Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment.

To analyze the transcriptional activity of the gene encoding the alpha subunit of the platelet integrin alpha(IIb)beta(3) during 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 the knock-in of the tk gene into the alpha(IIb) locus resulted in the knock-out of the alpha(IIb )gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the alpha(IIb) glycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from alpha(IIb)-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet alpha-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia. (Blood. 2000;96:1399-1408)

Ultrastructural analysis of bone marrow hematopoiesis in mice transgenic for the thymidine kinase gene driven by the alpha IIb promoter.

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the alphaIIb murine promoter of the gene encoding the alphaIIb-subunit of the platelet integrin alphaIIbbeta3 (Tropel et al, Blood 90:2995, 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 alphaIIb 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 alphaIIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.

Analysis of hematopoietic stem cell reprogramming with toxigenicity.

The molecular mechanisms by which a stem cell is committed to individual lineage are largely unknown. Two different models, though not mutually exclusive, are currently debated. The first describes the temporal and hierarchical coordination of lineage-specific transcriptional programs. The second suggests that multilineage genes are expressed in a self-renewing and undifferentiated cell prior to lineage commitment. To challenge these two models in in vivo-appropriate conditions, the expression of an exogenous toxigene was used to create transgenic animals in which an inducible, reversible cell knock-out at a specific stage of differentiation could be achieved. Both additional transgenesis using the megakaryocyte specific alphaIIb promoter and targeted transgenesis were used to express the herpes virus thymidine kinase (tk) gene in the megakaryocytic lineage. When the tk gene was targeted to the locus of the megakaryocyte-specific alphaIIb gene, a typical Glanzman thrombasthenic syndrome was created. Despite this bleeding disorder, the lack of expression of the alphaIIb gene did not affect the development of the mice. In both transgenic and targeted animals, all progenitor cells were sensitive to the effect of the gancyclovir (GCV), both in vivo and ex vivo. Long-term bone marrow cell cultures on stromal layers indicated that most of the very early progenitor cells expressed the enzyme. All the results obtained with this inducible toxic phenotype indicated that genetic programs that are in control of the expression of lineage-specific genes are operative in a totipotent stem cell prior to lineage commitment and strongly support the concept that stem cells express a multilineage transcriptome.

A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.

The 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 alpha subunit of the platelet integrin alphaIIb beta3 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 alphaIIb 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 alphaIIb 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 alphaIIb 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.

Dissecting megakaryocytopoiesis in vivo with toxigenes.

The genetic programs that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage remain poorly defined and require appropriate in vivo models. Using a cell-specific obliteration technique, a transgenic mouse model was produced where perturbations of megakaryocytopoiesis and platelet production may be induced on demand. This was achieved by targeting the expression of the herpes virus thymidine kinase (HSV-tk) to megakaryocytes using the regulatory regions of the gene coding for the alphaIIb gene, an early marker of megakaryocytopoiesis, which encodes the alpha subunit of the platelet integrin alphaIIb beta3. The HSV-tk gene is not toxic by itself, but sensitizes the target cell to the effect of ganciclovir (GCV), leading to the inhibition of DNA synthesis in dividing cells. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alphaIIb-tk) with GCV. After 10 days of treatment, the platelet number was reduced by greater than 96.5% and megakaryocytes were not detectable in the bone marrow (BM). After discontinuing GCV, BM was repopulated with megakaryocytes, and the platelet count was restored within seven days. The recovery was accelerated by the administration of interleukin 11. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic bipotent progenitor cells. The reversibility and facility of this system provide a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development, and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation.

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter.

The mechanisms that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage are largely unknown. Using a molecular approach to the study of megakaryocytopoiesis and platelet production, mice in which thrombocytopoiesis could be controlled were produced by targeting the expression of the herpes simplex virus thymidine kinase toxigene to megakaryocytes using the regulatory region of the gene encoding the alpha subunit of the platelet integrin alpha IIb beta 3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alpha IIbtk) with the antiherpetic drug ganciclovir (GCV). After 10 d of treatment, the platelet number was reduced by > 94.6%. After discontinuing GCV, the bone marrow was repopulated with megakaryocytes and the platelet count was restored within 7 d. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. The reversibility and facility of this system provides a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

A synthetic peptide with anti-platelet activity derived from a CDR of an anti-GPIIb-IIIa antibody.

