Biomedical subjects
Publications and source records attributed to J S Bennett.
Thrombosis can occur on the venous or the arterial side of the circulation. Each has different causes, requires a different diagnostic workup, and responds to different therapies. Venous thrombosis may be situational, but may also reflect inherited or acquired anticoagulation defects. Arterial thrombosis usually results from abnormalities in the blood vessel wall--most often atherosclerosis.
We report a case of Glanzmann thrombasthenia in a Pakistani child whose platelets express less than 10% of the normal amount of alphaIIb beta3 on their surface. Single-stranded conformation polymorphism analysis of the exons of the patient's alphaIIb and beta3 genes showed an abnormality in exon 4 of the beta3 gene. Direct sequence analysis showed that the patient was homozygous for a T --> G nucleotide substitution in this exon, resulting in the replacement of a highly conserved Leu at position 117 with Trp. Heterologous expression of alphaIIb beta3 containing the beta3 mutation in COS-1 cells confirmed the pathogenicity of the Leu117 --> Trp substitution and showed that it resulted in the intracellular retention of malfolded alphaIIb beta3 heterodimers. Additional site-directed mutagenesis at position 117 indicated that, although the smaller hydrophobic amino acid Val could be substituted for the wild-type Leu, the larger hydrophobic amino acids Trp and Phe or the charged amino acids Asp and Lys were not tolerated. These studies indicate that Leu117 in beta3 plays a critical role in attaining the correct folded conformation of alphaIIb beta3. These studies also suggest that the hydrophobic side chain of Leu117 is likely folded into the interior of beta3, where it serves to stabilize internal packing of the protein and determines its overall shape.
The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by alphavbeta3. alphavbeta3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an alphavbeta3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both alphavbeta3 and alphaIIbbeta3 confirmed that activated alphavbeta3 not activated alphaIIbbeta3 was responsible for the cellular adherence we measured. These studies indicate that alphavbeta3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated alphavbeta3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting alphavbeta3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.
Function of the platelet integrin alphaIIbbeta3 is regulated by agonist-generated signals interacting with its cytoplasmic tails. When alphaIIbbeta3 is expressed in Epstein-Barr virus-transformed B lymphocytes, stimulation of the cells with phorbol 12-myristate 13-acetate results in alphaIIbbeta3-mediated lymphocyte adherence to immobilized fibrinogen, as well as soluble fibrinogen binding to alphaIIbbeta3, indicating that agonists increase the affinity of alphaIIbbeta3 for fibrinogen in these cells. To address the contribution of the alphaIIb and beta3 cytoplasmic tails to this process, we mutated each tail and expressed the mutants in B lymphocytes. Truncation of the alphaIIb tail did not impair unstimulated or stimulated lymphocyte adherence to fibrinogen, regardless whether the truncation was proximal or distal to the conserved GFFKR sequence. However, deleting GFFKR or replacing it with alanines markedly reduced alphaIIbbeta3 expression due to impaired intracellular assembly of alphaIIbbeta3 heterodimers, probably due to a mutation-induced change in the conformation of alphaIIb. Introducing beta3 mutations known to impair alphaIIbbeta3 function in platelets into the cytoplasmic tail of beta3 in lymphocytes also impaired alphaIIbbeta3 function in these cells. These studies demonstrate that the cytoplasmic tail of alphaIIb is not required for alphaIIbbeta3 function in lymphocytes, although the presence of GFFKR in the alphaIIb tail is required for alphaIIb to interact with beta3. Additionally, they indicate that signals interacting with the beta3 cytoplasmic tail are responsible for the ability of agonists to stimulate alphaIIbbeta3 function.
A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.
The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.
The authors report the use of a tap water enema to displace, and thereby localize, a site of colonic bleeding that had remained fixed in the left lower quadrant of the abdomen and could not be accurately localized. In similar cases, this maneuver may help to localize colonic bleeds in a timely fashion, avoiding protracted imaging and obviating the need for additional diagnostic testing.
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.
Using Southern blot and fluorescence in situ hybridization analyses, we have shown that the novel human gene EPAG, expressed in association with the mitogenic activation of T and B cells in peripheral blood, maps to chromosome region Xq21-->q22. These data establish EPAG as a candidate gene in X-linked disorders.
