Myeloproliferative neoplasms (MPN) are hematological diseases associated with the Janus Kinase 2 (JAK2), calreticulin (CALR), and thrombopoetin receptor (MPL) genetic driver mutations. These mutations leads to a constitutive activation of the JAK-STAT pathway and results in the clonal proliferation and accumulation of myeloid cells in bone marrow and peripheral blood. Along with genetic alterations, inflammation is a key feature of MPN's pathogenesis, especially in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). The oncoinflammation status seems to contribute to immune cell activation, pro-coagulant state, and increased risk for thromboembolic events in MPN patients. In this context, potential alterations in platelets'immunophenotype may contribute to oncoinflammation and the occurrence of thromboembolic events in MPN patients. Therefore, the present study characterized the immunophenotype of peripheral blood platelets from patients with PV, ET, and MF and healthy subjects.

Peripheral blood from patients with PV (n = 19; 15 JAK2+, four with undetermined mutational status), ET (n = 10; seven JAK2+, one CALR+, two triple negative), MF (n = 14; 10 JAK2+, two CALR+, two MPL+), and controls (CTRL, n = 17) were collected using the 3.2% sodium citrate buffer. The platelet-rich plasma was obtained after centrifugation and platelets were immunophenotyped using the BD LSRFortessa™ flow cytometer to quantify the percent of labeled cells (%) and the median fluorescence intensity (MFI) for CD41a, CD62P, CD36, CD38, CD63, and CD154 (CD40L) markers. These markers are involved in platelet activation and pro-inflammatory status. Platelets'immunophenotype was evaluated under three conditions: without stimulation, after stimulation with calcium ionophore A23187 at 0.5 μM for 15 minutes and thrombin at 50 U/mL for 15 minutes.

At basal level, PV, ET, and MF patients'presented higher frequency of CD62P+, CD36+, and CD63+ platelets than the controls. The frequency of CD63+ platelets were higher in PV and ET patients than in MF patients. The frequency of CD38+ platelets was increased in MF patients, and the frequency of CD154+ platelets was higher in PV patients than in MF patients and controls. The stimuli with calcium and thrombin increased the frequency of CD154+ platelets in controls and in PV, ET, and MF patients and the frequency of CD62P+ platelets in PV and MF patients. In the controls and in PV patients, calcium and thrombin stimuli increased the MFI of the CD62P, CD63, and CD154 markers. In ET patients, calcium stimuli increased the MFI of CD63 and CD154 markers, and in MF patients, calcium increased the MFI of CD154. In PV patients, thrombin stimuli increased the MFI of CD62P and CD154 markers. In MF patients, thrombin increased the MFI of CD62P, CD36, and CD154 markers. Thrombin stimuli did not altered the expression of any markers in ET patients'platelets.

Platelets from MPN patients present alterations in the expression of activation markers at basal level and under stimuli with calcium and thrombin. These immunophenotypic alterations may contribute to oncoinflammation and to the pro-thrombotic status in PV, ET, and MF patients.

Coordination of Superior Level Staff Improvement (CAPES), #88887.688764/2022/00.