Biomedical subjects

M Poncz

Publications and source records attributed to M Poncz.

At least 55 records · Page 3Linked to original sources

PubMed1995

Agonist-stimulated ligand binding by the platelet integrin alpha IIb beta 3 in a lymphocyte expression system.

The ligand binding activity of the platelet integrin alpha IIb beta 3 is initiated by agonist-generated intraplatelet signals. We studied this process in vitro by expressing recombinant alpha IIb beta 3 in Epstein-Barr virus-immortalized B lymphocytes. We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing alpha IIb beta 3 to immobilized fibrinogen. Moreover, replacement of the transmembrane and cytoplasmic domains of the alpha and beta subunits of alpha IIb beta 3 with those of alpha L beta 2 significantly increased adherence, whereas replacement of only the cytoplasmic domains significantly decreased adherence. This suggests that transmembrane segments are involved in the agonist-induced modulation of alpha IIb beta 3 activity. Similar results were seen when the alpha IIb beta 3 activation-dependent monoclonal antibody PAC-1 was substituted for immobilized fibrinogen. We also found that the adherence of lymphocytes expressing beta 3 with either of the two alpha IIb/alpha L chimeras was similar to that of cells expressing alpha IIb beta 3, whereas the adherence of cells expressing alpha IIb with either of the two beta 3/beta 2 chimeras was substantially decreased, suggesting that the identity of the cytoplasmic domain of beta 3, but not of alpha IIb, is critical for alpha IIb beta 3 function. This report indicates that B lymphocytes contain signal transduction pathways involving protein kinase C that can increase the ligand binding activity of alpha IIb beta 3 and demonstrates the utility of these cells as an expression system for the study of agonist-stimulated alpha IIb beta 3 function.

Amino Acid Sequence↗

PubMed1995

Platelet glycoprotein IIb gene expression as a model of megakaryocyte-specific expression.

Glycoprotein (GP)IIb/IIIa is an integrin complex normally restricted in its expression to platelets and the megakaryocytes from which they are derived. This complex functions as a receptor for fibrinogen and other ligands and is involved in platelet aggregation. The receptor complex is expressed at high levels during final megakaryocyte differentiation. Further, while GPIIIa is expressed in other tissues as part of the vitronectin receptor, GPIIb is only expressed on maturing megakaryocytes and the platelets derived from them. Thus studies of the GPIIb gene may serve as a model of gene regulation during this process. Over the past several years, the genes for both GPIIb and IIIa have been cloned and analyzed. The GPIIb gene contains 30 exons over 18 kilobases (kb). The transcriptional start site has been determined and there does not appear to be a TATA-box immediately upstream of this site. Studies have been done to define regulatory elements upstream of the transcriptional start site. Most of these studies focused on the human promoter and on studies using megakaryocytic cell lines. These studies have defined several important tissue-specific promoter elements including a GATA454 site (454 basepairs upstream of the transcriptional start site that involves a GATA-binding consensus sequence), a GATA54 site and an Ets35 site (that involves an Ets-binding consensus sequence). Expression studies with megakaryocytic cell lines suggest that each of these sites effects expression approximately threefold. Further, an Ets510 site was also described that had a similar effect. While these studies were underway, we pursued studies of the rat 5'-flanking region using a rat primary marrow expression system. Qualitatively, our data support the human data; however, quantitatively, we found significant differences from the human studies done in cell lines. We found that the major tissue-specific promoter element was the GATA454 site. Mutations altering this site result in an approximately fiftyfold drop in expression. In comparison, eliminating the Ets510 site by truncation or point mutation had only a twofold effect on expression. Mutations at the Ets35 site did effect expression at a high level, decreasing expression approximately fifteenfold, while mutations at the GATA54 site effected expression by approximately ninefold. In addition, using 50 bp deletions, we have preliminarily defined two domains from -450 to -351 bp and -150 to -101 bp upstream of the transcriptional start site that effected expression. The former appears to contain a positive regulatory element, while the latter appears to be a silencer element.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia.

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.

Adult↗

PubMed1994

Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system.

Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 5'-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 5'-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 5'-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

Animals↗

PubMed1994

Structural requirements of platelet chemokines for neutrophil activation.

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb.

