Clinical relevance of nitrated beta 2-glycoprotein I in antiphospholipid syndrome: Implications for thrombosis risk.

Β2-Glycoprotein I (ß2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of ß2GPI was examined.The effects of nitrated (n)ß2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. ß2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. ß2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated ß2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nß2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma ß2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nß2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.

Presentation and outcome of idiopathic thrombocytopenic purpura in a single Australian centre.

Immune thrombocytopenia can be a therapeutic challenge with multiple first- and second-line treatment options. A change in idiopathic thrombocytopenic purpura (ITP) definition and classification in recent consensus guidelines suggests that past descriptions of ITP presentation and outcome may be outdated. In this single centre retrospective analysis of patients with thrombocytopenia from 1 January 2005 to 1 June 2010, 139 patients met current ITP diagnostic criteria. About 54/139 were new presentations of primary ITP. Six- and 24-month response rates were 39% and 30% respectively. About 26/54 patients did not require treatment at presentation: 15 were followed up for at least 6 months and none required treatment subsequently. These results suggest that almost half of all new primary ITP do not need treatment.

Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis.

INTRODUCTION: Neutrophil extracellular traps (NETs) are networks of extracellular fibres produced from neutrophil DNA with a pathogenic role in infection, thrombosis and other conditions. Reliable assays for measuring NETs are desirable as novel treatments targeting NETs are being explored for the treatment of these conditions. We compare a whole blood flow cytometry method with serum assays to measure NETs-associated markers in patients with sepsis and thrombosis. METHODS: Patients with deep venous thrombosis (n = 25), sepsis (n = 21) and healthy controls (n = 23) were included in the study. Neutrophil surface NETs markers were determined by flow cytometry on whole blood samples by gating of neutrophils stained for surface citrullinated histone (H3cit) and myeloperoxidase (MPO). Serum double-stranded (ds) DNA, MPO, myeloid-related protein, nucleosomes, DNAse, elastase, human high-mobility group box 1 and MPO-DNA complexes were quantified as circulating markers of NETs. RESULTS: Neutrophil NETs markers by flow cytometry and serum NETs markers were significantly higher in patients with thrombosis and sepsis compared with healthy controls. Neutrophil NETs markers significantly correlated with the serum marker dsDNA. CONCLUSION: Flow cytometry detection of neutrophil NETs markers is feasible in whole blood and correlates with serum markers of NETs. We propose the flow cytometry detection of MPO/H3cit positive neutrophils and serum dsDNA as simple methods to quantify cellular and extracellular NET markers in patients with thrombosis and sepsis.

Hematologic correlates of left atrial spontaneous echo contrast and thromboembolism in nonvalvular atrial fibrillation.

OBJECTIVES: This study examined the relation between left atrial spontaneous echo contrast, hematologic variables and thrombo-embolism in patients with nonvalvular atrial fibrillation. BACKGROUND: Left atrial spontaneous echo contrast is associated with left atrial stasis and thromboembolism in patients with nonvalvular atrial fibrillation. However, its hematologic determinants in patients with nonvalvular atrial fibrillation are unknown. METHODS: Clinical, hematologic and echocardiographic variables were prospectively measured in 135 consecutive patients with nonvalvular atrial fibrillation undergoing transesophageal echocardiography. RESULTS: Patients with left atrial spontaneous echo contrast (n = 74, 55%) had an increased fibrinogen concentration (p = 0.029), platelet count (p = 0.045), hematocrit (p = NS) and left atrial dimension (p = 0.005). Multivariate analysis showed that left atrial spontaneous echo contrast was independently related to hematocrit (odds ratio = 2.24, p = 0.002), fibrinogen concentration (odds ratio = 2.08, p = 0.008) and left atrial dimension (odds ratio = 1.90, p = 0.004) but not platelet count. It was also associated with left atrial thrombus (n = 15, p = 0.001) and with recent embolism (n = 40, p < 0.001). In 40 clinically stable outpatients without previous embolism, left atrial spontaneous echo contrast was significantly related to hematocrit (p = 0.005), fibrinogen concentration (p = 0.035) and left atrial dimension (p = 0.029) but not to coagulation factor VII, D-dimer, erythrocyte sedimentation rate, platelet count, plasma beta-thromboglobulin, plasma glycocalicin or glycocalicin index. CONCLUSIONS: Left atrial spontaneous echo contrast in patients with nonvalvular atrial fibrillation is independently related to hematocrit, fibrinogen concentration and left atrial dimension, indicating a relatively hypercoagulable state in addition to stasis. These findings support the hypothesis that left atrial spontaneous echo contrast is due to erythrocyte aggregation. Hematologic factors may contribute to its association with thromboembolism.

