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C N Chesterman

Publications and source records attributed to C N Chesterman.

At least 55 records · Page 3Linked to original sources
PubMed1996

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Blood Platelets↗
PubMed1996

Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias.

The diagnosis of idiopathic immune thrombocytopenia remains a clinical diagnosis based on the exclusion of other causes of immune and nonimmune thrombocytopenia. Measurement of platelet-associated Ig (PAIg), while sensitive, is nonspecific for the diagnosis of immune thrombocytopenia. Published experience of antigen capture assays (including monoclonal antibody immobilization of platelet antigens or MAIPA) suggest a high sensitivity and specificity (70% to 80%) in selected groups of patients. In a prospective evaluation of 158 patients with thrombocytopenia from all causes, we report a sensitivity of 51% and specificity of 80% for direct MAIPA assays. MAIPA was considerably better in discriminating immune from nonimmune thrombocytopenia than two assays of PAIgG. Antiplatelet antibodies detected by MAIPA were more frequently directed against the glycoprotein (GP) IIb/IIIa than the GP Ib/IX complex. Our experience suggests that MAIPA assays are useful in the laboratory assessment of thrombocytopenia, should be performed before therapy, and that some patients with 'nonimmune' thrombocytopenia may have genuine antiplatelet antibodies.

Antibodies, Monoclonal↗
PubMed1996

Fibroblast growth factor and heparin protect endothelial cells from the effects of interleukin 1.

Vascular endothelium is involved in both active and passive processes in haemostasis, but inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF) have been reported to convert the comparatively inert endothelial cell to an inflammatory state. Acidic fibroblast growth factor (aFGF) in the presence of heparin has effects opposite to IL-1 on cultured human umbilical vein endothelial cells (HUVEC); therefore, we have investigated the modulation of IL-1-induced effects by the c combination of aFGF and heparin (aFGF/heparin). First passage HUVEC were cultured for 6 days in the presence of 20% human serum with and without the addition of 625 pM human recombinant aFGF (hr aFGF) and 7 microM heparin. On day 5, recombinant IL-1 beta was included for 24 h. The following day the cells were washed and measurements made of the release of prostacyclin, von Willebrand factor, plasminogen activator inhibitor type 1, and thrombospondin, both in the resting state and following stimulation for 60 min with 1 U/ml thrombin. Tissue-type plasminogen activator was assayed in HUVEC lysates. Similar experiments were performed to assess effects on the expression of vascular adhesion molecule, intracellular adhesion molecule, and E-selectin using an ELISA on cells in situ. This study indicates that aFGF/heparin in the culture medium of HUVEC abrogates the measured responses to IL-1. These data imply that routine endothelial cell culture with aFGF/heparin may cause artefacts, the effects of FGF and Il-1 may involve common pathways, and FGF/heparin may offer an approach to design therapeutics to counter the adverse effects of IL-1.

Cell Adhesion Molecules↗
PubMed1996

Extracellular matrix is a source of mitogenically active platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A(s) (short splice version) is secreted, PDGF A(l) (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and epsilon-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions.

3T3 Cells↗
PubMed1996

Characterization of IgG Fc receptors on CD34 antigen-expressing cell lines (KG-1 and KG-1a).

Although Fc gamma receptors (Fc gamma R) on mature blood cells have been extensively studied, there are only limited data on Fc gamma R expression in the early haematopoietic progenitor cells. In this study, we used the stem cell antigen (CD34)-expressing cell line (KG-1) and its less differentiated subline (KG-1a) as a model for the study of Fc gamma R in the early haematopoietic progenitors. Flow cytometry and immunoprecipitation studies on KG-1 and KG-1a cells with anti-Fc gamma R mAb showed that Fc gamma RII is the only Fc gamma R expressed on the cell surface. Analysis of the steady-state levels of Fc gamma R mRNA in KG-1 and KG-1a cells using a quantitative in situ hybridization assay revealed the presence of only Fc gamma RII mRNA. On further analysis Fc gamma RIIA mRNA but no Fc gamma RIIB or Fc gamma RIIC transcripts were found in these cells; Fc gamma RIIA transcripts with and without the transmembrane exon were present in approximately equal amounts. These findings are surprisingly similar to those observed previously with Fc gamma R in platelets and megakaryocytic cells but different from those found with Fc gamma R in cells of other lineages. These data suggest that the Fc gamma R transcript distribution pattern observed in the early haematopoietic progenitors (KG-1 cells) is retained in later stages of haematopoietic differentiation only in cells of megakaryocytic lineage.

