Rac1 is essential for phospholipase C-gamma2 activation in platelets.
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.
Pleines
Irina
I
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Germany.
Elvers
Margitta
M
Strehl
Amrei
A
Pozgajova
Miroslava
M
Varga-Szabo
David
D
May
Frauke
F
Chrostek-Grashoff
Anna
A
Brakebusch
Cord
C
Nieswandt
Bernhard
B
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D037181:Lectins, C-Type / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D051966:Phospholipase C gamma / Q000378:metabolism*; D015539:Platelet Activation / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000819:agonists / Q000502:physiology; D011070:Poly I-C / Q000494:pharmacology; D013927:Thrombosis / Q000503:physiopathology; D020830:rac1 GTP-Binding Protein / Q000172:deficiency / Q000502:physiology*
C529518:CLEC-2 protein, mouse; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D051966:Phospholipase C gamma; D020830:rac1 GTP-Binding Protein; D011070:Poly I-C; D002118:Calcium