PKCalpha regulates platelet granule secretion and thrombus formation in mice.

Department of Physiology & Pharmacology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
Journal of Clinical Investigation (Impact Factor: 13.77). 02/2009; 119(2):399-407. DOI: 10.1172/JCI34665
Source: PubMed

ABSTRACT Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

  • [Show abstract] [Hide abstract]
    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 &amp Cellular Proteomics 09/2014; 13(12). DOI:10.1074/mcp.M114.038513 · 7.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background Calcium dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium-activated protease and kinase, the contribution of calcium-activated protein serine/threonine phosphatase in platelet activation is poorly understood.Objective To assess the role of catalytic subunit of protein phosphatase 2B (PP2B) or calcineurin in platelet function.ResultsHere, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein phosphatase 2B (PP2B-A) such as cyclosporine A (CsA) or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the β isoform of PP2B-A (PP2B-Aβ-/-) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aβ did not affect agonist-induced integrin αIIbβ3 inside-out signaling, but increased basal Src activation and outside-in αIIbβ3 signaling to p38 mitogen activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen induced increased p38 activation in PP2B-Aβ-/- platelets were blocked by Src inhibitor. Both PP2B-Aβ-/- platelets and PP2B-Aβ depleted human embryonal kidney 293 αIIbβ3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with β3 was dephosphorylated on Ser2152 in fibrinogen adhered wild type but not in PP2B-Aβ-/- platelets. In a FeCl3 injury thrombosis model, PP2B-Aβ-/- mice showed decreased time to occlusion in the carotid artery.Conclusion These observations suggest that PP2B-Aβ by suppressing outside-in αIIbβ3 integrin signaling limits platelet response to vascular injury.This article is protected by copyright. All rights reserved.
    Journal of Thrombosis and Haemostasis 10/2014; 12(12). DOI:10.1111/jth.12761 · 5.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelets. Although several platelets SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8-/- platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8-/- mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 11/2014; 290(3). DOI:10.1074/jbc.M114.602615 · 4.60 Impact Factor