Antigens, CD34↗
PubMed1996

Beta 2-glycoprotein I in thrombosis: evidence for a role as a natural anticoagulant.

Although the physiological role of beta2-glycoprotein (B2GPI) is unknown, in vitro evidence indicates that B2GPI may be a natural anticoagulant. In this study we have examined whether fluctuations of plasma B2GPI occur in in vivo coagulation. Serial measurements of B2GPI and other anticoagulant proteins were performed in 51 patients with thrombotic (group 1: six patients with disseminated intravascular coagulation (DIC), group 2: venous (n = 4) or arterial (n = 170 thrombosis) and non-thrombotic disease (group 3: 24 patients undergoing elective surgery). Reductions in plasma B2GPI levels were seen in most patients which were roughly proportional to the severity of their illness. Particularly striking reductions of B2GPI, protein C (PC) and antithrombin III (AT-III) (mean +/- 95% CI: 42.7 +/- 8.6%, 42.1 +/- 14.8%, 39.1 +/- 28.4% respectively) were seen in group 1. The reductions in plasma B2GPI were significantly greater in group 1 than in the other groups. Dilutional factors explain most of the reductions in B2GPI, PC and AT-III in groups 2 and 3, but contribute little to group 1. In conclusion, although B2GPI behaves as a 'negative acute phase reactant', the magnitude of reduction of plasma B2GPI levels, accompanied by reductions in other anticoagulant proteins in patients with DIC, suggests specific consumption of B2GPI in in vivo coagulation. This study provides further evidence that B2GPI is an anticoagulant of physiological importance.

Acute Disease↗
PubMed1996

Characterization of Fc gamma receptors on human megakaryocytes.

Megakaryocyte and platelet Fc gamma receptors (FcR) are of importance in the pathophysiology of immune complex-mediated thrombocytopenias such as heparin-induced thrombocytopenia. In this study, Fc gamma R proteins and mRNAs in normal human megakaryocytes were examined. Fc gamma R proteins were studied with immunocytochemical staining, dual colour flow cytometry and immunoprecipitation using monoclonal antibodies against Fc gamma R I, Fc gamma R II and Fc gamma R III. Fc gamma R mRNAs were measured with biotinylated cDNA of oligonucleotide probes using a novel quantitative in situ hybridization technique. Using these techniques, Fc gamma R II protein and mRNA, but not Fc gamma R I and Fc gamma R III proteins and transcripts were detected in megakaryocytes. Further, transcript analysis showed that megakaryocytes contain only the transcript of Fc gamma R IIA gene but no transcripts of Fc gamma R IIB nor Fc gamma R IIC genes; Fc gamma R IIA transcripts with and without the transmembrane (TM) exon are present in approximately equal proportions. In contrast, neutrophils and macrophages also contain Fc gamma R IIA transcript but Fc gamma R IIA transcript with the TM exon predominates suggesting cell lineage-specific Fc gamma R IIA expression. Fc gamma R IIA transcript lacking the TM exon predicts the presence of a potential soluble form of Fc gamma R in platelets and megakaryocytes which may have a physiological role as it can compete with the membrane-bound Fc gamma R IIA for binding of IgG-containing immune complexes and thus protect these cells from excessive binding and injurious effects of immune complexes.

Base Sequence↗
PubMed1996

Serotonin uptake, storage and metabolism in megakaryoblasts.

Serotonin (5-hydroxytryptamine, 5-HT) uptake, storage and metabolism in human megakaryoblastic cell line (Meg-01) which acts as a model for megakaryocyte precursors, megakaryoblasts were investigated by using biochemical (HPLC) and morphological (electron microscope, EM) techniques. Results showed that Meg-01 cells were able to take up 5-HT. The intracellular 5-HT level was 2.8 +/- 0.4 and 51.8 +/- 4.9 (1 h) or 59.0 +/- 4.4 (2 h) ng/10(6) cells, before and after incubation with 5-HT, respectively, but no dense bodies were visualized after incubation with excess 5-HT by electron microscope observation. This uptake was inhibited by 28% on pre-incubation with fluoxetine and 60% of 5-HT in the cells was released on incubation with reserpine. 5-Hydroxyindoleacetic acid (5-HIAA) concentration of Meg-01 cells was increased after incubation of the cells with 5-HT (0 and 17.6 +/- 2.1 or 19.9 +/- 1.9 ng/10(6) cells, before and after incubation 1 or 2 h, respectively). The study suggests that: (1) 5-HT uptake ability is well established in megakaryocytes precursors, megakaryoblasts and the uptake ability is affected by reserpine and fluoxetine; (2) however, the capacity to store the amine is not well developed in megakaryoblasts and (3) megakaryoblasts may contain monoamine oxidase (MAO) which converts 5-HT to 5-HIAA.