We studied the defect responsible for Glanzmann thrombasthenia in a patient whose platelets expressed < 5% of the normal amount of GPIIb-IIIa. Genetic and biochemical evidence indicated that the patient's GPIIIa genes were normal. However, DNA analysis revealed the patient homozygous for a G818-->A substitution in her GPIIb genes, resulting in a Gly273-->Asp substitution adjacent to the first GPIIb calcium-binding domain. To determine how this mutation impaired GPIIb-IIIa expression, recombinant GPIIb containing the mutation was coexpressed with GPIIIa in COS-1 cells. The GPIIb mutant formed stable GPIIb-IIIa heterodimers that were not immunoprecipitated by either of two heterodimer-specific monoclonal antibodies, indicating that the mutation disrupted the epitopes for these antibodies. Moreover, the GPIIb in the heterodimers was not cleaved into heavy and light chains, indicating that the heterodimers were not transported from the endoplasmic reticulum to the Golgi complex where GPIIb cleavage occurs, nor were the mutant heterodimers expressed on the cell surface. These studies demonstrate that a Gly273-->Asp mutation in GPIIb does not prevent the assembly of GPIIb-IIIa heterodimers, but alters the conformation of these heterodimers sufficiently to impair their intracellular transport. The impaired GPIIb-IIIa transport is responsible for the thrombasthenia in this patient.

Adenosine Diphosphate↗

PubMed1993

A comparative analysis of cDNA-derived sequences for rat and mouse beta 3 integrins (GPIIIA) with their human counterpart.

Alpha IIb beta 3 (GPIIb-IIIa), the platelet receptor for fibrinogen, is a member of the integrin superfamily. We have now cloned the mouse and rat beta 3 cDNAs. These data represent the first available non-human beta 3 sequences, allowing important comparative analyses. Both beta 3 sequences are highly homologous with human beta 3, well above average rodent-human protein homology of 79%. The ligand binding domains (aa) 109-171 and 204-229) are, respectively, 90% and 100% homologous. The beta 3 transmembrane and the cytoplasmic tail are surprisingly highly conserved, being 97% and 100% homologous, respectively, but share little homology with beta 1, or beta 2. This latter difference argues strongly in favor for a crucial beta 3-specific function for these domains. In conclusion, we present the first comparative analysis of beta 3 chains and demonstrate high overall homology. The biological implications of these comparisons are discussed.

Amino Acid Sequence↗

PubMed1993

Analysis of Gz alpha by site-directed mutagenesis. Sites and specificity of protein kinase C-dependent phosphorylation.

The G protein alpha subunit Gz alpha is a substrate for phosphorylation by protein kinase C. The phosphorylation has been documented both in human platelets and in vitro and is characterized by a high degree of selectivity in relation to other G protein alpha subunits. We have demonstrated previously by phosphoamino acid analysis and CNBr peptide mapping that phosphorylation occurs at a serine residue(s) within the NH2-terminal 53 residues of Gz alpha. In this study, we have examined the site of phosphorylation using site-directed mutagenesis. Gz alpha variants containing selected substitutions of alanine for serine residues were expressed in human kidney 293 cells, and the ability of each to be phosphorylated in response to phorbol 12-myristate 13-acetate was examined. A focus was placed on Ser25 and Ser27, the 2 serine residues within a sequence of Gz alpha used to obtain a phosphorylation-sensitive antibody. The results demonstrate that Ser27 is the primary site of phosphorylation. Conversion of Ser27 to an alanine resulted in a 65% decrease in incorporation of [32P] phosphate; conversion of Ser25 had no effect. Conversion of Ser16, which like Ser25 and Ser27 conforms to a consensus site for protein kinase C, resulted in a modest (15%) decrease. The conversion of both Ser16 and Ser27 resulted in an 80% suppression of incorporation. In addition to these results, we have extended studies of the subunit and kinase selectivity of phosphorylation in platelets. We show here that under conditions promoting phorbol 12-myristate 13-acetate-stimulated phosphorylation of Gz alpha in permeabilized platelets, Gq alpha is not phosphorylated. Moreover, Gi alpha, Gz alpha, and Gq alpha were not phosphorylated in response to analogues of cAMP or cGMP. Thus, only Gz alpha is phosphorylated in platelets and only in response to activation of protein kinase C.

Base Sequence↗

PubMed1993

Determinants of the intracellular fate of truncated forms of the platelet glycoproteins IIb and IIIa.

