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Biomedical subjects

B H Chong

Publications and source records attributed to B H Chong.

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

Serotonin stimulates megakaryocytopoiesis via the 5-HT2 receptor.

It is known that platelet alpha-granule constituents including platelet-derived growth factor (PDGF), platelet factor 4 (PF4) and transforming growth factor-beta (TGF-beta) can affect megakaryocytopoiesis. Serotonin, a platelet dense granule constituent has been shown to have a mitogenic effect on fibroblasts and smooth muscle cells but whether it has the same effect on megakaryocytes remains unclear. In this study, we investigated the effect of serotonin on megakaryocytopoiesis and the possible mechanism of its effect using the mouse plasma clot culture method. The results show that: (a) serotonin significantly stimulates megakaryocyte colony formation with maximum stimulation at 100 nM; (b) enhanced action is found between serotonin and interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) and PDGF; (c) ketanserin, a 5-HT2 receptor antagonist, blocks the mitogenic effect of serotonin on megakaryocytopoiesis; and (d) Meg-01 cells (a megakaryocyte cell line) express 5-HT2 receptors. This study demonstrates that serotonin has a mitogenic effect on megakaryocytopoiesis and this effect may be mediated via the 5-HT2 receptor which is known to be coupled to G protein. It is suggested that serotonin may also be involved in the feedback control of megakaryocytopoiesis.

Animals↗
PubMed1996

Activated protein C resistance--low incidence in glaucomatous optic disc haemorrhage and central retinal vein occlusion.

PURPOSE: Activated protein C (APC) resistance has recently been reported as conferring a sevenfold increase in the risk of venous thrombosis. It is linked to a genetic mutation in the factor V gene which occurs commonly (about 2% to 4% of the community have the mutation). Glaucoma patients with nerve fibre layer (NFL) haemorrhages on the optic disc and patients with central retinal vein occlusion (CRVO) were tested for APC resistance to determine if there was an association. METHODS: Twenty-three patients with glaucomatous NFL haemorrhages and 23 patients with CRVO were tested. The CRVO cases included 11 with relatively young age of onset (mean 45.1 +/- 6.9 years) without conventional vascular risk factors. Eighty randomly selected Red Cross blood donor samples and 33 staff members were tested as controls. Clotting times with and without exogenous APC were recorded and an APC ratio determined. Cases with APC resistance were tested to confirm that they had the factor V Leiden gene. RESULTS: No cases of APC resistance were identified in the glaucoma patients and only one of the younger CRVO patients tested positive, but four of 113 controls tested positive. The difference in prevalence between groups is not significant. The mean APC ratios for the three groups were very similar: NFL haemorrhages 5.46(+/- 1.62), CRVO 5.70(+/- 1.56), controls 5.34 ( +/- 1.19) p > 0.5. CONCLUSION: There was not clear association detected between glaucomatous NFL haemorrhages or CRVO and APC resistance in this sample of patients. This negative finding is important due its known association with venous thrombosis elsewhere in the body.

Adult↗
PubMed1996

Anticoagulation.

This review of anticoagulation discusses not only drugs that inhibit the formation of blood clots but also drugs that induce clot lysis. It includes warfarin, heparin and streptokinase as well as newer agents such as low molecular weight (LMW) heparins and recombinant tissue plasminogen activator (r-tPA).

Aged↗
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

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↗
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↗
PubMed1994

Plasma P-selectin is increased in thrombotic consumptive platelet disorders.

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P-selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P-selectin may be a useful new marker for thrombotic diseases.

Adult↗
PubMed1994

Clinical aspects of thrombocytopenias.

Severe thrombocytopenia is a common cause of bleeding. It may result from decreased platelet production by the bone marrow or increased peripheral platelet destruction-sequestration. In a patient with thrombocytopenia, it is important to identify the underlying cause by careful clinical evaluation and laboratory investigations so that appropriate treatment can be administered.

Adult↗
PubMed1993

Anticardiolipin antibodies block the inhibition by beta 2-glycoprotein I of the factor Xa generating activity of platelets.

Antiphospholipid antibodies, defined either by lupus anticoagulant (LA) activity or positive anticardiolipin immunoabsorbent assay (ACA) are associated with a predisposition to thromboses, recurrent fetal loss or thrombocytopenia. The mechanisms for these predispositions remain undefined. We have enriched immunoglobulin fractions from two patient plasmas to obtain antibodies with LA activity but no ACA, or conversely, with ACA positivity but no LA, in order to investigate in vitro characteristics which might explain a thrombotic propensity. beta 2-glycoprotein I (beta 2-GPI), the plasma cofactor required for ACA binding to negatively charged phospholipid, has previously been shown to inhibit prothrombinase generation in the presence of activated platelets (8). We now report that beta 2-GPI, at physiological concentrations, inhibits the generation of factor Xa in the presence of activated gel-filtered platelets. Further, ACA interferes with this inhibition, resulting in protracted, unopposed factor Xa generation. This interference with beta 2-GPI, a natural anticoagulant component of plasma, is potentially prothrombotic. LA immunoglobulins behave differently and inhibit factor Xa generation in a manner similar to beta 2-GPI. These findings provide the basis for a previously unsuspected mechanism for thrombosis in patients with aPL.

Adult↗
PubMed1993

Uses of heparin.

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Drug Administration Schedule↗
PubMed1993

The clinical usefulness of the platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia.

The platelet aggregation test is widely used for the diagnosis of heparin-induced thrombocytopenia (HIT), a potentially serious complication of heparin therapy. We have evaluated its sensitivity and specificity in comparison with those of the 14C-serotonin release test. The sensitivity of the platelet aggregation test was found to vary with the heparin concentration and the donor of the platelets used in the test. The optimal heparin concentrations were between 0.1 and 1.0 U/ml. Using these heparin concentrations, the mean sensitivity varied from 39% (with the least reactive platelets) to 81% (with the most reactive platelets). In comparison, the sensitivity of the release test ranged from 65% to 94%. The specificities of the platelet aggregation test were 82%, 90% and 100% for the following control groups: (1) non-thrombocytopenic patients given heparin, (2) patients with thrombocytopenia due to other causes, and (3) normal controls not given heparin, respectively. The corresponding specificities for the release test was 94%, 90% and 100%. The specificities can be further increased to 100% for all controls with the adoption of a two-point system which defines a positive result as one in which platelet aggregation occurs with a low heparin concentration (0.5 U/ml) but not with 100 U heparin/ml. For optimal results, a two-point platelet aggregation test should be performed with heparin concentrations of 0.5 and 100 U/ml and using platelets of more reactive donors.

Autoimmune Diseases↗
PubMed1993

Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants.

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid-dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]-iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]-ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2-GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

Adult↗