Impaired activation of platelets lacking protein kinase C-theta isoform.
ABSTRACT Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.
SourceAvailable from: Alastair Poole[Show abstract] [Hide abstract]
ABSTRACT: Upon activation by extracellular matrix components or soluble agonists, platelets release in excess of 300 active molecules from intracellular granules. Those factors can both activate further platelets and mediate a range of responses in other cells. The complex microenvironment of a growing thrombus, as well as platelets' roles in both physiological and pathological processes, require platelet secretion to be highly spatially and temporally regulated to ensure appropriate responses to a range of stimuli. However, how this regulation is achieved remains incompletely understood. In this review we outline the importance of regulated secretion in thrombosis as well as in 'novel' scenarios beyond haemostasis and give a detailed summary of what is known about the molecular mechanisms of platelet exocytosis. We also discuss a number of theories of how different cargoes could be released in a tightly orchestrated manner, allowing complex interactions between platelets and their environment.British Journal of Haematology 11/2013; 165(2). DOI:10.1111/bjh.12682 · 4.96 Impact Factor
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ABSTRACT: BackgroundFPD/AML is an inherited platelet disorder caused by germline RUNX1 mutation and characterized by thrombocytopenia, platelet function defect and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1-regulated genes involved in these defects. Methods Lumi-aggregometry, α- and dense- granule content and release, platelet ultrastructure and αIIbβ3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assay and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1. ResultsSevere decrease in platelet aggregation, together with defective αIIbβ3 integrin activation and combined αδ storage pool deficiency was found. However, while dense granules were markedly reduced, α-granule content was heterogeneous. A trend towards decreased platelet spreading was found, while β3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind NF-E2 promoter in primary megakaryocytes and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression. ConclusionsFPD/AML platelet function defect represents a complex trait and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights RUNX-1-target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function.This article is protected by copyright. All rights reserved.Journal of Thrombosis and Haemostasis 03/2014; DOI:10.1111/jth.12550 · 5.55 Impact Factor
Platelets 06/2014; DOI:10.3109/09537104.2014.926474 · 2.63 Impact Factor