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M Poncz

Publications and source records attributed to M Poncz.

At least 73 records · Page 4Linked to original sources
PubMed1991

A deletion in the gene for glycoprotein IIb associated with Glanzmann's thrombasthenia.

The platelet fibrinogen receptor is composed of a complex of glycoproteins (GP) IIb and IIIa on the surface of platelets. Deficient function of this receptor prevents normal platelet aggregation, resulting in Glanzmann's thrombasthenia (GT). In this paper, we describe a black thrombasthenic patient who is either homozygous or hemizygous for a deletion within the GPIIb gene. Initial Western blot analysis of platelet proteins from this patient did not detect any GPIIb, but did detect small amounts of GPIIIa of normal mobility. Quantitation of vitronectin receptor (VNR) demonstrated that this thrombasthenic patient had approximately 1.5-2 times the number of these receptors per platelet compared with controls, a finding that has previously been noted in other thrombasthenic patients with defects in GPIIb. Genomic Southern blot studies demonstrated a deletion in the GPIIb gene of approximately 4.5 kilobasepairs (kb). Analysis of the isolated GPIIb gene demonstrated that the deletion begins between two Alu repeats within intron 1 and ends in intron 9. Polymerase chain reaction (PCR) studies using platelet RNA and oligonucleotides directed to both the 5' and 3' ends of the GPIIb cDNA sequence easily detected GPIIb transcript, suggesting that the genomic deletion of exons 2-9 does not significantly decrease the level of the GPIIb mRNA. Sequence analysis of PCR-generated GPIIb cDNA showed that a cryptic AG splice acceptor sequence was being utilized, resulting in a transcript that contained a portion of introns 1 and 9, as well as having a deletion of exons 2-9. Unlike the GPIIb gene, the GPIIIa gene appears to be intact by Southern blot analysis. PCR studies using platelet RNA and oligonucleotides directed to the GPIIIa cDNA sequence demonstrated the presence of GPIIIa mRNA. In summary, the thrombasthenic state in this patient appears to be due to a GPIIb gene deletion resulting in an abnormal transcript and no detectable platelet GPIIb. Platelet GPIIIa levels were secondarily low presumably due to the known instability of GPIIIa in the absence of GPIIb.

Adolescent↗
PubMed1990

Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erythroleukemia cells.

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose alpha subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Gi alpha demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Gi alpha 1, Gi alpha 2, and Gi alpha 3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Gi alpha 2 much greater than Gi alpha 3 greater than Gi alpha 1. A HEL cell cDNA library screened with the Gi alpha antisera produced clones encoding Gi alpha 2 and Gi alpha 3 that had sequences similar to those reported from other sources. Gi alpha-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Gi alpha 2 and Gi alpha 3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Gi alpha family, all of which are candidates for interaction with receptors for thrombin and other agonists.

Adenylate Cyclase Toxin↗
PubMed1990

Structural and functional comparison of the genes for human platelet factor 4 and PF4alt.

Platelet factor 4 (PF4) is a 70 amino acid heparin-binding protein released from the alpha-granules of activated platelets. Its exact biologic function is not known, although PF4 is a member of a multigene family involved in chemotaxis, coagulation, inflammation, and cell growth. We previously cloned the cDNA for human PF4 from a human erythroleukemic (HEL) cell expression library. We now report the isolation and sequence determination of the gene for human PF4. This gene contains three exons and spans approximately 1,000 basepairs (bp). Concurrently, we have cloned a highly homologous gene that we have called PF4alt. We show that PF4 and PF4alt are non-allelic genes: the human PF4 gene is encoded on a 10 kilobasepairs (kb) EcoRI fragment, and its DNA sequence agrees with protein and cDNA data for PF4, while PF4alt is encoded in a polymorphic 3 or 5 kb EcoRI fragment. Compared with PF4, this gene has 14% DNA and 38% amino acid divergence in the signal peptide region, and 2.6% DNA and 4.3% amino acid divergence in the coding region of the mature protein. PF4alt contains three amino acid substitutions (P58----L, K66----E, and L67----H) near the C-terminus, in a region known to be critical for PF4 function. Primer extension studies show the 5'-untranslated region of PF4 is 73 bp long. A TATA box is present 30 bp 5' to the transcription start site. A 90 bp stretch of pyrimidines (including 53 consecutive thymidine residues) begins at -227 bp and is analogous to a similar region of 30 residues 5' to the rodent PF4 gene. This pyrimidine-rich region is absent from the PF4alt gene; however, DNA homology exists between the two human genes in the 5'- and 3'-flanking regions and extends for over 3.6 kb. Alternating purine/pyrimidine tracts occur both 5' and 3' to PF4 and PF4alt but do not define the endpoints of the gene duplication, which extend beyond these sequences at least at the 5' end. Northern blot analysis using gene-specific oligonucleotides and platelet RNA showed an 800 or 900 nucleotide (n) message for PF4 and PF4alt, respectively. Northern blot and primer extension studies show that steady-state platelet PF4 mRNA levels are approximately one magnitude greater than PF4alt mRNA levels. Thus, these studies demonstrate that PF4alt mRNA is expressed in platelets. Whether PF4alt protein is expressed remains to be determined, and the nature of its biologic function needs to be studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1990

