Differential expression of SDF-1 receptor CXCR4 in molecularly defined forms of inherited thrombocytopenias.

The SDF-1-CXCR4 axis plays an essential role in the regulation of platelet production, by directing megakaryocyte (MK) migration toward the vascular niche, thus allowing terminal maturation and proplatelet formation, and also regulates platelet function in an autocrine manner. Inherited thrombocytopenias (IT) comprise a spectrum of diverse clinical conditions caused by mutations in genes involved in platelet production and function. We assessed CXCR4 expression and SDF-1 levels in a panel of well-characterized forms of IT. Decreased surface CXCR4 levels were found in 8 of 27 (29.6%) IT patients by flow cytometry, including 4 of 6 patients with ANKRD26-RT, 3 of 3 patients with GPS and 1 of 6 patients with FPD/AML. Low CXCR4 levels were associated with impaired SDF-1-triggered platelet aggregation, indicating that this decrease is functionally relevant, whereas a normal platelet response was shown in patients harbouring preserved membrane CXCR4. Reduced CXCR4 was not due to decreased gene expression, as platelet RNA levels were normal or increased, suggesting a post-transcriptional defect. Increased ligand-induced receptor internalization was ruled out, as circulating SDF-1 levels were similar to controls. MK CXCR4 expression was normal, indicating that the defect in CXCR4 arises after the step of platelet biogenesis. In conclusion, the finding of defective CXCR4 expression specifically associated with certain IT disorders highlights the fact that abnormalities in several megakaryocytic regulators underlie IT pathogenesis and further reveal the heterogeneous nature of these conditions.

Abnormal regulation of soluble and anchored IL-6 receptor in monocytes from patients with essential thrombocythemia.

OBJECTIVE: In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction. MATERIALS AND METHODS: Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor-α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction. RESULTS: Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal. CONCLUSIONS: Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET.

Dysregulation of stromal derived factor 1/CXCR4 axis in the megakaryocytic lineage in essential thrombocythemia.

This study investigated the involvement of chemokines including stromal derived factor 1 (SDF-1), interleukin 8 (IL-8), growth-related oncogene alpha (GRO-alpha) and their receptors, CXCR4, CXCR2 and CXCR1 in essential thrombocythemia (ET), a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. Fifty-three ET patients were studied. Plasma levels of SDF-1, IL-8 and GRO-alpha, evaluated by enzyme-linked immunosorbent assay, and flow cytometric analysis of CXCR1 and CXCR2 on the platelet membrane, were found to be normal in ET patients. CXCR4 expression on platelet surface as well as platelet CXCR4 mRNA detected by real-time reverse transcription polymerase chain reaction, were decreased. Platelet CXCR4 internalization rate was normal while SDF-1-induced platelet aggregation was delayed, decreased or absent. Immunohistochemical staining revealed that megakaryocytes were also affected. CXCR4 decrease was not observed either in peripheral white blood cells or in circulating CD34(+) precursors. These results show that CXCR4 is decreased in the megakaryocytic lineage in ET, mainly due to a reduced CXCR4 production, and an abnormal platelet response to SDF-1. This report is the first to describe platelet and megakaryocytic CXCR4 deficiency in a human disease and the presence of this abnormality in a megakaryocytic-related illness highlights the important role of SDF-1/CXCR4 axis in platelet development.

JAK2V617F mutation in platelets from essential thrombocythemia patients: correlation with clinical features and analysis of STAT5 phosphorylation status.

OBJECTIVE: JAK2V617F mutation rate in granulocytes from essential thrombocythemia (ET) patients ranges from 12% to 57%. Our aim was to evaluate the frequency of this mutation in the megakaryocyte/platelet lineage, and to analyze its clinical associations in ET. In addition, we determined whether this mutation leads to constitutive phosphorylation of STAT5 in platelets. MATERIALS AND METHODS: Consecutive patients with ET were included and clinical features were retrospectively reviewed. Mutation detection was performed by allele specific RT-PCR (AS-RT-PCR) and Restriction fragment length polymorphism (RFLP) analysis of platelet RNA. Constitutive phosphorylation of STAT5 in platelets was studied by Western blot. RESULTS: Fifty patients were included, 24 (48%) were JAK2V617F-positive by both AS-RT-PCR and RFLP. Patients with the mutation were older, had significantly higher hemoglobin levels, and lower platelet counts. Besides, higher frequency of thrombotic events was found in JAK2V617F-positive patients younger than 60, 53% vs. 4%, P = 0.0008. In addition, constitutive STAT5 phosphorylation was not detected in platelets from 12 patients. CONCLUSIONS: The frequency of the JAK2V617F mutation in platelets was similar to that reported in granulocytes in the literature, suggesting this mutation does not occur as an isolated event in the megakaryocyte lineage. If confirmed in a larger study, the observed higher frequency of thrombosis in patients younger than 60 might be a useful predictive marker for thrombosis in this subset of patients. Even though this mutation has been predicted to constitutively activate the JAK2 kinase, spontaneous phosphorylation of STAT5 does not seem to be a frequent finding in platelets from ET patients.

PDGF-A, PDGF-B, TGFbeta, and bFGF mRNA levels in patients with essential thrombocythemia treated with anagrelide.

Plasmatic levels of PDGF-AB, TGFbeta1, and bFGF are increased in patients with essential thrombocythemia (ET) while intraplatelet levels are low for PDGF, normal for TGFbeta, and elevated for bFGF. To evaluate the contribution of gene expression to the dysregulated cytokine levels, we studied platelet PDGF-A, PDGF-B, TGFbeta1, and bFGF mRNA in ET patients before and during anagrelide treatment. We found decreased PDGF-A and PDGF-B, increased TGFbeta1, and normal bFGF mRNA levels. During treatment, mRNA levels remained decreased for PDGF-A, were increased for PDGF-B and normal for TGFbeta1. In untreated patients, protein expression of PDGF paralleled its mRNA levels while different patterns of RNA and protein were found for TGFbeta1 and bFGF.