Autoimmune thrombocytopenia.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets coated with mainly antibodies against platelet GPIIb/IIIa and GPIb/IX are destroyed in the spleen. Recent evidence suggests that platelets are also destroyed by cytotoxic T cells. The diagnosis is made by exclusion for other causes of thrombocytopenia. As routine blood counts are becoming more available, many mild cases of ITP (platelets >30 x 10(9) L(-1)) are being diagnosed and they usually do not require treatment. In patients with platelet counts persistently <30 x 10(9) L(-1), treatment with corticosteroids, and/or intravenous immunoglobulin (IVIG) or anti-D may be required. The primary goal of treatment is to maintain the platelet count at a safe level with minimal side effects. After 3-6 months, if spontaneous remission has not occurred and if the side effects are significant, splenectomy is recommended. This is the single most effective treatment of ITP. The refractory patients who fails splenectomy and subsequently first- and second-line therapies, is a management dilemma. Therapeutic options are limited, available treatments potentially toxic and the chances of sustained response low. Observation with no active treatment is a reasonable option. With the increased availability of the thrombopoietic agents in the future, there may be a good prospect of keeping the platelet counts of these refractory patients at a safe long-term level with one of these drugs.

Activation of tumour cell ECM degradation by thrombin-activated platelet membranes: potentially a P-selectin and GPIIb/IIIa-dependent process.

The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of >2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

Heparin-induced thrombocytopenia.

Thrombocytopenia is a common adverse effect of heparin therapy which may occur with bovine or porcine heparin when it is given either intravenously or subcutaneously. There are two main clinical types: a common, mild thrombocytopenia of early onset, probably due to the platelet proaggregating effect of heparin itself and a severe delayed-onset thrombocytopenia caused by an immune mechanism. Patients with mild thrombocytopenia due to heparin are usually asymptomatic. However, patients with severe thrombocytopenia may develop thromboembolic complications which often result in disastrous sequelae such as limb gangrene necessitating amputation or death. The thromboembolic complications may be attributed to an IgG heparin-dependent platelet antibody which induces thromboxane production and platelet aggregation. The diagnosis of heparin-induced thrombocytopenia is made clinically and may be confirmed by the demonstration of the heparin-dependent antibody in vitro by platelet aggregometry or [14C] serotonin release. In patients with delayed-onset severe thrombocytopenia, heparin should be stopped and warfarin commenced. Antiplatelet drugs and/or dextran may also be added. Recently low molecular weight heparin has been used with some success. Conversely, heparin may be continued in patients with asymptomatic mild thrombocytopenia provided the patients' condition and the platelet counts are closely monitored.

Advances in the diagnosis of idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) remains a clinical diagnosis made by the exclusion of other causes of thrombocytopenia. It is based on the patient's history, physical examination, and complete blood cell count, as well as examination of the blood film. Over the last four decades, a number of platelet antibody tests have been developed to aid the diagnosis of ITP. They can be divided chronologically into three groups. Phase I assays measure a functional change in control platelets after incubation with test serum. Because their sensitivity and specificity are low, they are no longer used to diagnose ITP. Phase II assays measure platelet-associated IgG by three different approaches. They lack the ability to differentiate between pathologic and nonpathologic platelet-associated IgGs. These assays are sensitive (80% to 90%) but their specificity is too low for them to be diagnostically useful. Phase III assays are the latest development in platelet serology testing. They measure glycoprotein-specific platelet antibodies by different approaches, namely, immunoblot, immunoprecipitation, and glycoprotein immobilization. Despite their high specificity, they suffer from low sensitivity (47% to 60%), which must be improved if they are to be clinically useful for the diagnosis of ITP.

