Biomedical subjects
Publications and source records attributed to B H Chong.
We sought to test the hypothesis that intracoronary blood sampling in patients with angiographically demonstrated coronary artery disease could be performed using the multifunction probing catheter (Schneider, Bulach, Switzerland), without causing ex vivo platelet activation.
Patients with immune heparin-induced thrombocytopenia (HIT) possess antibodies that bind to a complex of platelet factor 4 (PF4) and heparin. We observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, we are able to provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd = 7-30 nM) and polystyrene adsorbed PF4 alone (Kd = 20-70 nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. We conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and therefore most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (suboptimal) promotes recognition of this epitope.
OBJECTIVE: To investigate that ginsenosides (GS) promotes proliferation of hematopoietic cells by virtue of the intracellular signaling pathway. METHODS: Three human megakaryocytic cell lines and Northern blot analysis, electrophoretic mobility shift assay (EMSA), antibody gel supershift assay and UV-radiation cross-linking were used. RESULTS: After treatment with GS, GATA-2 mRNA levels in CHRH-288, Meg-01 and MO7e cells were increased by 1.84-, 2.43- and 1.52-fold, respectively, while GATA-1 mRNA levels remained as low as pretreatment. GATA binding activities were elevated in all the three GS treated cell lines. The GATA binding complex appeared as a band with an approximate molecular weight of 50,000 corresponding to the size of GATA-2 protein. CONCLUSIONS: The intracellular signaling pathway initiated by GS was involved in transcription factor GATA-2. GATA-2 might play the key role in the GS induced upregulation of megakaryopoiesis.
Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to characterize the binding site of GPIb-IX-specific quinine-dependent antibodies. Antibody binding to Chinese hamster ovary cells or mouse L cells stably transfected with various combinations of the three genes (Ibalpha, Ibbeta, or IX) that encode this complex was detected using flow cytometry, monoclonal antibody-specific immobilization of platelet antigens assay, and differential adsorption studies. IgG in sera from 15 patients with quinine-induced thrombocytopenia binding to the cells, in the presence of quinine, showed three distinct patterns. Group 1 sera contained at least two antibody populations, one which binds to GPIbalpha and another which recognizes GPIX. Group 2 sera contained an antibody which binds drug dependently to GPIX, and Group 3 sera contained an antibody which recognizes a quinine-dependent epitope on GPIbalpha. Thus, the quinine-dependent antibodies fall into two distinct populations that bind to GPIbalpha and GPIX independently. Using proteases which cleave GPIbalpha at specific sites, we have shown that the GPIbalpha-specific antibody binds to an 11-amino acid (283 to 293) region. Peptide inhibition studies provide confirmatory evidence that this region contains the epitope for the GPIbalpha-specific quinine-dependent antibody.
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Early diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.
Heparin-induced thrombocytopenia (HIT) type II is an immune-mediated reaction that generally occurs 5 to 14 days after initiation of heparin therapy. It is characterized by a severe decline in platelet count (by >50%) in association with a new thromboembolic complication. Type II HIT is mediated by antibodies, usually of the IgG class. The antibodies cause platelet activation in the presence of heparin or other polysulfated saccharides. The antigen in type II HIT frequently is a complex of platelet factor 4 (PF4) and heparin. In addition to stimulating platelet activation, type II HIT antibodies bind to PF4/heparin complexes on the surface of endothelial cells, leading to activation of the cells. Concomitant activation of platelets and endothelial cells together with enhanced thrombin generation are likely causes for the thromboembolic events. The percentage of patients receiving heparin who develop type II HIT antibodies is higher than the percentage of patients who develop clinical symptoms characteristic of type II HIT. Therefore, it is currently not indicated to screen asymptomatic patients for the development of HIT antibodies. If clinical symptoms of HIT appear, the clinical diagnosis should be confirmed, when possible, by a laboratory test for HIT antibodies. Assays for detection of HIT antibodies are either functional assays, which measure heparin-dependent platelet activation, or antigen assays based on the enzyme-linked immunosorbent assay (ELISA) technique. A reasonable approach to applying these laboratory tests is to use one assay technique as a screening test and reserve the other for testing samples that had negative screening results when the diagnosis of HIT is strongly suspected.
A rapidly acting anticoagulant that can either inhibit thrombin generation or inhibit thrombin itself is the optimum therapy for acute thrombosis associated with heparin-induced thrombocytopenia (HIT). In this review, the newer treatment approaches that fulfill this requirement are discussed. These newer treatments include hirudin and argatroban, direct thrombin inhibitors, and danaparoid, which inhibits thrombin generation. Preliminary outcome results from the extensive compassionate-use program for danaparoid in HIT and from a recently completed randomized clinical trial that compared danaparoid with dextran in patients with HIT are provided. Based on these data, danaparoid appears to be a useful and safe replacement for heparin in patients who develop HIT.
