PKCα regulates platelet granule secretion and thrombus formation in mice
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
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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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 7

In vivo thrombus formation is impaired in the absence of PKCα, but tail bleeding time is normal.

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In vivo thrombus formation is impaired in the absence of PKCα, but tail ...
(A–D) Mice were either Prkca–/– or littermate-matched wild-type controls. Platelets were labeled in vivo with Alexa Fluor 488, as described in Methods. (A) Platelets (green) composited with bright field images (black/white) of the cremaster arteriole were viewed, and images were acquired using a digital CCD camera (SensiCam II; Cooke Corp.) with a 640 × 480 pixel array. Original magnification, ×40. (B) Traces shown are median integrated platelet fluorescence of 15 thrombi induced in 3 or more mice for each group. Fluorescent intensity of platelets in arbitrary units is presented as a function of time. (C) Data are presented as a scatter diagram. Horizontal bar represents mean of 15 thrombi induced in at least 3 mice for each group. There is a significant reduction in thrombus intensity in Prkca–/– in comparison with WT controls (mean Prkca+/+, 652200; mean Prkca–/–, 279600; P < 0.05). (D) Time to reach peak thrombus size also significantly differed from WT controls, with mean data shown by the horizontal bars (mean Prkca+/+, 68.1 seconds; mean Prkca–/–, 115.2 seconds; P < 0.01). (E) Mice were anesthetized and a transverse incision made with a scalpel at a position where the diameter of the tail was 2.25 to 2.5 mm. The tail was immersed in normal saline (37°C) in a hand-held test tube. The time from incision to cessation of bleeding was recorded, and mean times are shown as horizontal bars. No significant difference was seen comparing WT mice (mean 140 seconds) with Prkca–/– (mean 117 seconds; P > 0.05).