The nature of a disorder producing moderate to severe bleeding after minor trauma, venipuncture, and surgery was studied in 3 families of American cocker spaniel dogs. In the 5 affected dogs tested, platelet counts and measurements of plasma coagulant function and von Willebrand factor were normal. However, bleeding times were prolonged in 4 of the 5 affected dogs tested, and platelet aggregation in response to ADP and collagen was consistently abnormal in 3, suggesting that the bleeding disorder was due to abnormal platelet function. Measurements of 14C-serotonin uptake and retention by the affected platelets were normal. However, their ADP content was decreased, while their ATP content was normal, resulting in a mean ATP/ADP ratio of 8.32, compared to a mean ratio of 1.9 in normal canine platelets. Electron microscopy revealed that the number and appearance of the dense granules in the affected platelets were indistinguishable from those of normal controls. These studies suggest that this bleeding disorder results from a deficient delta-granule storage pool of ADP; given the normal serotonin uptake and retention by affected platelets and the apparently normal number of dense granules, the ADP deficiency may be the consequence of a selective defect in delta-granule ADP transport. Additional studies of this unique platelet disorder will provide an opportunity to understand the mechanism of adenine nucleotide storage in platelet delta-granules.
We have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, we have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. We hypothesize that epag functions as an early signal that helps mediate the activation of T cells.
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.
Integrin adhesion receptors participate in two-way transfer of information across the plasma membrane. For example, cytoplasmic events, such as activation of protein kinase C, cause an increase in the fibrinogen (Fg) binding affinity of the extracellular domain of integrin alpha IIb beta 3 ("inside-out signaling"). Conversely, ligand binding to alpha IIb beta 3 results in the generation of intracellular signals. We used anti-LIBS2, an anti-beta 3 monoclonal antibody, to understand potential mechanisms of this bidirectional signaling. Anti-LIBS2 bound to alpha IIb beta 3 with low affinity (Kd = 7.4 microM), and mimicked inside-out signaling by promoting Fg binding. The affinity of anti-LIBS2 binding was increased 20-fold (Kd = 326 nM) by addition of an Fg-mimetic synthetic peptide, RGDS. Thus, anti-LIBS2 and ligands (Fg and Fg-mimetic peptides) bind cooperatively to integrin alpha IIb beta 3, indicating a functional linkage between the ligand-binding site and the antibody-binding site. The anti-LIBS2-binding site was mapped by its binding to proteolytic and recombinant fragments of the beta 3 subunit. The epitope was located within an 89-residue region immediately adjacent to the transmembrane domain and 400 residues carboxyl-terminal to the known ligand-binding site(s). Electron microscope images of rotary shadowed ternary complexes of Fg, anti-LIBS2, and alpha IIb beta 3 revealed that the ligand-binding site and anti-LIBS2 epitope are separated by about 16 nm. This indicates that propagated long distance conformational changes can occur in alpha IIb beta 3. Such changes are likely to be involved in the bidirectional signaling function of this integral membrane protein.
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.
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The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.
A hemostatic plug is initially composed of an adherent layer of platelets, but it is subsequent platelet aggregation that stems the flow of leaking blood. Unlike platelet adhesion--which is a passive process--platelet aggregation is active, requiring ongoing platelet metabolism and stimulation by one or more specific agonists.
A 24 year old male with a history of eczema, recurrent mild infections, and thrombocytopenia consistent with the Wiskott-Aldrich syndrome (WAS) presented with a mediastinal mass, generalized lymphadenopathy, splenomegaly, and severe thrombocytopenia. Studies of immune function including immunoglobulin levels and T-cell subsets were normal. Furthermore, his T lymphocytes proliferated normally in response to phytohemagglutinin, concanavalin A, and the combination of neuraminidase/galactose oxidase. However, their proliferative responses to anti-CD43 antibody and periodate were diminished, consistent with the clinical diagnosis of WAS. An initial inguinal lymph node biopsy surprisingly revealed Kaposi sarcoma. However, following splenectomy to increase the platelet count, biopsy of the mediastinal mass revealed T-cell large cell lymphoma. Studies of biopsied tissue for the presence of Epstein-Barr virus and cytomegalovirus were negative, as were studies of blood, including the polymerase chain reaction, for the presence of the human immunodeficiency virus (HIV). This is the first report of Kaposi sarcoma arising in a patient with a congenital immunodeficiency syndrome. Although Kaposi sarcoma can arise in the face of the severe immunosuppression that follows allograft transplantation and in patients infected with HIV, we postulate that longevity in the face of mild immunosuppression was the major factor in the development of Kaposi sarcoma in this patient.