Decreased CXCR4 mRNA Levels and Abnormal SDF-1 Response in Platelets from Essential Thrombocythemia.

Abstract

Chemokines are soluble mediators implicated in a wide range of physiologic and pathologic processes including megakaryocytic development and platelet production. Previously, we found decreased levels of CXCR4, the only known receptor for the chemokine SDF-1, on platelet membrane from essential thrombocythemia (ET) patients, a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. In the present work we sought to evaluate the expression levels of platelet CXCR4 mRNA, the functional consequences of CXCR4 decrease in ET platelets as well as whether the receptor reduction was also extended to other lineages. We studied the expression levels of CXCR4 mRNA in platelets from ET patients by semi-quantitative RT-PCR. For semi-quantification, co-amplification of a GAPDH internal control sequence was performed. Functional studies consisted in turbidimetric evaluation of platelet aggregation induced by SDF-1 and/or ADP. In order to evaluate CXCR4 expression on leukocyte membrane, white cell subpopulations were individualized by the addition of specific MoAbs directed against CD45 labeled with PerCP and CD3 and CD14 labeled with FITC. Data were expressed as relative fluorescence intensity (RFI, ratio between the mean fluorescence intensity of CXCR4 antibody and the corresponding isotype control). The ratio between CXCR4 mRNA and the housekeeping gene GADPH in platelets from patients, 0.16 (0.1–0.24) was significantly decreased compared to controls, 0.35 (0.18–0.86), n=11, p=0.0002 (Wilcoxon signed rank sum test). Functional studies in response to SDF-1 showed a biphasic, dose-dependent full platelet aggregation in PRP in normal controls. On the contrary, abnormalities in SDF-1 response were observed in all 8 patients evaluated. Our results showed that platelet response to SDF-1 in ET patients closely paralleled that of ADP. Three patients did not respond either to SDF-1 or to ADP. From the remaining five patients, three had a delay in the second wave of aggregation induced by SDF-1 and the other two displayed only a primary wave of aggregation. These patients had a normal response to ADP when standard concentrations were used (2 mmol/L), but displayed a decreased response compared to normal controls when challenged with suboptimal doses of this agonist (0.8 to 1.2 mmol/L). CXCR4 RFI in granulocytes from patients was found elevated, 11.1 (2.22–22.74) compared to normal controls, 7.54 (1.73–9.52), p=0.036 (n=7). Similar results were found in CD3-positive lymphocytes, 40.0 (6.78–67.91) from patients and 17.23 (5.22–29.97) from normal controls, p=0.036 (Mann-Whitney Wilcoxon Rank Sum Test). On the contrary, CXCR4 on monocyte membrane was normally expressed, RFI from patients, 49.81 (1.17–118.28), and normal controls, 74.23 (3.0–80.49), p=0.8. These results indicate that the decrease in CXCR4 membrane expression on ET platelets is related to a reduced CXCR4 mRNA, and also to the abnormal platelet response to SDF-1. Besides, flow cytometric studies on leukocyte subpopulations demonstrated that CXCR4 decrease is restricted to the megakaryocytic lineage. Deregulation of SDF-1/CXCR4 axis in ET patients could be implicated in the megakaryocytic alterations seen in this illness and its presence in a megakaryocyte-related disease highlights the important role of SDF-1/CXCR4 axis in platelet development.