Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma.

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03-1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.

Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia.

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.

Overexpression of chemokine receptor CXCR2 and ligand CXCL7 in liver metastases from colon cancer is correlated to shorter disease-free and overall survival.

Our aim was to analyze the potential role of chemokine receptors CXCR2 and CXCR4 signalling pathways in liver metastatic colorectal cancer (CRC) relapse. CXCR2, CXCR4, and their chemokine ligands were evaluated in liver metastases of colorectal cancer in order to study their correlation with overall and disease-free survival of patients having received, or not received, a neoadjuvant chemotherapy regimen. Quantitative RT-PCR and CXCR2 immunohistochemical staining were carried out using CRC liver metastasis samples. Expression levels of CXCR2, CXCR4, and their ligands were statistically analyzed according to treatment with neoadjuvant chemotherapy and patients' outcome. CXCR2 and CXCL7 overexpression are correlated to shorter overall and disease-free survival. By multivariate analysis, CXCR2 and CXCL7 expressions are independent factors of overall and disease-free survival. Neoadjuvant chemotherapy increases significantly the expression of CXCR2: treated group 1.89 (0.02-50.92) vs 0.55 (0.07-3.22), P = 0.016. CXCL7 was overexpressed close to significance, 0.40 (0.00-7.85) vs 0.15 (0.01-7.88), P = 0.12. We show the involvement of CXCL7/CXCR2 signalling pathways as a predictive factor of poor outcome in metastatic CRC. 5-Fluorouracil-based chemotherapy regimens increase the expression of these genes in liver metastasis, providing one explanation for aggressiveness of relapsed drug-resistant tumors. Selective blockage of CXCR2/CXCL7 signalling pathways could provide new potential therapeutic opportunities.

iTRAQ-based proteomic profiling of human serum reveals down-regulation of platelet basic protein and apolipoprotein B100 in patients with hematotoxicity induced by chronic occupational benzene exposure.

Benzene is an important industrial chemical and an environmental contaminant, but the pathogenesis of hematotoxicity induced by chronic occupational benzene exposure (HCOBE) remains to be elucidated. To gain an insight into the molecular mechanisms and developmental biomarkers for HCOBE, isobaric tags for relative and absolute quantitation (iTRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) were utilized. Identification and quantitation of differentially expressed proteins between HCOBE cases and healthy control were thus made. Expressions of selected proteins were confirmed by western blot and further validated by ELISA. A total of 159 unique proteins were identified (≥95% confidence), and relative expression data were obtained for 141 of these in 3 iTRAQ experiments, with fifty proteins found to be in common among 3 iTRAQ experiments. Plasminogen (PLG) was found to be significantly up-regulated, whereas platelet basic protein (PBP) and apolipoprotein B100 (APOB100) were significantly down-regulated in the serum of HCOBE cases. Additionally, the altered proteins were associated with the molecular functions of binding, catalytic activity, enzyme regulator activity and transporter activity, and involved in biological processes of apoptosis, developmental and immune system process, as well as response to stimulus. Furthermore, differential expressions of PLG, PBP and APOB100 were confirmed by western blot, and the clinical relevance of PBP and APOB100 with HCOBE was validated by ELISA. Overall, our results showed that lowered expression of PBP and APOB100 proteins served as potential biomarkers of HCOBE, and may play roles in the benzene-induced immunosuppressive effects and disorders in lipid metabolism.

Experimental thermal lesions induce beta-thromboglobulin release from activated platelets.

