Platelet function in polycythemia vera : studies of agonist and cytokine induced platelet activation

University dissertation from Stockholm : Karolinska Institutet, -

Abstract: Polycythemia vera (PV) is a malignant myeloproliferative disorder characterized by an increased red cell mass and most often accompanied by leukocytosis and thrombocytosis. The pathogenic mechanisms behind PV are still largely unknown. Studies using X-chromosome linked gene probes have revealed that circulating platelets, erythrocytes, monocytes and polymorphonuclear neutrophils (PMN) are derived from the malignant clone in the vast majority of PV patients. Changes in the malignant stem cell could therefore be inferred from results of studies of PV platelet and PMN function and biochemical changes. Previous studies of PV-PMN and monocytes implied a reduced oxidative response to surface-dependent stimuli, such as fMLP, but a normal response to phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC). The decreased oxidative response was associated with an impaired activation of phospholipase D (PLD). One of the purposes of the present studies was to investigate if the same could be true for PV platelets, since a defect in all three cell lineages could imply a defect in the malignant stem cell. Aggregation was indeed reduced in PV platelets after stimulation with a cell surface receptor stimulus, platelet activating factor (PAF), whereas PMA elicited a normal response. However, release of ß-thromboglobulin (ß-TG) induced by PAF in vitro was not reduced in PV platelets, indicating normal function of the PAF receptor. In accordance with a normal release of ß-TG, flow cytometry showed norrnal upregulation of a granule protein CD 62 after stimulation with PAF and PMA, but no increased expression on unstimulated platelets. Quantitative analysis of the fibrinogen receptor glycoprotein lIb/IIIa (GPIIb/IIIa) indicated a lower number of receptors on resting PV platelets compared with healthy controls. The number of receptors was upregulated after stimulation with PAF and PMA but never to the amount expressed on the platelets of healthy controls. Existing GPIIb/IIIa receptors in PV, however, bound fibrinogen after stimulation, indicating that they were functional. Immunoblot against GPIIIa could not reveal any apparent differences in amounts between PV and control platelets. There was no sign of preactivated PV platelets. The increase in cytosolic calcium in PV platelets stimulated with PAF was similar to that in healthy controls, indicating normal activation of phospholipase C (PLC). Furthermore, generation of phosphatidylethanol induced by thrombin and PMA in the presence of ethanol was similar in PV and control platelets, suggesting a normal PLD function. There was no apparent reduction of cellular amounts of either FAK, Syk, rhoA, Grb2 or Shc in PV platelets. Tyrosine phosphorylation in resting platelets as well as after stimulation with thrombin, PAF and PMA was also alike in PV and control platelets. Since no mutation of cytokine receptors has been identified in PV and the interaction between cytokines and their specific receptors is normal, post receptor abnormalities in hematopoietic cells could explain the hypersensitivity to cytokines in PV. Hematopoietic cell phosphatase (HCP) negatively regulates cytokine induced stimulation. Expression of HCP in enriched CD34+ bone marrow cells, granulocytes, platelets and Iymphocytes did not differ between controls, PV, ET and CML patients. HCP protein expression in PV granulocytes and platelets was also similar to that seen in healthy controls. Expression of the thrombopoietin receptor, c-mpl, as detected by immunoblotting, was similar to that in controls in half of the patients investigated. In the other patients, expression was markedly reduced. Patients with abnormally low amount of c-mpl were more often treated with bone marrow suppressive regiments than c-mpl positive patients. Thrombopoietin induced phosphorylation of JAK2 was normal in c-mpl positive patients but lacking in patients negative for c-mpl on immunoblots. ISBN 91-628-3442-8

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