PKCα regulates platelet granule secretion and thrombus formation in mice
Olga Konopatskaya, … , Johan W.M. Heemskerk, Alastair W. Poole
Olga Konopatskaya, … , Johan W.M. Heemskerk, Alastair W. Poole
Published January 19, 2009
Citation Information: J Clin Invest. 2009;119(2):399-407. https://doi.org/10.1172/JCI34665.
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Research Article Hematology

PKCα regulates platelet granule secretion and thrombus formation in mice

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 PKCα and PKCβ and novel isoforms PKCδ and PKCθ. Here, we used a genetic approach to definitively demonstrate the role played by PKCα in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca–/– mice, and PKCα 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 PKCα 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 PKCα ablation on thrombus formation and granule secretion may implicate PKCα as a drug target for antithrombotic therapy.

Authors

Olga Konopatskaya, Karen Gilio, Matthew T. Harper, Yan Zhao, Judith M.E.M. Cosemans, Zubair A. Karim, Sidney W. Whiteheart, Jeffery D. Molkentin, Paul Verkade, Steve P. Watson, Johan W.M. Heemskerk, Alastair W. Poole

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Figure 1

PKCα plays a major role in regulating inside-out signaling to integrin αIIbβ3 but no significant role in outside-in signaling or in adhesion to fibrinogen or collagen.

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PKCα plays a major role in regulating inside-out signaling to integrin α...
(A) Washed platelets from WT or Prkca–/– mice were labeled with PE–anti-αIIbβ3 antibody and stimulated with CRP (5 μg/ml) or thrombin (1 U/ml) for 15 minutes; immunofluorescence intensity was measured by flow cytometry. The data presented are geometric means as percentages of basal nonstimulated levels. Error bars represent SEM. n = 3. *P < 0.05. (B–D) Washed platelets from WT or Prkca–/– mice were added to CRP- or fibrinogen-coated coverslips, and the levels of spreading and adherence were analyzed. (B) Representative images of murine platelets 40 minutes after addition to CRP- or fibrinogen-coated surfaces as indicated. Images were taken under oil immersion. Original magnification, ×63. Scale bars: 5 μm. (C and D) Time course of WT and Prkca–/– platelet adherence (number of platelets estimated per field of view; upper panel) and spreading (as assessed by measurement of cell surface area; lower panel) on CRP-coated (C) and fibrinogen-coated (D) surfaces. Data shown are mean ± SEM. n = 4.