Time courses of beta-thromboglobulin release, and protein extravasation after thermal inflammation of human skin was compared to neurogenic inflammation induced by histamine iontophoresis. Beta-TG and protein levels were sampled by intradermal microdialysis. Three microdialysis membranes were inserted at each stimulation site. The collected samples were measured photometrically for protein by the Coomassie blue method and for beta-TG by ELISA. Heat stimuli of 40°C and 47°C, and histamine iontophoresis were inflicted directly above the membranes. In vitro recovery rates of beta-TG and bovine albumin were measured using a latex chamber. Recovery rates at a continuous flow rate of 3 µl/min for bovine albumin ranged between 20% and 35%; those for beta-TG ranged between 14% and 17%. Heat stimulation at 40°C showed a slight but insignificant increase of beta-TG during the stimulation period. Temperatures of 47°C produced a significant, long-lasting increase of beta-TG, whereas histamine iontophoresis did not. Protein extravasation corresponded with beta-TG release; a long-lasting significant increase during and after the burn lesion, and only an initial increase of protein extravasation during the 40°C heat stimulus. Long lasting heat stimuli to the skin induced beta-TG release from platelets, whereas histamine iontophoresis although provoking protein extravasation and neurogenic flare, did not. Using microdialysis techniques we detected beta-TG release during an inflammatory response. We conclude that local platelet activation is induced by the heating stimulus. Platelet mediators, such as beta-TG might contribute to the subsequent inflammatory process which is also characterized by mechanical and heat hyperalgesia.

Increased concentration of platelet-derived chemokines in serum of patients with delayed pressure urticaria.

BACKGROUND: Delayed pressure urticaria (DPU) is a distinct form of urticaria, characterized by marked dermal swelling, deep inflammatory infiltrate and systemic symptoms. Little is known about inflammatory mediators involved in this disease. OBJECTIVE: To investigate secretion of platelet-specific chemokines, platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG) during the course of DPU. METHODS AND MATERIAL: Plasma concentrations of PF-4 and beta-TG were measured in eight adult DPU patients and in 15, age- and sex-matched, healthy controls. RESULTS: Plasma PF-4 and beta-TG concentration scores were significantly higher in the DPU group as compared with the control subjects. CONCLUSION: The present study, as well as an earlier contribution, suggest that distinct platelet activity may be identified in different types of urticaria. In contrast to chronic idiopathic urticaria, chronic urticaria with a positive response to autologous serum skin testing, and acute urticaria, delayed pressure urticaria may be associated with increased secretion of platelet chemokines, similar to that observed in cold urticaria.

Inflammatory response and platelet activation after off-pump coronary artery bypass surgery.

BACKGROUND: Cardiac surgery induces a systemic inflammatory activation and alterations in the hemostatic cascade. The responses contribute to postoperative complications but may also have protective effects. We investigated the relationship between inflammation, hemostasis and bleeding after off-pump coronary artery bypass surgery (OPCAB). METHODS: Ten OPCAB patients were included in a prospective descriptive study. Selected markers of inflammation (IL-6, IL-8, PMN-elastase, C3a, and SC5b-9), and hemostasis (platelet count, ss-thromboglobulin, anti-thrombin, D-dimer and fibrinogen) were measured before and immediately after surgery. Postoperative bleeding was registered. RESULTS: Inflammatory variables did not alter significantly during surgery while ss-thromboglobulin concentrations increased and anti-thrombin and fibrinogen decreased. There were significant postoperative correlations between PMN-elastase and ss-thromboglobulin (r=0.82, p=0.004), between PMN-elastase and fibrinogen (r=0.69, p=0.03) and between C3a and ss-thromboglobulin (r=0.71, p=0.02). In addition, there were significant inverse correlations between postoperative bleeding and pre- and postoperative fibrinogen levels (r=-0.76, p=0.011 and r=-0.84, p=0.002 respectively), between bleeding and postoperative ss-thromboglobulin levels (r=-0.66, p=0.04) and between bleeding and postoperative PMN-elastase (r=-0.75, p=0.01). CONCLUSIONS: The results give further evidence for an association between the inflammatory response and hemostasis after cardiac surgery.

Monocyte-derived CXCL7 peptides in the marrow microenvironment.

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The recombinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty.

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1beta, IL-8, beta-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.

Platelet activity measured by plasma levels of beta-thromboglobulin and platelet factor 4 in seasonal allergic rhinitis during natural pollen exposure.

