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Biomedical subjects

J S Bennett

Publications and source records attributed to J S Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1992

Disorders of platelet function.

Platelets provide for primary hemostasis by forming a hemostatic plug at sites of vascular damage. They also provide a surface for the assembly of the coagulation protein complexes that generate thrombin, serve as a nidus for fibrin clots, and secrete factors involved in wound repair. Normal platelet function can be divided into four phases: adhesion, aggregation, secretion, and expression of procoagulant activity. Platelet adhesion initiates plug formation as platelets adhere to the connective tissue at the edges of a wound within seconds after vascular damage. When damage occurs in regions of slow blood flow, platelets adhere to subendothelial collagen, fibronectin, and laminin. However, when damage occurs in regions of rapid flow, platelet adhesion requires the presence of subendothelial von Willebrand factor (vWf) and a specific platelet receptor, the glycoprotein Ib/IX (GPIb/IX) complex. Following initial adhesion, platelets aggregate to complete the formation of a hemostatic plug. Platelet aggregation requires active platelet metabolism, platelet stimulation by agonists such as ADP, thrombin, collagen, or epinephrine; the presence of calcium or magnesium ions and specific plasma proteins such as fibrinogen or vWf; and a platelet receptor, the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. Thus, platelet stimulation results in the generation of intracellular second messengers that transmit the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or vWf) then binds to GPIIb/IIIa and crosslinks adjacent platelets to produce platelet aggregates. Platelet stimulation also results in platelet secretion and the elaboration of platelet procoagulant activity. During secretion, substances are released to propagate the aggregation response and to promote wound healing; the expression of procoagulant activity localizes thrombin generation to the site of vascular damage. Disorders of platelet function can be divided into those of congenital and those of acquired origin. Although congenital disorders are uncommon, acquired disorders are encountered frequently in clinical practice. Congenital absence of GPIb/IX and GPIIb/IIIa results in the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT), respectively. Each is an autosomal recessive bleeding disorder in which absence of a protein complex renders the affected platelets incapable of undergoing either vWf-mediated adhesion (BSS) or fibrinogen-mediated aggregation (GT).(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex Hormones↗
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

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↗
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

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

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex.

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.

Adenosine Diphosphate↗
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

Chromosomal localization of the genes for the vitronectin and fibronectin receptors alpha subunits and for platelet glycoproteins IIb and IIIa.

The integrins, a family of related membrane receptors involved in cell-cell and cell-matrix interactions, are heterodimeric complexes of alpha and beta subunits. To begin to understand the evolution of these complexes, we studied the genomic organization of several alpha and beta integrin subunits. Using both somatic cell hybrids and an in situ hybridization technique, we have determined the chromosomal location of the genes for the alpha subunits of the vitronectin receptor (VNR alpha), the fibronectin receptor (FNR alpha), and for the alpha subunit of the platelet glycoprotein IIb/IIIa complex, GPIIb. In addition, we have determined the chromosomal location of the gene for the beta subunit of the GPIIb/IIIa heterodimer, GPIIIa. Our studies indicate that the alpha subunits do not localize to a single locus, but that each is found on a different chromosome. The gene for VNR alpha is located on chromosome 2, the gene for FNR alpha is on chromosome 12q11----13, and the gene for GPIIb is on chromosome 17q21----23. In contrast to the chromosomal dispersion of the alpha subunits, the genes for GPIIb and GPIIIa are physically close, with the gene for GPIIIa also located on chromosome 17q21----23. These studies indicate that the genes for the alpha subunits of the integrin family have been dispersed during evolution while GPIIb and GPIIIa are in close physical proximity. This physical proximity of GPIIb and GPIIIa may be involved in the concurrent expression of these proteins by megakaryocytes, and may result in linkage disequilibrium between these two genes, which would limit the use of restriction length polymorphisms in linkage studies of GPIIb/IIIa abnormalities in small kindreds.

Animals↗
PubMed1988

Changes in peripheral blood cells in mice after injection with benzo(a)pyrene during pregnancy.

Pregnant C3H/Anfcum mice were injected ip with 150 micrograms benzo(a)pyrene (BP)/g body weight at the second trimester (12 days). Quantitative and differential changes were assayed in the peripheral blood leukocytes and erythrocytes at various times before and after mating and treatment. Within 5 days after injection, a 2- to 4-fold reduction in leukocytes was observed when compared to controls [corn oil (vehicle for BP)-injected pregnant females] which persisted into the 10th postpartum day. The erythrocytes were also significantly reduced but not to the same degree (1.2- to 1.5-fold). Depression in white blood cells is attributed to lymphocyte depletion since the granulocytes were virtually unchanged and the lymphocyte to granulocyte ratio, ordinarily greater than 2 was 1 or less than one. No change in monocytes was observed and none of the cell populations, including the erythrocytes, appeared to be abnormal (e.g., no increase in reticulocytes). A moderate reduction (1.5-fold) in erythrocytes and leukocytes also occurred in the controls (vs virgin females). Pregnancy also led to transient decreases in medium sized lymphocytes and 3- to 4-fold increase in small lymphocytes shortly after mating to about 3 days before parturition. These results show that, although pregnancy depresses the leukocyte profile, exposure to BP exacerbates this change, and preferentially affects the lymphocytes. These blood profile changes may have important health consequences in the mother and her progeny.

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

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↗
PubMed1987

The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa.

The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium-dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

Blood Platelets↗