Impaired activation of platelets lacking protein kinase C- isoform

Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood (Impact Factor: 10.45). 02/2009; 113(11):2557-67. DOI: 10.1182/blood-2008-07-169268
Source: PubMed


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.

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    • "Similarly, the role for PRKCQ is debatable (Harper & Poole, 2009). Nagy et al (2009) showed that platelet activation and secretion is impaired in Prkcq À/À animals, whereas another study suggested an enhancement of alpha granule secretion, but not dense granule secretion, at low levels of GPVI stimulation (Harper & Poole, 2009). These discrepancies may hint at the complex nature of control of secretion in platelets, with responses varying depending on agonist and degree of stimulation. "
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    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 · 4.71 Impact Factor
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    • "GPVI fragment with molecular mass matching remnant murine GPVI fragment by Western blot analysis (Fig. 2D)[19]. "
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    ABSTRACT: Introduction: Platelet Glycoprotein (GP)VI is a member of the immunoglobulin superfamily expressed only on platelets, and is the major signalling receptor for collagen. Histone deacetylase inhibitors (HDACi) are anti-cancer agents used for the treatment of haematological malignancies, and we examined the effects of administration of HDACi to mice on platelet function including responses to agonists including collagen related peptide (CRP). Materials and methods: C57BL/6 mice were injected with two structurally different HDACi, panobinostat and romidepsin, for three days and platelet receptor levels and responses to agonists were assessed by flow cytometry and western blot. Results: Platelets from mice treated with either HDACi were impaired in their ability to respond to CRP, but not thrombin or adenosine diphosphate (ADP). HDACi treatment increased acetylation of megakaryocytic GPVI, resulting in loss of intact (~60-65-kDa) GPVI and formation of ~10-kDa remnant GPVI. Circulating platelets had reduced surface and total expression of GPVI. Platelets from mice treated with HDACi had impaired GPVI signalling following treatment with CRP, resulting in inhibition of Syk phosphorylation and activation, and the final common pathways of platelet activation. Conclusions: Administration of HDACi in vivo may ablate platelet responses to agonists and platelet function.
    Thrombosis Research 05/2013; 131(6). DOI:10.1016/j.thromres.2013.02.013 · 2.45 Impact Factor
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    • "However, it is important to point out that T cells also express other isoforms of PKC (Bauer et al., 2000). Furthermore, although most studies have so far focused on PKC-θ function in T cells, there is evidence supporting that PKC-θ is also expressed and play a role in other tissues including muscle (Kim et al., 2004; Benoit et al., 2009; Paoletti et al., 2010), platelets (Nagy et al., 2009; Harper and Poole, 2010; Cohen et al., 2011), natural killer (NK) cells (Aguilo et al., 2009), and likely mast cells (Kempuraj et al., 2005). Therefore, inhibition or targeting of PKC-θ for immunotherapeutic treatments may also affect other tissues in addition to T cells. "
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    ABSTRACT: PKC-θ is selectively enriched in T cells and specifically translocates to immunological synapse where it mediates critical T cell receptor signals required for T cell activation, differentiation, and survival. T cells deficient in PKC-θ are defective in their ability to differentiate into inflammatory effector cells that mediate actual immune responses whereas, their differentiation into regulatory T cells (Treg) that inhibits the inflammatory T cells is enhanced. Therefore, the manipulation of PKC-θ activity can shift the ratio between inflammatory effector T cells and inhibitory Tregs, to control T cell-mediated immune responses that are responsible for autoimmunity and allograft rejection. Indeed, PKC-θ-deficient mice are resistant to the development of several Th2 and Th17-dependent autoimmune diseases and are defective in mounting alloimmune responses required for rejection of transplanted allografts and graft-versus-host disease. Selective inhibition of PKC-θ is therefore considered as a potential treatment for prevention of autoimmune diseases and allograft rejection.
    Frontiers in Immunology 08/2012; 3:225. DOI:10.3389/fimmu.2012.00225
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