The platelet glycoproteins GPIIb and GPIIIa are integral membrane proteins and form calcium-dependent heterodimers in the endoplasmic reticulum (ER). In the absence of heterodimer formation, GPIIb and GPIIIa are retained in the ER and degraded. To produce soluble forms of these proteins, we truncated each at a site just proximal to its transmembrane anchor and expressed the mutants in COS-1 cells. We found that both truncated GPIIIa (GPIIIatr) and GPIIIatr were secreted by the transfected cells. However, GPIIbtr was retained by the cells and was immunoprecipitated as a doublet with a 115,000 molecular weight protein. Incubation of transfected cells with the calcium ionophore A23187 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) failed to induce appreciable GPIIbtr secretion, suggesting that formation of intracellular calcium complexes was not a factor in GPIIbtr retention. Further, immunoblotting of immunoprecipitated GPIIbtr and GPIIIatr revealed that the chaperone binding protein (BiP) was associated with each, arguing that BiP alone was not responsible for GPIIbtr retention. These studies indicate that the intracellular retention of GPIIIa involves sequences located in the transmembrane or cytoplasmic domains of the molecule. GPIIb contains an additional retention signal located in the extracellular portion of the molecule whose effect is abrogated by formation of a GPIIb-IIIa heterodimer. This signal may be involved in the fate of nascent GPIIb monomers and the generation of correctly configured GPIIb-IIIa heterodimers.

Amino Acid Sequence↗

PubMed1993

Agonist receptors and G proteins as mediators of platelet activation.

Recent studies have helped to define the earliest events of signal transduction in platelets, particularly those involved in the generation of second messengers. The best-understood of these events are those which involve guanine nucleotide binding regulatory proteins. G proteins are heterotrimers comprised of alpha, beta and gamma subunits, each of which can exist in multiple forms. Some, but not all, of the known variants of G alpha are substrates for ADP-ribosylation by pertussis toxin, a modification which disrupts the flow of information from receptor to effector. The G proteins that have been identified in platelets to date are Gs, Gi1, Gi2, Gi3, Gz and Gq. Gs and one or more of the Gi family members regulate cAMP formation by adenylylcyclase. Gi may also be responsible for the pertussis toxin-sensitive activation of phospholipase C which occurs when platelets are activated by thrombin. Gq is thought to be responsible for the pertussis toxin-resistant activation of phospholipase C by TxA2. Gz does not have an established role, but has the unique property of being phosphorylated by protein kinase C during platelet activation. Recent efforts to clone the receptors that interact with G proteins in platelets have been successful for epinephrine, thrombin, TxA2 and platelet activating factor. Each of these resembles other G protein-coupled receptors, being comprised of a single polypeptide with 7 transmembrane domains. In the case of thrombin, receptor activation is thought to involve a unique mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Peptides corresponding to the tethered ligand can mimic the effects of thrombin, while antibodies to the same domain inhibit platelet activation. Shortly after activation, thrombin receptors become resistant to re-activation by thrombin. This desensitization, which appears to be due to a combination of proteolysis, phosphorylation and internalization, provides a potential mechanism for limiting the duration of thrombin-initiated signals in platelets.

Adenosine Diphosphate↗

PubMed1992

Genes for beta-thromboglobulin and platelet factor 4 are closely linked and form part of a cluster of related genes on chromosome 4.

The small inducible gene (SIG) family encodes related proteins that are involved in the overlapping processes of coagulation, inflammation, immune response, and wound repair. This family contains two branches, termed CXC and CC, which are distinguished by whether or not the first two of four conserved cysteine residues are separated by an additional amino acid residue. All of the CXC SIGs map to chromosome 4, including those encoding beta-thromboglobulin (beta TG) and platelet factor 4 (PF4), both of which are expressed by megakaryocytes in a tissue-specific fashion. Both of these latter two genes have been previously reported to be duplicated, there being a PF4 and a PF4alt gene, and a beta TG1 and beta TG2 gene. We now show by pulse-field gel electrophoresis (PFGE) that the beta TG genes are closely linked to the PF4 genes and to other previously mapped CXC SIGs, namely IL8 (encoding interleukin-8), GRO1 (encoding a cytokine also called melanoma growth-stimulatory activity), and two related genes GRO2 and GRO3, on a single 700-kb Sfil fragment localized to chromosome bands 4q12-q13. The only CXC SIG not linked to this cluster is that encoding gamma-interferon-induced 10-Kd protein (INP10), which has been previously localized to 4q21. Analysis of lambda genomic clones demonstrate that the beta TG1 and PF4 genes are separated by less than 7 kb, and the beta TG2 and PF4alt genes by approximately 5 kb. Within each beta TG/PF4 duplication, the beta TG-like gene is upstream of its linked PF4-like gene. Thus, the beta TG/PF4 genes appear to form a close-linked complex expressed in a megakaryocyte-specific fashion. Further genomic studies may provide additional insights into the regulation of the tissue-specific expression of the beta TG/PF4 gene complex, while further analysis of the linked CXC SIG cytokine family may provide further understanding of their evolutionary history.

