ROLE OF novel PKCs IN PLATELET PRODUCTION AND FUNCTION

Protein kinase C (PKC) is a family of serine-threonine kinase involved in many cellular function, including cell death, proliferation and differentiation (Corbalán-García S et al. 2006). In particular PKCε and PKCδ could be considered the yin and yang of novel PKCs, because of their antithetical role in many cellular mechanisms such as proliferation, apoptosis, tumor growth and cardioprotection. PKCε is largely considered as an oncogene because of its anti-apoptotic and pro-proliferation functions (Newton PM and Messing RO 2011), conversely PKCδ generally slows proliferation, induces cell cycle arrest and apoptosis (Steinberg SF 2004). In the heart, they are among the most widley expressed PKC isozymes and they play an antithetical role in the ischemic-reperfusion preconditioning process (i.e.: enhancing role forPKCε and inhibiting role for PKCδ (Chen L et al. 2001). Moreover, the importance of these two PKC isoforms in pathological conditions has been proven by clinical trials based on specific PKCε and PKCδ inhibitor peptides (Mochly-Rosen D et al. 2011). Platelets are the smallest blood cells with a primary physiological role in hemostasis. They are produced by megakaryocytes as anucleated cells that contain proteins and mRNA derived from megakaryocyte. Platelets retain also the protein synthesis machinery, therefore, platelet mRNAs can be efficiently translated during platelet life, during around 10 days. PKCs have been established as important regulators of several platelet functions. The physiological expression of the different PKC isoforms varies significantly in mature platelets, with relevant differences in humans and mice. Concerning novel PKC isoforms, it has been demonstrated that PKCδ is expressed in human platelets and regulates the activation response to GPVI agonists and adhesion to collagen (Hamm CW et al. 1987). This behavior of PKCδ in humans is similar to the one described for PKCε in mice, where it plays a relevant role in the activatory signaling cascade emanating from the GPVI receptor (Pears CJ et al. 2008). On the other hand, PKCε expression and function in human platelets is still very controversial (Crosby D and Poole AW. 2003; Muruggapan S et al. 2004; Beunsuceso S et al. 2005; Pears CJ et al. 2008). On these basis, during the first period of my doctoral fellowship, I focused my research on PKCε expression in human platelets, exploring whether its levels could be associated to platelet hyper-reactivity and related diseases (i.e. acute myocardial infarction), in order to clarify PKCε role in platelet function. The results of my research showed that the majority of HD-derived platelets did not express PKCε. This is in agreement with previous data (Gobbi G et al. 2007) describing the down-modulation of PKCε expression in in vitro human megakaryocytopoiesis from day 6 onward of TPO-driven MK differentiation of CD34 precursors. Platelets play a central role in the genesis and propagation of atherothrombosisis and the development of platelet thrombus is a critical, final phase in myocardial infarction. I demonstrated that in human platelets, PKCε is selectively de novo expressed in myocardial infarction, but not in stable coronary artery disease patients, and its expression returns negative after 15 days of follow-up after the acute event. Additionally, by functional experiments, I demonstrated that PKCε-transfected normal human platelets enhance their adhesion properties to collagen-coated surfaces under physiologically high shear forces. Myocardial infarction patients express PKCε mRNA at significantly higher frequency than healthy donors and stable coronary artery disease. Considering the dimensions of the first intronic sequence of the PKCε gene, that would virtually preclude the persistence of a potential PKCε pre-mRNA in the platelet, my findings suggest that platelet generations produced before the acute event of myocardial infarction might retain PKCε-mRNA that is not down-regulated during terminal MK differentiation. An alternative explanation would be an anticipated release of platelets, before physiological PKCε down-modulation. This possibility is however unlikely, as PKCε down-modulation takes place around day 6 of in vitro MK differentiation, that would be too early. Besides, the analysis conducted on the reticulated platelets of some myocardial infarction patients, randomly selected from the recruited cohort, did not show any difference in terms of PKCε expression as compared to mature platelets, excluding the possibility that the appearance of PKCε positive platelets in myocardial infarction patients could be selectively