Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

Carubbi, Cecilia; Mirandola, Prisco; Mattioli, Maria; Galli, Daniela; Marziliano, Nicola; Merlini, Piera Angelica; Lina, Daniela; Notarangelo, Francesca M.; Cozzi, Marzia; Gesi, Marco; Ardissino, Diego; Marco, Luigi De; Vitale, Marco; Gobbi, Giuliana

doi:10.1371/journal.pone.0046409

Cited by 12 publications

(22 citation statements)

References 51 publications

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“…on May 11, 2018. by guest www.bloodjournal.org From megakaryocytopoiesis and its forced expression impairs PLT generation 18 and function, 32 it must be considered that human-but not mouse-platelets express other novel PKC isoforms (namely PKCd). The goal now will be to understand whether these 2 PKC isoforms parallel in the 2 species, with obvious consequences on our approach to human PLT disorders such as thrombocytopenia or thrombocytemia.…”

Section: Discussionmentioning

confidence: 99%

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Gobbi

Mirandola

Carubbi

et al. 2013

Blood

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• PKCe, regulating RhoA activity, is a critical mediator of proplatelet formation. • PKCe shut down results inRhoA expression levels that are incompatible with normal platelet generation.During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKC«), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKC« in proplatelet formation. By immunofluorescence we observed that PKC« colocalizes with a/b-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKC« expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKC« inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKC« inhibition. Collectively, these data indicate that by regulating RhoA activity, PKC« is a critical mediator of mouse proplatelet formation in vitro. (Blood. 2013;122(7):1305-1311) IntroductionDefective platelet numbers can compromise proper wound healing and cause bleeding, whereas excessive platelet production or activation can lead to thrombosis. As a result, platelet homeostasis is a tightly regulated process. The process of generating thousands of platelets from a single megakaryocyte is characterized by several morphologically distinct stages that involve extensive cytoskeletal remodeling. In particular, megakaryoctyes, following polyploidization and prior to platelet release, form elongated cellular processes called proplatelets (proPLT).1 Perturbations in proPLT formation lead to alterations in platelet morphology and thrombocytopenia, 2,3 emphasizing the importance of this step. The marginal microtubule band, actin-based cytoskeleton, and spectrin-based membrane skeleton are all prominent components of the cytoskeletal remodeling that occurs during platelet formation. 4 Specifically, dynamic tubulin polymerization is required for the proper platelet morphology, assembly of the marginal microtubule coil, proPLT extensions and microtubule band formation.4-8 Various mutations in genes that regulate actin polymerization and turnover cause thrombocytopenia due to altered formation and morphology of proPLT exten...

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Section: Discussionmentioning

confidence: 99%

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Gobbi

Mirandola

Carubbi

et al. 2013

Blood

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“…Among PKC isoforms, we have previously demonstrated that PKCepsilon is over-expressed in platelets from myocardial infarction (MI) patients compared to subjects with stable coronary artery disease and healthy controls. We also showed that PKCepsilon-expressing platelets display higher levels of surface activation markers and enhanced adhesion to sub-endothelial collagen, thus suggesting that higher levels of this kinase account for platelets' increased reactivity (7).…”

Section: Introductionmentioning

confidence: 64%

“…Platelet were purified from whole blood sample as we previously described (7). Blood samples were centrifuged at 160 g for 20 min at RT, to obtain platelet rich plasma (PRP).…”

Section: Platelet Purificationmentioning

confidence: 99%

“…Expression of PKCepsilon mRNA was analyzed as previously described (7). Briefly, purified platelets were treated with an appropriate amount of TRIzolTM (Invitrogen) for cell lysis and then processed for RNA extraction, following manufacturer's protocol.…”

Section: Rna Extraction and Real Time Pcrmentioning

confidence: 99%

“…PKCepsilon protein expression was analyzed as previously described (7). Briefly, purified platelets were treated with an appropriate amount of cell lysis buffer (50 mM TrisHCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF) supplemented with fresh protease inhibitors and protein concentration was determined using BCATM protein assay kit (Pierce, Rockford, IL, USA).…”

Section: Protein Extraction and Western Blotmentioning

confidence: 99%

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Platelet expression of PKCepsilon oncoprotein in myelofibrosis is associated with disease severity and thrombotic risk

Masselli¹,

Carubbi²,

Pozzi³

et al. 2017

Ann. Transl. Med.

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Background: Myelofibrosis (MF) is the most aggressive Philadelphia-negative chronic myeloproliferative neoplasm (MPN) with high morbidity and mortality due to thrombo-hemorrhagic complications and leukemic transformation. MF is characterized by profound alterations of megakaryocytopoiesis, with consequent abnormalities in platelet number and function. We recently showed that the overexpression of the oncoprotein PKCepsilon plays a key role in the aberrant differentiation of MF megakaryocyte clone and that its levels correlate with disease burden. Moreover, our group previously demonstrated that PKCepsilon is over-expressed in platelets from patients with acute myocardial infarction (MI) and accounts for their increased reactivity. On these bases, we investigated here the activation state and PKCepsilon expression of MF platelets, testing potential correlations with thrombotic risk and disease aggressiveness.Methods: Platelets were isolated from peripheral blood samples of MF patients and healthy donors (HDs).Patients were stratified according to the IPSS/DIPSS risk category and history of cardiovascular events.Platelet activation was assessed by flow cytometry. PKCepsilon mRNA and protein levels were determined by real time-PCR and western blot.Results: MF platelets circulate in an activated status and display significantly higher levels of PKCepsilon compared to HDs. In MF patients, PKCepsilon platelet levels were associated with high-risk disease as well as with a positive history of major cardiovascular events.Conclusions: PKCepsilon is configuring as the common denominator of neoplastic transformation and thrombus formation in MF. Overall, our data pinpoint PKCepsilon as a potential novel biomarker of disease aggressiveness and thrombotic risk in this hematologic neoplasm.

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PKCε promotes human Th17 differentiation: Implications in the pathophysiology of psoriasis

et al. 2018

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PKCε is implicated in T cell activation and proliferation and is overexpressed in CD4 -T cells from patients with autoimmune Hashimoto's thyroiditis. Although this might induce the suspicion that PKCε takes part in autoimmunity, its role in the molecular pathophysiology of immune-mediated disorders is still largely unknown. We studied PKCε expression in circulating CD4 -T cells from patients with psoriasis, a skin disorder characterized by an increased amount of Th17 cells, a CD4 subset that is critical in the development of autoimmunity. Although the mechanisms that underlie Th17 differentiation in humans are still unclear, we here show that: (i) PKCε is overexpressed in CD4 -T cells from psoriatic patients, and its expression positively correlates with the severity of the disease, being reduced by effective phototherapy; (ii) PKCε interacts with Stat3 during Th17 differentiation and its overexpression results in an enhanced expression of Stat3 and pStat3(Ser727); iii) conversely, when PKCε is forcibly downregulated, CD4 -T cells show lower levels of pStat3(Ser727) expression and defective in vitro expansion into the Th17-lineage. These data provide a novel insight into the molecular mechanisms of Th17 cell polarization that is known to play a crucial role in autoimmunity, pinpointing PKCε as a potential target in Th17-mediated diseases.

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Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

Cited by 12 publications

References 51 publications

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Platelet expression of PKCepsilon oncoprotein in myelofibrosis is associated with disease severity and thrombotic risk

PKCε promotes human Th17 differentiation: Implications in the pathophysiology of psoriasis

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