Submitted May 17, 2006
Accepted August 4, 2006
PKC
regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation
Giordano Pula, Kai Schuh, Keiko Nakayama, Keiichi I Nakayama, Ulrich Walter, and Alastair W. Poole*
University of Bristol, UK
Institute for Clinical Biochemistry, Wuerzburg, Germany
Tohoku University, Japan
Kyushu University, Japan
* Corresponding author; email: a.poole{at}bris.ac.uk.
Protein kinase C
(PKC) has been shown by pharmacological approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKC-/- platelets, we show that the mechanism did not involve altered inside-out signaling to integrin 
IIb
3 and did not affect early signaling events downstream of GP VI, since there was no difference in tyrosine phosphorylation of PLC
2 between wild type and PKC-/- platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly however there was marked enhancement of filopodia generation in PKC-/- platelets upon adhesion to collagen, compared to wild type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation, by increasing platelet-platelet contact. We show that the critical effector for PKC is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKC physically interacts with VASP constitutively, and regulates its phosphorylation on Ser157. In VASP-/- platelets, the enhancement of filopodia generation, actin polymerisation and platelet aggregation by rottlerin is ablated. PKC is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.