BACKGROUND: Upon activation, platelets release mediators with potent inflammatory properties in IgE-mediated immune responses. Moreover, the atopic state leads towards functional abnormalities of these cells. OBJECTIVE: The aim of our study was to examine the degree of activation of circulating platelets in patients with seasonal allergic rhinitis (SAR) during the symptomatic period, to improve the understanding of platelet function in atopy. SUBJECTS AND METHOD: Plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) - specific markers of platelet activation were measured by enzyme-linked immunoassay in 20 patients suffering from SAR and in 15 healthy, nonatopic subjects. RESULTS: There were no significant differences in peripheral blood platelet numbers and plasma levels of beta-TG and PF4 in SAR patients when compared with control subjects. CONCLUSION: It seems that increased in vivo platelet activity, assessed by measuring plasma beta-TG and PF4, may not occur during allergic inflammation that is associated with SAR.

Prospective double-blind randomized study of the effects of four intravenous fluids on platelet function and hemostasis in elective hip surgery.

A prospective randomized double-blind study was performed to determine the effects of three colloids, Haemaccel, Gelofusine and albumin, and also saline on platelet activation, platelet aggregation (induced by adenosine diphosphate (ADP), epinephrine, collagen) platelet agglutination by ristocetin and other hemostatic variables in 55 patients undergoing primary unilateral total hip replacement. The fluids were administered according to normal clinical practice and assessments were made immediately before, at the end, and 2 h after the end of surgery. Surgery was accompanied by thrombin generation (increases in thrombin/antithrombin III complex, prothrombin F1 +2 fragment) platelet activation (betaTG) and compromised coagulation. Generally, the platelet activation appeared to result in platelet desensitization and brought about a persistent reduction in platelet aggregation to ADP and epinephrine, irrespective of the fluid used. Additionally, Haemaccel and Gelofusine inhibited ristocetin-induced platelet agglutination and albumin inhibited collagen-induced platelet aggregation. Gross inhibitory effects of Haemaccel that had been predicted from an earlier in vitro study did not occur. Particular fluids had selective additional effects on the hemostatic system. Albumin infusion served to maintain plasma albumin at normal concentrations postsurgery. The two gelatin preparations, Haemaccel and Gelofusine, maintained plasma viscosity. All three colloids led to a transient increase in activated partial thromboplastin time postsurgery and also a transient fall in the concentration of factor VIII, which were accompanied by a transient increase in bleeding time, but there was no measurable increase in blood loss. Inhibition of platelet aggregation by certain colloids may provide additional protection against the increased thrombotic risk in patients following major surgery.

Protease action and generation of beta-thromboglobulin-like protein followed by platelet activation.

Beta-thromboglobulin (beta-TG) is a platelet-specific protein present in the alpha-granules and secreted into the surrounding medium on cell activation. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of platelet releasate after inhibition of metalloproteinases with ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetra acetic acid (EGTA) showed disappearance of an 8.0-kDa band. In the absence of the cation chelators, a 48-kDa band disappeared and concurrently, the 8.0-kDa band intensity increased suggesting that the former may be the immediate precursor of the latter. The Western blot stained using specific antibodies, isolated from single-cell clones of hybridoma, against 8.0 kDa protein recognized not only 48 and 8.0 kDa bands but few others too. The data suggests that one or more high molecular weight protein is released from alpha-granules and is broken down into smaller fragments after release to form beta-thromboglobulin (beta-TG)-like proteins by the action of metal dependent proteases.

Chemokine production by human megakaryocytes derived from CD34-positive cord blood cells.