Thrombopoietin (TPO) is the primary hematopoietic growth factor involved in the regulation of platelet production. Although the kidney, liver, bone marrow (BM), and spleen have been identified as the major sources of TPO production, the precise cellular location of TPO mRNA expression in these tissues remains unknown. We have identified the cells expressing TPO mRNA in the human kidney, liver, and BM using an in situ hybridization assay. In the BM of individuals with normal platelet counts, the hybridization signal was too weak to allow identification of the TPO mRNA expressing cells. However, in thrombocytopenic subjects with aplastic anemia, postchemotherapy marrow aplasia, and immune thrombocytopenia, the stromal cells showed strong TPO mRNA expression. In the human subjects with normal platelet counts, the cells of the proximal convoluted tubules of the kidney showed consistent positive staining whereas the signal in the cells of the distal convoluted tubules was less consistent. Strong hybridization signal was also evident in the hepatocytes. The hybridization signal in the spleen, even in thrombocytopenic subjects, was too weak to allow confident identification of the cells expressing TPO mRNA. In all subjects, the interstitial cells and endothelial cells of the liver and spleen, the renal peritubular cells, and the hematopoietic precursor cells of the BM showed no TPO mRNA expression. Our data suggest that TPO mRNA expression in the human BM may be modulated by platelet mass.
Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose-dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.
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Thrombocytopenia is a common finding in malaria. In clinical trials, recombinant macrophage colony-stimulating factor (M-CSF) causes a reversible, dose-dependent thrombocytopenia, and high M-CSF has been reported in autoimmune thrombocytopenias. P-selectin, which is secreted into the plasma following platelet/endothelial activation or damage, is elevated in certain consumptive thrombocytopenic disorders. The relationships between thrombocytopenia, M-CSF and P-selectin were analysed in 63 patients with severe (n = 13) or uncomplicated (n = 26) P. falciparum (PF) or P. vivax (PV) malaria (n = 24). On admission, 69% of PF patients and 75% of PV patients were thrombocytopenic (platelets < 150 x 10(9)/l). M-CSF was elevated in PF (3021 +/- 1844 pg/ml) and PV (2602 +/- 1668 pg/ml) patients, compared to controls (589 +/- 200 pg/ml). The platelet count was inversely correlated with M-CSF in PF (r = -0.681), and in PV malaria (r = -0.548). Elevated P-selectin was found in severe PF malaria, but not in PV malaria. Severe PF malaria was associated with marked thrombocytopenia, very high M-CSF, elevated P-selectin and compelling evidence of disseminated intravascular coagulopathy (DIC). Platelet counts, M-CSF and P-selectin returned to control values in 7-14 days. These data suggest that elevated M-CSF in malaria, by enhancing macrophage activity, may result in increased macrophage-mediated platelet destruction. Further, platelet/endothelial activation or damage, as measured by P-selectin, or DIC could intensify thrombocytopenia in severe PF malaria, but does not appear to contribute to thrombocytopenia in uncomplicated PF or PV malaria.
Platelet-derived growth factor (PDGF) is a potent chemotactic and mitogenic factor implicated to play important roles in a variety of normal and pathophysiologic settings. We investigated PDGF receptor expression on human megakaryocytes and several megakaryocytic cell lines (CHRF, DAMI, Meg-01, M-07e) using enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical staining. Both PDGF receptor subtypes were identified on CHRF, DAMI, and Meg-01 cells by ELISA; PDGF beta-receptor levels exceeded alpha-receptor levels. Flow cytometry revealed that beta-receptor levels on CHRF and DAMI cells exceeded those on Meg-01 cells, and that M-07e expressed neither receptor. Immunocytochemical staining confirmed these findings and determined that bone marrow megakaryocytes also expressed PDGF receptors. Exposure of megakaryocytes to PDGF-BB dramatically induced the expression of the immediate-early gene, c-fos, within 30 min. Moreover, PDGF-BB significantly stimulated CHRF proliferation and colony formation. The present study demonstrates the presence of functional PDGF receptors on human megakaryocytes and their ability to mediate a mitogenic response.
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.
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Previous studies suggested that the cross-reactivity rates of low molecular weight (LMW) heparins with the antibody in heparin-induced thrombocytopenia (HIT) are more than 80%, whilst that of the LMW heparinoid (Orgaran) is relatively low at about 10%. These earlier studies were limited either in the number of patients studied, or in investigating only a single drug. They were also inadequate due to non-standardisation of testing conditions. This study compares three LMW heparins (Fragmin, Clexane, Fraxiparin) and a heparinoid (Orgaran) in their cross-reactivity rates with the HIT antibody. The sera of 45 HIT patients were tested using platelet aggregometry under standardised conditions. The cross-reactivity rates are: 7% (Orgaran), 89% (Fragmin), 83% (Clexane) and 86% (Fraxiparin). Although there are no controlled trials to determine the in vivo cross-reactivity rates of LMW heparins and heparinoid, the low in vitro cross-reactivity rate of the latter favours its use in HIT. Nevertheless, the therapeutic use of these drugs in HIT should be preceded by exclusion of in vitro cross-reactivity.
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.
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.