Autoradiography↗

PubMed1991

Study of the endoproteolytic cleavage of platelet glycoprotein IIb using oligonucleotide-mediated mutagenesis.

The precursor of platelet membrane glycoprotein IIb (GPIIb) undergoes endoproteolytic cleavage into heavy and light chains post-translation. Endoproteolysis occurs within a 17-amino acid stretch of the precursor that contains 4 arginine residues, 3 in dibasic sequences [Lys-Arg (855-856) and Arg-Arg (858-859)] and a single arginine at 871. To determine the site of GPIIb cleavage and its role in the function of the glycoprotein IIb/IIIa heterodimer, we mutated arginine 856, the di-arginine sequence 858-859, and arginine 871 and coexpressed the mutants with glycoprotein IIIa (GPIIIa) in COS-1 cells. Each GPIIb mutant formed recombinant GPIIb-IIIa heterodimers, but mutants lacking arginine at 856 or 858-859 failed to undergo cleavage. Nevertheless, heterodimers containing the uncleaved GPIIb were expressed on the cell surface. Because endoproteolysis most often occurs after arginines in dibasic sequences, we next expressed GPIIb mutants containing lysine at 856 or aspartic acid at 855 with GPIIIa. Both mutants were cleaved and surface-expressed, indicating that the dibasic sequence at 858-859, but not at 855-856, is required for GPIIb cleavage. Lastly, we tested the function of GPIIb-IIIa containing uncleaved GPIIb by measuring adhesion of transfected cells to immobilized fibrinogen. We found no difference in the adhesion of cells expressing either wild-type or mutant GPIIb, indicating GPIIb-IIIa heterodimers containing uncleaved GPIIb maintain their ability to interact with fibrinogen.

Amino Acid Sequence↗

PubMed1991

Effect of deletion of glycoprotein IIb exon 28 on the expression of the platelet glycoprotein IIb/IIIa complex.

We have isolated from an HEL cell cDNA library an alternatively spliced transcript for the platelet membrane glycoprotein IIb (GPIIb) that resulted from the deletion of the 34 amino acids of exon 28 of the GPIIb gene. Confirming an earlier report, we also detected this transcript in platelet mRNA. To determine the consequences of exon 28 deletion on the expression of the GPIIb/IIIa heterodimer, we expressed cDNA for GPIIb-28 in COS-1 cells, either individually or simultaneously with a cDNA for GPIIIa. When recombinant GPIIb-28 was expressed alone, it did not acquire resistance to the enzyme endo-beta-N-acetylglucosaminidase H, was not cleaved into heavy and light chains, and was not transported to the cell surface. However, when recombinant GPIIb-28 was coexpressed with recombinant GPIIIa, GPIIb/IIIa heterodimers were assembled. Nevertheless, these heterodimers failed to complete posttranslational processing and were degraded intracellularly. Exon 28 contains one site for Asn-linked glycosylation. To determine if loss of this glycosylation site was responsible for the effects of exon 28 deletion, we removed the site from the exon 28 of intact GPIIb by oligonucleotide-mediated mutagenesis. However, absence of the carbohydrate appended to exon 28 did not prevent normal GPIIb/IIIa heterodimer expression. Our studies indicate that absence of the amino acids encoded by GPIIb exon 28 sufficiently perturbs the quaternary configuration of the GPIIb/IIIa heterodimer to impair its subsequent intracellular transport and processing. They also indicate that this alternatively spliced form of GPIIb mRNA, although present in megakaryocytes, is unlikely to make a significant contribution to the GPIIb/IIIa complexes expressed on platelets.

