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

Abstract

Background: PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ-/- T cells exhibit reduced activation and PKCθ-/-mice are resistant to autoimmune disease, making PKCθ 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 PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ 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 αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ-/- platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ-/- platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 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 PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and α IIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors. © 2008 Hall et al.