Cells, Cultured↗
PubMed1995

Urokinase binding and catabolism by Hep G2 cells is plasminogen activator inhibitor-1 dependent, analogous to interactions of tissue-type plasminogen activator with these cells.

The adherent human hepatoma cell line Hep G2 exhibits receptor mediated endocytosis and catabolism of tissue-type plasminogen activator.plasminogen activator inhibitor type-1 (t-PA.PAI-1) complexes formed when exogenous t-PA combines with endogenous PAI-1 in the extracellular matrix. To determine whether the other major PA, urokinase (u-PA), which also complexes with PAI-1, is metabolised via the same mechanism, 125I-labelled high (hmw) and low (lmw) molecular weight forms of u-PA were incubated with Hep G2 cells at 4 degrees C for 2 hr in the absence and presence of a 100-fold excess of unlabelled ligand in order to detect specific binding. Both hmw and lmw 125I-u-PA formed complexes with PAI-1 and these bound specifically and with high affinity (apparent Kd 3.9 and 4.1 nM, with Bmax 78 x 10(3) and 83 x 10(3) binding sites/cell respectively). Binding by each form of radiolabelled u-PA was inhibited in a dose-dependent fashion by unlabelled t-PA, hmw-u-PA, lmw-u-PA, and by monoclonal anti-PAI-1 antibody. At 37 degrees C, bound hmw and lmw 125I-u-PA.PAI-1 complexes were internalised and degraded rapidly. These findings indicate that the specificity of the previously described receptor which mediates PAI-1 dependent catabolism of t-PA by Hep G2 cells extends to complexes of u-PA with this inhibitor.

Antibodies, Monoclonal↗
PubMed1995

Effects of cyclosporin A and dexamethasone on haemostatic and vasoactive functions of vascular endothelial cells.

Glucocorticoids reduce prostaglandin synthesis in cultured vascular endothelium, but their effects on other haemostatic functions are unclear. We examined the effects of dexamethasone and cyclosporin A (CSA) on cultured human umbilical vein endothelial cells (HUVEC). One, 10 and 50 micrograms/ml CSA and 1 microgram/ml dexamethasone (Dx) were added to the culture medium for 3 h, 3 days and 6 days and compared with HUVEC cultured in medium and serum alone. After assay of accumulated release of tissue type plasminogen activator (t-PA) and endothelin 1 (ET), cells were stimulated with 1 U/ml of human thrombin for 1 h and medium collected for RIA of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), thrombospondin (TSP), von Willebrand factor (vWf) and ELISA of plasminogen activator inhibitor 1 (PAI-1). CSA at 1 microgram/ml modestly reduced release of prostacyclin (PGI2) but had no reproducible effects on other metabolites. CSA at 10 and 50 micrograms/ml inhibited cell growth and thrombin stimulated release of PGI2 in a time- and dose-dependent manner. Inhibition of other endothelial metabolites was also observed at CSA 10 > micrograms/ml. Dexamethasone 1 microgram/ml reduced both cell number and PGI2 release and increased thrombin stimulated release of vWf, TSP and PAI-1 with increases in t-PA and endothelin 1 in the medium. CSA 1 microgram/ml and dexamethasone 1 microgram/ml together were additive in reducing PGI2 release and increasing PAI-1 secretion. These observations suggest a role for endothelial dysfunction in the hypertensive and thrombotic complications observed in steroid treated patients with CSA potentially contributing to such complications.

6-Ketoprostaglandin F1 alpha↗
PubMed1995

Quantitation of soluble and membrane-bound Fc gamma RIIA (CD32A) mRNA in platelets and megakaryoblastic cell line (Meg-01).

Fc gamma receptors (Fc gamma Rs) are glycoproteins on platelet surface that bind IgG-containing immune complexes. However, excessive binding of immune complexes leads to platelet activation and thrombosis or increased platelet clearance and thrombocytopenia. In this study, Fc gamma R transcripts in platelets and megakaryoblastic cell line (Meg-01) were investigated using specifically designed oligonucleotides and a new quantitative in situ hybridization assay. Platelets and Meg-01 cells were found to express only Fc gamma RII transcripts. Of Fc gamma RIIA mRNA isoforms (Fc gamma RIIA, B and C), Fc gamma RIIA mRNA predominates in these cells. Platelets and Meg-01 cells contain both alternative spliced forms of Fc gamma RIIA mRNA, those with and without the transmembrane (TM) exon and both forms were present in near equal amounts. In contrast, Fc gamma RIIA transcript with the TM exon predominates in neutrophils and monocytes, suggesting that the splicing of the TM exon is under lineage-specific control.