Atypical presentation of Wiskott-Aldrich syndrome: diagnosis in two unrelated males based on studies of maternal T cell X chromosome inactivation.

Congenital thrombocytopenia may occur in isolation or accompanied by eczema and immunodeficiency, as part of the X-linked hereditary Wiskott-Aldrich syndrome (WAS). Because the clinical and immunologic picture of WAS is variable, particularly early in life, definite diagnosis cannot always be made in cases with a negative family history. Two unrelated males with sporadic congenital thrombocytopenia had only questionable immunologic abnormalities as infants, making them clinically indistinguishable from cases of isolated thrombocytopenia, although one developed episodic neutropenia and the other began to manifest a multisystem autoimmune disease at 2 years of age. Evaluation of X chromosome inactivation in the T cells of both patients' mothers showed each of these women to have the same highly skewed X chromosome inactivation pattern seen in carriers of typical familial WAS. A T-cell defect was subsequently directly demonstrated in the second patient, whose lymphocytes failed to proliferate to periodate and anti-CD43. Taken together, these data suggest the presence of T cell immunodeficiency consistent with WAS in these patients. Furthermore, their mothers were found to have a very high likelihood of being carriers, lending support to the diagnosis of a hereditary disease in these boys and making possible genetic prediction in other family members and subsequent pregnancies.

Dosage Compensation, Genetic↗
PubMed1990

The genomic organization of platelet glycoprotein IIIa.

The platelet membrane glycoprotein (GP) IIb/IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix interactions, contains binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Absence or defects of this receptor result in the platelet bleeding disorder Glanzmann's thrombasthenia. In this report, we describe the isolation of genomic DNA coding for the entire mature GPIIIa protein. Mature GPIIIa is encoded by 14 exons which range in length from 90 to 3618 base pairs, which are contained within an approximately 46-kilobase (kb) stretch of genomic DNA on chromosome 17. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. The coding region of the GPIIIa gene is identical with the previously described cDNA sequence except for three silent substitutions. One substitution creates a TaqI site which may be the site of a known GPIIIa polymorphism. A second substitution eliminates a SmaI site. Aside from the start of the first exon described, which begins at the second base of the first codon of the mature protein, there is no correlation between the organization of the exons in this gene and proposed functional domains of the protein based on analysis of the primary amino acid sequence. The less frequently used polyadenylation signal AAATTAAA was present at the 3'-end of the major RNA transcript. Recently, an alternatively processed GPIIIa transcript has been described. We demonstrate that this transcript results from nonsplicing of the final intron. The description of the GPIIIa gene organization should be of importance in understanding the evolution of the integrin family of receptors and should be useful in the molecular biology analysis of thrombasthenic patients who have a defect in the GPIIIa gene.

Amino Acid Sequence↗
PubMed1990

Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein.

Platelet factor 4 (PF4) is a 70 amino acid protein released from the alpha-granules of platelets after activation. The exact biologic function of this protein is unknown. We have constructed an expression vector for recombinant PF4 (rPF4) in the T7-based promoter vector pT7-7 to better study the relationship between PF4 structure and function. The protein was expressed in Escherichia coli and purified to homogeneity by heparin-agarose affinity chromatography and reverse-phase high-performance liquid chromatography. Purity of protein was confirmed by immunoblot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which resulted in a single component with a molecular weight of 8,000 daltons. The amino acid composition and sequence of the N-terminal 20 residues showed that rPF4 is identical to PF4 prepared from human platelets (hPF4), except for an N-terminal initiating methionine residue. Immunoblots revealed that rPF4 and hPF4 bound polyclonal anti-hPF4 equally well, while chemotaxis experiments demonstrated similar potencies as neutrophil attractants. Dose-dependent neutrophil chemotactic responses and competitive studies with polyclonal anti-hPF4 antiserum further demonstrate similar chemotactic properties of the two PF4 species. In conclusion, our data show that this recombinant protein and the native protein appears to have similar immunologic, heparin-binding, and chemotactic properties. The chemotactic properties of hPF4 appear to be entirely intrinsic to the protein and not due, in part, to any contaminating protein. Furthermore, our expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure/function relationships of this biologically important protein.

