Platelet activation at sites of
vascular injury is triggered through different signaling pathways leading to activation of
phospholipase (PL) Cbeta or
PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and
thrombus formation. PLCbeta
isoenzymes are activated downstream of
G protein-coupled receptors (GPCRs), whereas
PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet
collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of
small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of
inositol 1,4,5-trisphosphate (IP(3)) and intracellular
calcium mobilization suggesting inappropriate activation of
PLCgamma2 despite normal
tyrosine phosphorylation of the
enzyme. Rac1 ( -/- ) platelets displayed defective
thrombus formation on
collagen under flow conditions which could be fully restored by co-infusion of
ADP and the TxA(2) analog
U46619, indicating that impaired GPVI-, but not
G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two
collagen-dependent arterial
thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent
PLCgamma2 activation in platelets and that this is critical for
thrombus formation in vivo.