Abstract
Introduction Thrombotic complications occurring in MPNs patients have been shown to be associated with increased levels of platelet-derived microparticles (MP). The JAK2 mutational status and allele burden level increase the expression of these MP. Recently, MP have been implicated as prognostic factor and a marker for the presence of acute coronary syndromes, strokes and thromboembolism. The increase in CD36- platelet receptor expression (GPIV) is shown to be due to the platelet activation mechanism. The CD36 receptor plays a role in the fixation of the platelet on phosphatidylserine and in the recruitment of the MP into the thrombi. The CD36 signalling is important for the formation of a normal thrombus or complete platelet activation. Method This retrospective study included 157 patients with MPNs, CML, HES as well as 10 controls and 21 non haematological patients with thrombosis. The group of MPNs patients included 25 patients with chronic myeloid leukaemia (CML), 5 patients with HES and 127 patients with MPNs Ph negative (essential thrombocythemia, polycythemia vera and idiopathic myelofibrosis). For all MPNS patients it was assessed mutational status- JAK, CALR and for CML patients the disease status based by BCR/ABL expression. Blood samples were taken fasting, on citrate tube and were processed less than 4 hours in the lab. Whole blood sample was used for platelet receptors expression and platelet poor plasma (PPP) obtained by centrifuged blood at 2500G was used for MP expression The platelet receptors assessed were: CD42a,CD42b, CD41, CD61 and CD36. Sample reading was performed on BD FACS Canto II platform, FACS Diva software 7. Calibration for microparticle size was performed using Mega-mix-SSC Plus (BioCytex) for diameters of 0.16 μm, 0.20 μm, 0.24 μm and 0.5 μm for MFI setting of fluorescents SPHERO Calibration Particles 8 peaks were used, compensation was done using BD Comp-Beads and titration used poor plasma in platelets from a healthy donor. The integral microparticles were defined as positive VPD450 and the subsets were defined as CD41 + / CD61 + derived microparticles, epithelial derived microparticles such as CD31 + / CD41- / CD61- / CD45- / GlyA-, microparticles derived from erythrocytes such as GlyA + / CD31- / CD41- / CD61- / CD45- and microparticles derived from leukocytes such as CD45 + / CD31- / CD41- / CD61- / GlyA-. The platelet response was assessed by PFA200 method using ADP/Col and EPI/COL cartridge. Results The expression of CD41 is lower in MPNs patients cmpared with nonhematological(NH) patients with thrombosis, p-0.005. No differences compared with healthy volunteers. There are no differences in CD61 expression between MPNs and Nhpatients with thrombosis or healthy volunteers but we obtain a lower variation coefficient for both receptors in MPNs patients compared with controls, p=0.01 suggested a higher homogenicity The CD42a expression is higher in MPNs and CML group compared with healthy volunteers, p=0.004, the variation coefficient is higher in MPNs patients-higher heterogenicity-p=0.05. There are no differences for CD42b. Conclusions The MPNs JAK positive patients have higher number of MP compared with MPNs JAK negative patients The expression of CD36 on platelet membrane and MP platelet derived, is higher in MPNs patients, especially in JAK positive group; This expression decreases in MPNs group with thrombosis. This decrease of expression could be a marker for higher risk of thrombosis? We have to check this finding in a large group of patients The CD41/CD61 and CD 42a/CD42b receptors are lower expressed in MPNs and CML groups on the platelet membrane and PM platelet derived. The low expression does not correlate with thrombosis presence or JAK2/CALR mutation The low expression of CD41/CD61 and CD42a/CD42b is positive correlated with platelet response in PFA 200 assessing Higher risk of thrombosis in MPNs patient seems to be associated with JAK2 mutation especially those with high expression of allele burden and higher expression of CD 36. Acknowledgments This research was supported by UEFSCDI Romania center grant to PN-III-P2-2.1-PED-2016-1315 project -PARTE-MPN
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No relevant conflicts of interest to declare.
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