Amino Acid Sequence↗
PubMed1990

Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing.

Recently, the full-length primary amino acid sequence for human glycoproteins (GP) IIb and IIIa have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPIIb/IIIa receptor with the corresponding rodent receptor. The rodent rGPIIb/IIIa receptor differs from the human receptor, having low affinity for R.G.D-containing oligopeptides and not binding at all to the C-terminus of the gamma chain of human fibrinogen. We describe the structure of rodent platelet GPIIb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N-terminal sequence L.N.L.D, agreeing with the consensus derived from other integrin family alpha heavy chains. All 18 cysteine residues occur at positions conserved in human GPIIb and the vitronectin receptor alpha subunit VNR alpha. The putative calcium-binding domains of the GPIIbs have a high level of amino acid identity (92%), supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPIIb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPIIb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPIIb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPIIb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPIIb gene.

Amino Acid Sequence↗
PubMed1990

Organization of the gene for platelet glycoprotein IIb.

The glycoprotein (GP) IIb/IIIa heterodimer functions as a receptor for fibrinogen, von Willebrand factor, and fibronectin on activated platelets; it is dysfunctional in the bleeding diathesis Glanzmann's thrombasthenia. This receptor is a member of the integrin family, which includes homologous membrane receptors involved in a number of different cell-cell and cell-matrix adhesive interactions. Knowledge of the sequence and organization of the GPIIb and GPIIIa genes will help in understanding evolutionary relationships and functional homologies of this family of adhesion protein receptors and will facilitate analysis of molecular defects responsible for thrombasthenia. Using the GPIIb cDNA as a probe, we have isolated overlapping genomic clones encompassing the entire coding region, the 5'- and 3'-untranslated sequences, and the immediate flanking regions for the GPIIb gene. The gene spans approximately 17.2 kilobases (kb); all but approximately 2.6 kb of intronic DNA sequence has been determined. The GPIIb gene contains 30 exons whose demarcations do not correlate with previously suggested functional domains. Two intron/exon borders have the rare GC splice donor sequence instead of the consensus GT sequence. There are at least seven complete and three partial AluI sequence repeats within the intron sequences. RNase protection, S1 nuclease analysis, and primer extension studies using human erythroleukemia (HEL) cell RNA and platelet RNA map a major transcription start site 32 base pairs (bp) 5' to the beginning of the coding region; however, there are no canonical consensus TATA or CAAT boxes in the region immediately 5' to the proposed cap site. The immediate 5'-flanking sequence of rodent GPIIb demonstrates complete identity near the proposed cap site with its human counterpart, but again, no TATA or CAAT boxes are apparent.

Amino Acid Sequence↗
PubMed1988

A large deletion encompassing the entire alpha-like globin gene cluster in a family of northern European extraction.

We describe a new deletional form of alpha thalassemia segregating in three generations of a family of northern European origin. A full-term female girl had hypochromic, microcytic anemia since early infancy associated with delayed language development, slow growth and weight gain. Hematologic studies suggested the presence of alpha thalassemia. Gene-blotting studies showed no abnormal alpha-like globin gene fragments; however, studies of inheritance of informative polymorphic restriction fragments using zeta, alpha and 3'-alpha-hypervariable region (3'-HVR) probes showed evidence for an extensive deletion encompassing the entire alpha-like globin gene cluster. The 3' breakpoint of this deletion maps beyond the 3'-HVR, a region implicated as a hot spot for the generation of other large deletional events within the alpha-like cluster. The 5' breakpoint maps at least 10 kilobases (kb) 5' to the zeta-globin gene. The minimum size estimate for this deletion is greater than 47 kilobases.

Child, Preschool↗
PubMed1988

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

Amino Acid Sequence↗
PubMed1988

Non-random association of the Rsa I polymorphic site 5' to the beta-globin gene with major sickle cell haplotypes.