Antigens, CD↗
PubMed1995

Recombinant PDGF enhances megakaryocytopoiesis in vitro.

The effect of recombinant platelet-derived growth factor (PDGF) on both murine and human megakaryocyte colony formation was studied in the plasma clot culture system. PDGF significantly stimulates megakaryocyte colony formation in a dose-dependent manner. The minimum concentration which had a significant stimulating effect on colony forming unit megakaryocyte (CFU-MK) was 10 ng/ml and maximum stimulation occurred at 50 ng/ml. The effect of PDGF was compared with that of interleukin (IL)-3, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and acid fibroblast growth factor (aFGF) on megakaryocyte colony formation. The results showed that megakaryocyte colony stimulating activity of PDGF was slightly higher than those of GM-CSF and aFGF, but lower than those of IL-3, IL-6 and EPO. The effect of PDGF in combination with IL-3 or IL-6 on megakaryocyte colony formation was also investigated. No synergistic action was found between PDGF and IL-3 or IL-6, but an additive effect was observed with IL-3 plus IL-6. We also studied the effects of PDGF in combination with anti-IL6, anti-IL-3 or anti-GM-CSF antibody. The increase of megakaryocyte colony formation induced by PDGF was partially inhibited by anti-IL-6 or anti-GM-CSF antibody but not by anti-IL-3 antibody. These results indicate that PDGF is a positive regulator for megakaryocytopoiesis in vitro and IL-6 and GM-CSF may play a role in the mechanism whereby PDGF stimulates megakaryocytopoiesis.

Animals↗
PubMed1995

Single amino acid mutation of Fc gamma receptor is associated with the development of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is mediated by a heparin-dependent antibody/platelet factor 4/heparin complex binding to platelets via the Fc gamma receptor (type IIA). A single base polymorphism at position 131 of Fc gamma RIIA changes the native arginine to histidine. In the presence of murine monoclonal IgG1 the former phenotype (Fc gamma RIIAArg131) is functionally characterized by strong platelet aggregation (high responder) and the latter (Fc gamma RIIAHis131) by poor aggregation (low responder). In the presence of human IgG2 the opposite response is observed. It has recently been shown that the heparin-dependent antibody is predominantly of this subclass. We hypothesize that a relationship exists between Fc gamma RIIAHis131 and the development of HIT. We studied 24 normal individuals and 20 HIT patients using VM58, a murine monoclonal IgG1, to characterize the phenotype by platelet aggregrometry, and PCR products, amplified with primers bordering the Fc gamma RIIA polymorphism and hybridized with oligonucleotide probes specific for the single base mutation, to determine the genotype. The distribution of phenotypes and genotypes in the two populations differed, with a greater prevalence of the Fc gamma RIIAHis131 allele in the HIT patient population. Homozygous Fc gamma RIIAArg131 individuals were absent from this group. We conclude that the presence of the Fc gamma RIIAHis131 allele is associated with a predisposition to HIT.

Adult↗
PubMed1995

Acidic and basic fibroblast growth factors have comparable effects on the haemostatic function of vascular endothelium.

Acidic and basic fibroblast growth factor (aFGF and bFGF respectively) are closely related mitogens (55% homology) of the heparin binding growth factor family. Reports of the relative potency of these growth factors and the ability of heparin to potentiate the activity of bFGF are conflicting. We have examined the effect of heparin and human recombinant aFGF and bFGF on basal and thrombin challenged release of metabolites from cultured human umbilical vein endothelial cells (HUVEC). Culture supernatant was assayed for thrombospondin, prostacyclin and PAI-1 and cell lysates were analysed for t-PA. aFGF and bFGF were equipotent in regulating ther release of all metabolites studied, except thrombin stimulated release of PGI2 where bFGF was more potent than aFGF in the absence of heparin. Heparin potentiated the mitogenic and metabolic effects of both bFGF and aFGF. However, heparin was not essential for the expression of the biological activity of FGF.

Animals↗
PubMed1994

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation.

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin-catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.

Amino Acid Sequence↗
PubMed1994

Quantitation of Fc gamma RII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization.

We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.

Blood Platelets↗