45 and 25% identity at the amino acid level.

Tissue-specific expression of the platelet GPIIb gene.

One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage. Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis. One gene of interest is the glycoprotein IIb (GPIIb) gene; GPIIb, the alpha subunit of the platelet cytoadhesin GPIIb-IIIa, is produced in megakaryocytes at an early stage of the differentiation, whereas the other subunit of this complex, GPIIIa, is expressed in other cells. For these reasons, the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors. A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments. Within this region, we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays. One region, centered at -54, is similar to a nuclear factor E1-binding site, and a region located at position -233 contains a CCAAT motif. Two domains centered at positions -345 and -540, respectively, bind proteins that are present in megakaryocytic cells and nonrelated cells as well. Finally, two other domains, located at positions -460 and -510, interact with proteins that are only present in megakaryocytic cells. In addition, deletion of the region containing these two domains results in a significant decrease of the promoter activity. It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors.

Isolation of the human platelet glycoprotein IIb gene and characterization of the 5' flanking region.

Platelet membrane glycoprotein (GP) IIbIIIa complex functions as a receptor for fibrinogen, von Willebrand factor and fibronectin, and mediates adhesive reactions of platelets. The gene for the GPIIb subunit is only active in megakaryocytic cell type. We have isolated this gene from a genomic library. The GPIIb gene was characterized by restriction mapping and sequencing of the 5' and 3' regions containing the first and the last exons. The transcription start site and the polyadenylation signal were identified. From these data we deduced that the gene spans a region of 22 kb and that the mRNA contains a leader sequence of 32 nucleotides. At the 3' end the last exon encodes the 19 amino acids corresponding to the cytoplasmic domain of the GPIIb light chain. Upstream the transcription start site, two sequences are homologous to consensus binding sites of the nuclear factors SP1 and CP2. Two inverted repeats were also identified in this region.

Expression of the fibrinogen genes in rat megakaryocytes.

A variety of evidence suggests that megakaryocytes synthesize fibrinogen and comparative immunochemical and structural studies indicate that fibrinogen produced in or associated with megakaryocytes may be different than fibrinogen produced in the liver. Two studies have reported that the gamma' chain, which is produced from the gamma chain gene by alternative splicing, is absent from fibrinogen produced in the megakaryocyte. Since there is only a single gene for each of the three fibrinogen chains the reported structural differences suggest different mechanisms for production of hepatic and megakaryocytic fibrinogen. We have begun an investigation of the varying mechanisms for expression of the fibrinogen genes by examining the structure of fibrinogen mRNA's in the two tissues. Fibrinogen mRNA's of identical length are found in both liver and megakaryocytes. Furthermore, despite the reported absence of the gamma' chain in platelet-associated fibrinogen, we have used a probe specific for the alternative spliced region of the gamma' mRNA to clearly demonstrate this chain in megakaryocyte mRNA. These studies indicate that the gamma' mRNA is either not translated in platelets or that the gamma' chain is unable to associated with the alpha and beta chains to form a mature molecule.

The transcription factor GATA-1 regulates the promoter activity of the platelet glycoprotein IIb gene.

Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. We have previously shown that a fragment extending 643 base pairs upstream the transcription start site of the human GPIIb promoter was able to control the tissue-specific expression of the CAT gene in transfection experiments. Four potential GATA-binding sites, located at positions -463, -376, -243, and -54 are present within this fragment. Gel shift analysis revealed that nuclear extracts from the erythroleukemic cell line HEL contain a DNA-binding protein that recognizes these GATA sites. Using an antiserum raised to an hydrophilic region of the transcription factor GATA-1, the HEL GATA-binding protein was found to be GATA-1. Point mutations of the different GATA sites indicated that they did not equally contribute to GPIIb promoter activity. The -463 GATA motif located in an enhancer region is essential for full transcription activity and was found to be dominant upon the other GATA motifs. When this site is mutated, the -54 GATA site appears to be essential for the remaining CAT activity. These results indicate that the transcription factor GATA-1 plays an important role in the regulation of the transcription of the megakaryocyte specific GPIIb gene.