There are three main African haplotypes associated with the sickle mutation on chromosome 11. We have examined an Rsa I polymorphism 550 bp 5' to the beta-globin gene to study the degree of linkage disequilibrium between this Rsa I site and the three haplotypes. This Rsa I site is contained within the 10.3 kb or less area of randomization separating the 5'- and 3'-haplotype clusters. The beta S-containing chromosomes of the Benin and Senegal haplotypes are not cut, while those of the Central African Republic are cleaved by Rsa I at this site. Possible explanations of these findings are discussed.

Anemia, Sickle Cell↗
PubMed1988

Globin gene expression in hereditary persistence of fetal haemoglobin and (delta beta) (0)-thalassaemia.

Recent advances in molecular and cell biology have led to major insights into understanding the regulation of globin gene expression in erythroid cells. Studies on HPFH and related syndromes have contributed much to this knowledge. Although no single explanation has emerged concerning the mechanism by which the deletional HPFH and (delta beta)(0)-thalassaemia affect HbF expression, they have led to new testable hypotheses to explain how different deletions result in similar phenotypes. The non-deletional HPFH and related syndromes have been the subject of recent intense interest, and a number of these disorders are now known to be associated with single-base changes in the promoter regions of one or the other fetal globin genes. If these base changes are causally related to the observed phenotypes, the molecular basis by which they increase HbF and simultaneously decrease HbA production remains to be determined. In addition, it has been recognized that other single-base changes 5' to the fetal-globin genes are associated with and may affect the phenotypic expression of certain forms of sickle-cell disease and beta-thalassaemia. Increased HbF production and high G gamma: A gamma ratios result in these individuals only under conditions of erythropoietic stress. The relationship between these point mutations and the cell biology of erythroid development clearly needs to be better delineated. Studies on HbF expression in humans will no doubt lead to elucidation of the mechanisms controlling the regulated expression of globin gene switching in normal and disease states, and hopefully, result in the design of molecular strategies for the amelioration of many of the human haemoglobinopathies.

Animals↗
PubMed1987

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb.

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated alpha- and beta-subunits. The present study was designed to compare the cDNA-derived protein sequences of the alpha-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the alpha-subunit of the FnR (FnR alpha) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR alpha-subunit (VnR alpha) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR alpha. Translation of these sequences showed that the FNR alpha, the VnR alpha, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca2+-binding domains of about 30 amino acids that have sequence similarities to other Ca2+-binding proteins. The identity among the protein sequences of the three receptor alpha-subunits ranges from 36.1% to 44.5%, with the Ca2+-binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication.

Amino Acid Sequence↗
PubMed1987

DNA methylation in hereditary persistence of fetal hemoglobin (HPFH-2).

Recent studies show that the region of DNA brought into close proximity to the fetal gamma-globin genes in deletional forms of hereditary persistence of fetal hemoglobin (HPFH) is selectively hypomethylated (and presumably active) in normal erythroid tissue. This region is normally located approximately 100 kilobases (kb) 3' to the fetal genes. The continued expression of fetal hemoglobin in adult life in these forms of HPFH has been ascribed to the effect of this erythroid-specific region in altering local chromosomal structure and allowing transcription. Because transcriptional activity is often associated with hypomethylation, we have examined the methylation status of the gamma-globin genes and the truncated psi beta gene on the HPFH chromosome to determine whether juxtaposition of this erythroid-specific region results in a generalized hypomethylation of the globin gene region upstream of the deletion breakpoint. Genomic DNA purified from nucleated red blood cells (nRBC) from a patient with HPFH-2/beta O thalassemia was digested with Msp I or Hpa II, and the methylation pattern determined on the HPFH chromosome by using secondary cleavage with restriction enzymes which span the deletion breakpoint. These studies show that in nRBC the HPFH-2 chromosome is hypomethylated in the 3'-juxtaposed region (3'JR) and in the region of the gamma-globin genes. In contrast, Msp I sites near the truncated psi beta-globin gene remain methylated, suggesting that only a subset of CpG sites upstream from the 3'JR become hypomethylated in HPFH-2. This subset of sites corresponds to those which are normally hypomethylated when fetal genes are active. The continued high level of fetal globin expression is, therefore, not associated with a generalized hypomethylation upstream from the deletion junction.

DNA↗
PubMed1987

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors.

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.

Amino Acid Sequence↗