A monoclonal antibody, AC7, directed against the RGD (Arg-Gly-Asp) binding site on the GpIIIa subunit of the platelet fibrinogen receptor, interacts only with activated platelet. In order to identify the regions of AC7 that interact with the receptor, cDNA sequences of AC7 immunoglobulin heavy and light chain variable regions were determined. Among the six complementarity-determining regions (CDRs) of AC7, the CDR3 heavy chain (H3) contains homology to the RGDF sequence within fibrinogen. A synthetic peptide encompassing the H3 region (H3, RQMIRGYFDV) inhibited platelet aggregation and fibrinogen binding to platelet (IC50 = 700 microM). The inhibitory potencies of modified H3 peptides suggest that the RGYF sequence within the H3 peptide mimic the receptor recognition sequence in fibrinogen.

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.

Lack of transcription and expression of the alpha IIb integrin in human early haematopoietic stem cells.

The glycoprotein IIb, the alpha subunit of the platelet integrin GPIIb-IIIa, is a marker of megakaryocyte, but the stage of its expression during haematopoiesis remains controversial. We have examined the expression of GPIIb protein and alpha IIb mRNA in early human normal stem cells. We have purified stem cell expressing the CD34 surface marker (CD34+ fraction) and selected among this population quiescent cells (CD34+ MF(R) fraction). We have failed to detect GPIIb protein and alpha IIb mRNA in the pluripotential (CD34+ MF(R)) cells, even with polymerase chain amplification. Therefore alpha IIb transcription and GPIIb protein expression seemed to follow the commitment of the pluripotential cell in the megakaryocyte lineage.

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.

Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.

We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.

Megakaryocytic and erythrocytic lineages share specific transcription factors.

Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines.

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs.

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.

In vitro studies of the effect of buflomedil on platelet responsiveness.

The effect of buflomedil (Fonzylane; Laboratoire Lafon, Maisons-Alfort, France) on platelet function, a drug used clinically for the treatment of peripheral vascular diseases, was investigated in vitro. The compound significantly inhibits epinephrine-induced aggregation at the micromolar level. At higher doses (approximately 1 mM), a weak inhibition of ADP- and collagen-induced aggregation was observed; at these concentrations, buflomedil inhibits granular secretion and the interaction of fibrinogen with its receptor on platelet. Further investigations indicate that the drug affects calcium uptake at the membrane level and inhibits the binding of [3H]-yohimbine to the same extent as observed with phentolamine. The IC50 determined from competition binding assays was 1 +/- 0.5 microM. This value was consistent with the affinity constant approximated for the binding of [3H]-buflomedil to non-stimulated platelets. Taken-together, these results indicate that the vasoactive compound buflomedil is a weak antiaggregating agent which exhibits alpha 2-adrenergic antagonistic properties.

Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3.

The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the alpha subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the beta subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-bp GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.

cDNA clones for human platelet GPIIb corresponding to mRNA from megakaryocytes and HEL cells. Evidence for an extensive homology to other Arg-Gly-Asp adhesion receptors.

Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb-IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypeptide chains of GPIIb are described. A number of clones were isolated from lambda gt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, lambda IIb1, from HEL cells, and lambda IIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. The identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the alpha subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences.

Megakaryocytic development in liquid cultures of cryopreserved leukocyte stem cell concentrates from chronic myelogenous leukemia patients.

The proliferation and differentiation of human megakaryocytes in liquid culture has been obtained using cryopreserved light-density blood cell concentrates from chronic myelogenous leukemia (CML) patients. A large number of megakaryocytes, representing 20%-60% of total cells cultured, developed after 12-14 days in liquid cultures supplemented with human plasma, while fetal calf serum supported the development of cells of the megakaryocytic lineage poorly. Ploidy studies showed the presence of 8N and 16N cells in human plasma-supplemented cultures while very few cells with DNA content greater than 4N were found in those supplemented with fetal calf serum. Using the FACS IV cytofluorometer, 1-2 X 10(6) megakaryocytes/h were sorted after immunolabeling of the human plasma-cultured cells with a monoclonal antibody reacting against the platelet glycoprotein complex IIb-IIIa. Thus, cryopreserved CML blood stem cell concentrates seem to offer a reproducible source of human megakaryocytes that retain their capacity to proliferate and differentiate in liquid cultures. These megakaryocytes can be used for the study of platelet glycoprotein biosynthesis as well as the regulation of megakaryocytopoiesis.