ascribed to newly formed platelets. These results suggest that the ectopic expression of PKCε in platelets could be used as a marker of probability to anticipate the acute event in patients at risk. Since many pathological conditions depend on an abnormal platelet function, the study of molecular mechanisms involved in platelet activation and thrombus formation, has always been a matter of great interest. On the other hand, many diseases are related to abnormal platelet production. Thrombocytopenia is a major clinical problem encountered across a number of conditions, including immune thrombocytopenic purpura, myelodysplastic syndromes, chemotherapy, aplastic anaemia, human immunodeficiency, virus infection, complications during pregnancy and delivery, and surgery. Patients with a low platelets number, are at increased risk of spontaneous bleeding or hemorrhage and, to prevent them, they are treated with platelets transfusion. However the use of apheresis-equivalent units derived from human donors, shows several limitations and challenges with platelet preparation and storage technologies , such as clinically significant immunogenicity, associated risk of sepsis and inventory shortages due to high and 5-days shelf life (Thon JN et al. 2014). On these basis, two strategy are possible: i) to potentiate an in vitro platelet producing system to obtain platelets for infusion and ii) to develop pharmacological treatment able to modulate in vivo megakaryocytopoiesis and platelet production. Therefore, a deeper understanding of molecular mechanisms that control megakaryocytopoiesis is a key element to regulate in vitro and in vivo platelet production for clinical applications. PKCε is involved in human and mouse megakaryocytopoiesis. In human CD34+-derived MKs, PKCε is down-modulated in the later phases of differentiation and its forced overexpression reduces cell polyploidization and platelet production via Bcl-xL up-regulation (Gobbi G et al 2007). Regarding to PKCδ although it is well established that PKCδ is present in human platelets regulating their function, little is known about its expression during megakaryocytopoiesis Given this background, during the second period of my doctoral fellowship I sought to demonstrate whether PKCδ expression in human platelet could be the expression of a specific modulation of this isoform during megakaryocyte differentiation, similarly to what described for PKCε. Using in vitro model of human megakaryocytopoiesis, I found that, conversely to PKCε levels, PKCδ is constantly expressed and its forced down-modulation results in reduced MKs differentiation and platelet release via Bcl-xL up-regulation and Bax down-modulation Finally, given the growing interest of the scientific community on ameliorating in vitro platelet production for transfusion purposes, I studied whether the modulation of PKCε/PKCδ balance could affect platelet production in vitro. The importance of PKCε and PKCδ balance in human thrombopoiesis is additionally proven by my findings in pathological conditions, ie. myeloproliferative disorders. Primary myelofibrosis is a chronic myeloproliferative neoplasm characterized by bone marrow hyperplastic MKs with an impaired capacity to generate proplatelets in vitro. Interestingly, I found that PMF-CD34+-derived megakaryocytes show an imbalance between PKCδ/ PKCε expression, with an increase in PKCε levels- in agreement with our more recently data (Masselli et al. In press) – and a decrease in PKCδ levels, than those from healthy subjects. Moreover, these expression levels of PKCs are associated with similar imbalance between their down-stream effectors Bcl-xL and Bax, respectively. Finally, using a pharmacological inhibitor and activator of PKCδ and PKCε function, I obtained a modulation of in vitro platelet production. The concomitant PKCδ inhibition and PKCε activation reduces platelet release; conversely, PKCδ activation associated with PKCε inhibition clearly demonstrates the possibility to potentiate platelet production. Collectively, these data suggest that novel PKCs i) should be adequately expressed in human circulating platelets and PKCε ectopic expression could be associated to cardiovascular deseases, suggesting its possible use as a risk marker; ii) have a crucial role during normal human megakaryocytopoiesis and platelet production, through a mechanism involving apoptotic pathway (specifically Bcl-xL and Bax); iii) might be modulated, as protein expression levels, during in vitro megakaryocytopoiesis and by modifying their rate is possible to revise platelet production, suggesting future strategy for platelet diseases theraphy and infusion-aimed platelet expansion.