Pathophysiology and laboratory testing of heparin-induced thrombocytopenia.

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.

New treatment options for heparin-induced thrombocytopenia.

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.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is not only a common but also a potentially serious drug adverse effect. Unlike other drug-induced thrombocytopenias, HIT does not usually cause bleeding, but instead causes thrombosis. Thrombosis in HIT can lead to limb gangrene (requiring leg amputation) or even death. HIT is mediated by an antibody that recognizes an epitope on the platelet factor 4 (PF4)-heparin complex. The antibody-PF4-heparin complex binds to FcgammaRII receptors on the platelet surface and cross-links the receptors. This induces intense platelet activation and platelet aggregation, and simultaneously activates blood-coagulation pathways. These changes are probably the basis of the thrombotic events in HIT. Diagnosis of HIT should be made mainly on clinical criteria but should be confirmed whenever possible by laboratory tests, particularly in patients with comorbid conditions, in whom the diagnosis of HIT cannot be made with certainty without testing. The tests for HIT antibodies are either immunoassays (e.g. ELISA), or functional tests, (e.g. 14C-serotonin release assay). Once a clinical diagnosis of HIT is made, heparin should be ceased immediately and treatment with an alternative anticoagulant (such as danaparoid, r-hirudin or argatroban) commenced. This should continue for at least 5 days unless the diagnosis of HIT is subsequently proven to be incorrect. Warfarin should also be commenced when the patient is clinically stable and thrombosis is under control. There should be an overlap of a few days between warfarin and the alternative anticoagulant therapy.

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.

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.

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.

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.

Heparin-induced thrombocytopenia: IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid.

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.

Identification of PDGF receptors on human megakaryocytes and megakaryocytic cell lines.

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.

Plasma macrophage colony-stimulating factor and P-selectin levels in malaria-associated thrombocytopenia.

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.

Prospective randomised open-label comparison of danaparoid with dextran 70 in the treatment of heparin-induced thrombocytopaenia with thrombosis: a clinical outcome study.

AIM: To compare clinical outcomes in a randomised comparison of treatment with danaparoid sodium (a heparinoid), or dextran 70, for heparin-induced thrombocytopaenia (HIT) plus thrombosis. METHODS: Forty-two patients with recent thrombosis and a clinical diagnosis of probable HIT who presented at ten Australian hospitals during a study period of six and one half years were randomly assigned to open-label treatment with intravenous danaparoid or dextran 70, each combined with oral warfarin. Thirty-four patients (83%) had a positive platelet aggregation or 14C-serotonin release test for HIT antibody. Twenty-five received danaparoid as a bolus injection of 2400 anti-Xa units followed by 400 units per hour for 2 h, 300 units per hour for 2 h, and then 200 units per hour for five days. Seventeen received 1000 mL dextran 70 on day one and then 500 mL on days 2-5. Patients were reviewed daily for clinical evidence of thrombus progression or resolution, fresh thrombosis or embolism, bleeding or other complications. The primary trial endpoint was the proportion of thromboembolic events with complete clinical resolution by the time of discharge from hospital. RESULTS: With danaparoid, there was complete clinical recovery from 56% of thromboembolic events compared to 14% after dextran 70 (Odds Ratio 10.53, 95% Confidence Interval 1.6-71.4; p = 0.02). Clinical recovery with danaparoid was complete or partial in 86% of thromboembolic events compared with 53% after dextran 70 (Odds Ratio 4.55, 95% Confidence Interval 1.2-16.7; p = 0.03). Overall clinical effectiveness of danaparoid was rated as high or moderate in 88% of patients compared with 47% for dextran 70 (p = 0.01). One patient given danaparoid died of thrombosis compared with three patients given dextran 70. The platelet count returned to normal after a mean of 6.7 days with danaparoid and 7.3 days with dextran 70. There was no major bleeding with either treatment. CONCLUSION: danaparoid plus warfarin treatment for HIT with thrombosis is effective, safe, and superior to dextran 70 plus warfarin.

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.