To clarify the roles of megakaryocytes and platelets in the responses associated with infection and inflammation, we examined the effects of interleukin (IL) 1, the common mediator of the inflammatory process, on the development and secretory functions of megakaryocytes generated from CD34(+)cord blood cells under stimulation with thrombopoietin (TPO). The addition of IL-1alpha did not influence the generation, endomitosis or expression of surface makers of megakaryocytes, compared with TPO alone. However, IL-1alphaenhanced the ability of megakaryocytes to produce IL-8 and growth-regulating oncogene-alpha(GRO-alpha) in the presence of TPO. In contrast, the production of regulated on activation with normal T cell expressed and secreted (RANTES), platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were not potentiated. A flow cytometric analysis and a reverse transcription-polymerase chain reaction analysis revealed IL-1 receptor type I (IL-1RI) expression of megakaryocytes generated by TPO. Moreover, the addition of an anti-IL-1RI monoclonal antibody significantly decreased the TPO plus IL-1alpha-induced secretion of IL-8 by the cultured megakaryocytes to the level attained by TPO alone. These results suggest that the production of IL-8 and GRO-alpha (but not RANTES), PF4 and beta-TG, by megakaryocytes is potentiated by signalling through IL-1RI with the aid of TPO. Thus, megakaryocytes and platelets may play an important role in the development of inflammation via chemokine release.

Protein kinase C inhibitors enhance G-protein induced phospholipase A2 activation in intact human platelets.

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AlF4-, a known unspecific activator of G-proteins. Both stimuli induced the liberation of [3H]AA, the release of beta-thromboglobulin (beta-TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced beta-TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and beta-TG released by thrombin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and calphostin C) increased the release of [3H]AA and inhibited beta-TG release and aggregation induced by AlF4- but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation of beta-TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, beta-TG and aggregation in AlF4--stimulated platelets. These results indicate the presence of a G-protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.

Establishment and characterization of an immature human megakaryoblastic cell line, MEG-A2.

We have established a novel human megakaryoblastic cell line, designated as MEG-A2, from a patient with megakaryoblastic crisis of Philadelphia (Ph) chromosome positive chronic myelogenous leukemia. MEG-A2 cells showed positive phenotypes for periodic acid Schiff and alpha-naphthylbutyrate esterase reactions, but were negative for myeloperoxidase and naphthol ASD chloroacetate esterase reactions. Flow cytometric analyses of cell surface markers revealed that MEG-A2 cells had a low level of GP IIb/IIIa expression as well as apparent expressions of CD4, CD7, CD13, CD33 and CD34 antigens, but no expression of GP Ib nor glycophorin A. Stimulation with phorbol 12-myristate 13-acetate (PMA) dramatically increased the expression of megakaryocyte-related markers such as HPL-3, J15, Pit-1, Y2/51 and AN51 in MEG-A2 cells. The PMA-stimulation also induced expression of platelet peroxidase (PPO) in MEG-A2 cells on electromicroscopic observation. Proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or erythropoietin were observed, and the expression of GP IIb/IIIa was increased by stimulation with GM-CSF, IL-3, erythropoietin and interleukin-6 (IL-6). Protein S mRNA expression was seen in cultured cells on Northern blot analysis. Expression of platelet factor 4 mRNA was induced in PMA-stimulated cells, and a marked accumulation of protein was observed in the culture medium. In conclusion, a new cell line, MEG-A2, belongs to the relatively immature megakaryocytic lineage and has markedly increased megakaryocytic characteristics with PMA stimulation.

Increased thrombin-antithrombin III complexes after 1 h of physical exercise.

Concentrations of thrombin-antithrombin III (TAT) complexes in plasma were previously reported to be increased after a 100-km run, while fibrinopeptide A (FPA) concentration remained unchanged. Thus, antithrombin III appears to neutralize thrombin generated during running and prevents fibrin formation. To determine the clinical relevance of these findings, we compared the effects of exhaustive running (1 h, n = 10) on the plasma concentrations of prothrombin fragments F1 and F2, TAT, FPA, and beta-thromboglobulin with the effects of recreational jogging (1 h, n = 10) and exhaustive bicycling on an ergometer (1 h, n = 8). Prothrombin fragments F1 and F2 and TAT concentrations increased significantly in each group. The most significant increase in TAT concentration was measured in the running group (from 1.72 +/- 0.49 to 3.61 +/- 1.03 ng/ml, P < 0.001). The best correlation was found between the postexercise TAT and lactate concentrations (r = 0.62, n = 28, P < 0.001). Mean FPA concentrations after exercise did not exceed normal values in any of the three groups analyzed. An increase in beta-thromboglobulin concentration was measured in the running and in the cycling group. Thus, thrombin is formed, in particular, when associated with anaerobic metabolism, and platelets are activated during high-intensity exercise.