Base Sequence↗

PubMed1991

Identification of Gz alpha as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes.

G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein alpha subunit referred to as Gz alpha. Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different Gz alpha-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the Gi alpha family. Northern blotting with a Gz alpha-specific probe prepared from retinal Gz alpha showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding Gz alpha was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of Gi alpha 2' the most abundant of the Gi alpha species present in platelets. The complete sequence of the platelet homolog to Gz alpha was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal Gz alpha. The paucity of Gz alpha protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize Gz alpha from these cells or that Gz alpha appears at a later stage in megakaryocyte development than does Gi alpha.

Base Sequence↗

PubMed1991

Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes.

The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.

Alleles↗

PubMed1991

Characterization of the human beta-thromboglobulin gene. Comparison with the gene for platelet factor 4.

beta-Thromboglobulin (beta TG) is a platelet alpha-granule protein that is released in large amounts following platelet activation. This protein has approximately 70% amino acid identity with another platelet alpha granule protein, platelet factor 4 (PF4), and approximately 30% identity with other members of a family of proteins encoded by the small inducible genes (SIG) that are involved in the overlapping processes of inflammation, wound repair, and coagulation. Thus, the isolated beta TG gene is of interest in that it may further increase our understanding of beta TG's biological role, the regulation of its megakaryocytic-specific nature of expression, and the evolution and divergence of the SIG family. Using a beta TG cDNA isolated from a DAMI cell cDNA library, we cloned the corresponding beta TG gene. The gene is 1,139 base pairs (bp) long, and, like other members of the SIG family, the beta TG gene is divided into 3 exons. Two major transcriptional start sites were defined by primer extension analysis of platelet RNA, and, based on the more commonly used start site, the 5'-untranslated region is 87 bp. A TATA box is present beginning 32 bp upstream to this site. The first exon contains the 5'-untranslated region as well as the signal peptide. The second exon begins 6 bp 3' to the homologous site in PF4, and the third exon begins at a position homologous to that in PF4. Interestingly, the beta TG and PF4 genes have little detectable homology in the flanking or intronic sequences. In particular, a pyrimidine tract 5' to both the rat and human PF4 genes is not present in the upstream region of the beta TG gene. Genomic Southern blot analysis suggests that, as with the PF4 gene, there are multiple copies of the beta TG gene in the human genome. Chromosomal localization using polymerase chain reaction analysis of human/hamster somatic cell hybrids demonstrated that the beta TG gene, like the PF4 gene, is located on chromosome 4. It is, therefore, possible that the beta TG and PF4 gene form a single genomic locus on chromosome 4 whose members become coordinately activated during megakaryocyte differentiation.

Amino Acid Sequence↗

PubMed1991

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.

Adenosine Diphosphate Ribose↗

PubMed1991

Elevated common acute lymphoblastic leukemia antigen expression in pediatric immune thrombocytopenic purpura.

Bone marrow examination is often performed in thrombocytopenic children to distinguish immune thrombocytopenic purpura (ITP) from acute leukemia. We describe a patient with thrombocytopenia and 50% common acute lymphoblastic leukemia antigen (CALLA) positivity in his marrow who was subsequently shown to have ITP. CALLA (CD10) is a surface antigen found in early B-lymphocytes and is elevated in most cases of childhood acute lymphoblastic leukemia (ALL). This case prompted us to prospectively study the frequency of immature lymphocyte populations in children with ITP. Fourteen patients with acute ITP and five with other conditions were studied. The two groups were comparable with respect to age: ITP mean, 4.3 (range 0.3-15.5) years; control mean, 5.8 (0.6-13.8) years. The ITP group had a significantly higher percentage of CD10 positive bone marrow lymphocytes (p = 0.007). Five of the 10 patients younger than 4 years of age in the ITP group had CD10 levels of greater than 30%, which is in the leukemic range, whereas none of the control patients had a CD10 levels of greater than 17% (p = 0.003). There was good correlation between CD10 positivity and B4 positivity indicating that both of these markers arise from the same population of immature B-lymphocytes. None of the ITP patients who were older than 4 years had a CD10 level of greater than 30%. We conclude that it is common to have an increase in the proportion of immature lymphocytes in the marrow of young children with ITP. The cause of this increase in CD10 positive cells is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