Isoform-specific functions of protein kinase C: the platelet paradigm.
ABSTRACT Platelets are central to haemostasis and thrombosis. Many key steps in platelet activation and aggregation are regulated by members of the PKC (protein kinase C) family. Multiple isoforms of PKC are expressed in platelets, and evidence is emerging that different isoforms play distinct roles in the platelet activation process. This may, in part, be regulated by isoform-specific interactions between PKC family members and other intracellular signalling molecules, such as tyrosine kinases, or the actin cytoskeleton regulator, VASP (vasodilator-stimulated phosphoprotein). The contributions of individual PKC isoforms can be addressed directly in platelets from knockout mouse models, which are providing key insights into the physiological function of PKC isoform diversity and can be a valuable complimentary approach to more commonly used pharmacological analyses. Using knockout mouse models, recent reports have demonstrated the importance of PKCbeta and PKCtheta in integrin-dependent platelet spreading, and also a novel role for PKCdelta in regulating filopodial formation, highlighting the utility of such models to investigate the functions of specific PKC isoforms in a physiological process that is significant to our understanding of cardiovascular disease.
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ABSTRACT: Platelet hyperactivity often occursd in hypertensive patients and is a key factor in the development of cardiovascular diseases including thrombosis and atherosclerosis. Nifedipine, an L-type calcium channel blocker, is widely used for hypertension and coronary heart disease therapy. In addition, nifedipine is known to exhibit an antiplatelet activity, but the underlying mechanisms involved remain unclear. Several transcription factors such as peroxisome proliferator-activated receptors (PPARs) and nuclear factor kappa B (NF-κB) exist in platelets and have an ability to regulate platelet aggregation through a non-genomic mechanism. The present article focuses on describing the mechanisms of the antiplatelet activity of nifedipine via PPAR activation. It has been demonstrated that nifedipine treatment increases the activity and intracellular amount of PPAR-β/-γ in activated platelets. Moreover, the antiplatelet activity of nifedipine is mediated by PPAR-β/-γ-dependent upon the up-regulation of the PI3K/AKT/NO/cyclic GMP/PKG pathway, and inhibition of protein kinase Cα (PKCα) activity via an interaction between PPAR-β/-γ and PKCα. Furthermore, suppressing NF-κB activation by nifedipine through enhanced association of PPAR-β/-γ with NF-κB has also been observed in collagen-stimulated platelets. Blocking PPAR-β/-γ activity or increasing NF-κB activation greatly reverses the antiplatelet activity and inhibition of intracellular Ca(2+) mobilization, PKCα activity, and surface glycoprotein IIb/IIIa expression caused by nifedipine. Thus, PPAR-β/-γ- dependent suppression of NF-κB activation also contributes to the antiplatelet activity of nifedipine. Consistently, administration of nifedipine markedly reduces fluorescein sodium-induced vessel thrombus formation in mice, which is considerably inhibited when the PPAR-β/-γ antagonists are administrated simultaneously. Collectively, these results provide important information regarding the mechanism by which nifedipine inhibits platelet aggregation and thrombus formation through activation of PPAR-β/-γ- mediated signaling pathways. These findings highlight that PPARs are novel therapeutic targets for preventing and treating platelet-hyperactivity-related vascular diseases.BioMedicine. 12/2014; 4:24.
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ABSTRACT: Knowledge of the identity and quantity of expressed proteins of a cell type is a prerequisite for a complete understanding of its molecular functions. Mass spectrometry (MS)-based proteomics has allowed the identification of the entire protein complement of yeast and the close-to-complete set of proteins expressed in mammalian cell lines. Using recent technological advances we here characterize the proteome of murine platelets, key actors in mediating hemostasis and thrombosis. We accurately measured the absolute protein concentrations of thirteen platelet proteins by SILAC-Protein Epitope Signature Tags (PrESTs) and used them as reference points to estimate the copy number of all proteins of the platelet proteome. To distinguish contaminants such as plasma or erythrocyte proteins from true platelet proteins, we monitored protein abundance profiles across multiple purification steps. In total, we absolutely quantified 4,400 platelet proteins, with estimated copy numbers ranging from less than ten to about a million per cell. Stoichiometries derived from our data correspond well with previous studies. Our study provides a close-to-complete reference map of platelet proteins, which will be useful to the community, for instance for interpreting mouse models of human platelets diseases.Molecular & Cellular Proteomics 09/2014; · 7.25 Impact Factor