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Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

Hall KJ, Harper MT, Gilio K, Cosemans JM, Heemskerk JW, Poole AW - PLoS ONE (2008)

Bottom Line: Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced.These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation.PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT

Background: PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/-) T cells exhibit reduced activation and PKCtheta(-/-) mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIb)beta(3) in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed.

Methodology/principal findings: In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIb)beta(3) activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/-) platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/-) platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIb)beta(3) activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced.

Conclusions/significance: These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

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Related in: MedlinePlus

PKC isoforms are not upregulated in PKCθ−/− mice.Platelets lysates from wild-type (WT) or PKCθ−/− (KO) mice were assessed for PKC isoform expression by SDS-PAGE and western blotting using specific antibodies for PKCα, -β, -δ, -θ and -ε. Membranes were stripped and re-probed for α-tubulin as indicated to ensure equal loading of protein. Blots are representative of three independent experiments.
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pone-0003277-g001: PKC isoforms are not upregulated in PKCθ−/− mice.Platelets lysates from wild-type (WT) or PKCθ−/− (KO) mice were assessed for PKC isoform expression by SDS-PAGE and western blotting using specific antibodies for PKCα, -β, -δ, -θ and -ε. Membranes were stripped and re-probed for α-tubulin as indicated to ensure equal loading of protein. Blots are representative of three independent experiments.

Mentions: In order to be confident that any differences seen between PKCθ−/− and wild-type (WT) platelets were due to loss of PKCθ, and not due to altered expression of other PKC isoforms, we assessed the expression of the major PKC isoforms in platelets by western blotting. In addition to PKCθ, mouse platelets strongly express PKCα, -β, -δ, and -ε. No difference in expression of these isoforms was seen in PKCθ−/− platelets relative to WT platelets (Fig. 1). The blotting membranes were stripped and re-probed for α-tubulin, to ensure equal loading of proteins between samples (Fig. 1, lower panels).


Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

Hall KJ, Harper MT, Gilio K, Cosemans JM, Heemskerk JW, Poole AW - PLoS ONE (2008)

PKC isoforms are not upregulated in PKCθ−/− mice.Platelets lysates from wild-type (WT) or PKCθ−/− (KO) mice were assessed for PKC isoform expression by SDS-PAGE and western blotting using specific antibodies for PKCα, -β, -δ, -θ and -ε. Membranes were stripped and re-probed for α-tubulin as indicated to ensure equal loading of protein. Blots are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533697&req=5

pone-0003277-g001: PKC isoforms are not upregulated in PKCθ−/− mice.Platelets lysates from wild-type (WT) or PKCθ−/− (KO) mice were assessed for PKC isoform expression by SDS-PAGE and western blotting using specific antibodies for PKCα, -β, -δ, -θ and -ε. Membranes were stripped and re-probed for α-tubulin as indicated to ensure equal loading of protein. Blots are representative of three independent experiments.
Mentions: In order to be confident that any differences seen between PKCθ−/− and wild-type (WT) platelets were due to loss of PKCθ, and not due to altered expression of other PKC isoforms, we assessed the expression of the major PKC isoforms in platelets by western blotting. In addition to PKCθ, mouse platelets strongly express PKCα, -β, -δ, and -ε. No difference in expression of these isoforms was seen in PKCθ−/− platelets relative to WT platelets (Fig. 1). The blotting membranes were stripped and re-probed for α-tubulin, to ensure equal loading of proteins between samples (Fig. 1, lower panels).

Bottom Line: Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced.These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation.PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT

Background: PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/-) T cells exhibit reduced activation and PKCtheta(-/-) mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIb)beta(3) in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed.

Methodology/principal findings: In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIb)beta(3) activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/-) platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/-) platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIb)beta(3) activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced.

Conclusions/significance: These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

Show MeSH
Related in: MedlinePlus