Platelet activation and prostacyclin release in essential hypertension.

To evaluate platelet activation thromboxane A2 (TxA2) and beta-thromboglobulin (beta TG) were used as markers and in addition we studied the biosynthesis of prostacyclin. Synthesis of TxA2 and prostacyclin was assessed by measurement of urinary metabolites. Fifteen untreated hypertensive patients (HT) and 15 age-matched normotensive controls (NT) were investigated at rest, during and after exercise. HT patients were re-examined after 3 months on enalapril. During basal conditions there was no difference in the excretion of Tx-M, PGI-M or beta TG between the groups. During strenuous exercise HT exhibit a significantly higher increase in prostacyclin synthesis (162%) compared to NT (76%). The levels of beta TG increased with 82% in the HT and 24% in the NT group, Tx-M increased with 27% and 23% respectively. Treatment with the ACE-inhibitor enalapril did not significantly alter these findings. These results indicate that there is no evidence of basal platelet activation in early essential hypertension. Strenuous exercise leads to some increase in Tx-M in both groups, with no pronounced differences between the groups. Hypertensive patients exhibit a significantly increased prostacyclin response to exercise which could be due to differences in vessel-wall reactivity. Enalapril seems to exert no effect on platelet activation or on prostacyclin biosynthesis.

Modification of platelet function by isosorbide dinitrate in patients with coronary artery disease.

The effect of a four weeks oral treatment with 100 mg isosorbide dinitrate (ISDN) daily on platelet function was evaluated in 40 patients (aged 40-65 years) with proven coronary artery disease. Isosorbide dinitrate decreased platelet reactivity to ADP (p less than 0.001), increased platelet sensitivity to PGI2 (p less than 0.01) while the production of TXB2 from exogenous arachidonic acid substrate and from endogenous substrate were both significantly reduced. Circulating platelet aggregates as measured by the Wu-test were markedly reduced (p less than 0.001) but there was little change in the plasma concentration of the platelet proteins beta-thromboglobulin and platelet factor 4. Overall, platelet activation correlated with smoking, hypertension and a family history of coronary artery disease. The reduced platelet activation seen during treatment with isosorbide dinitrate may contribute to the therapeutic benefit seen with this drug in patients with coronary artery disease.

Studies of in vitro megakaryocytopoiesis in adult immune thrombocytopenic purpura (ITP).

In vitro megakaryocytopoiesis was studied in 8 patients with chronic immune thrombocytopenia (ITP). A significant increase of megakaryocyte (MK) colony formation was observed in 5/5 patients studied. Furthermore, the serum of these 8 patients was able to enhance MK colony formation by normal marrow cells. This effect was neither due to a decrease of inhibitors of megakaryocytopoiesis such as betathromboglobulin (beta TG) nor to the IgG fraction of patients' serum. In addition, the level of interleukin 6, which is above all a stimulus for MK maturation, was found within the normal range in 8/8 patients tested. These data suggest that in chronic ITP there is an increase of MK progenitor cell number which may be due to an increased level of MK colony-stimulating activity.

Biosynthesis and secretion of beta-thromboglobulin by a human megakaryoblastic cell line (MEG-01).

The de-novo synthesis and secretion of beta-thromboglobulin (BTG) by a human megakaryoblastic cell line (MEG-01) were studied by measuring and immunoblotting of BTG in culture supernatant and immunoprecipitation of radiolabeled BTG synthesized after incubation with [35S]methionine. It was demonstrated that BTG synthesized by MEG-01 was secreted into culture media in a monomer form having a molecular weight of 8,800. Furthermore, we purified BTG from culture medium of MEG-01 with a heparin affinity column and compared BTG from MEG-01 with that from normal platelets. The molecular weights of BTG purified from both sources were 8,800 using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results provide direct evidence for the synthesis and secretion of BTG by megakaryocytes.