Integrin alpha2beta1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cgamma2 in platelets: role of Src kinase and Rac GTPase.
BACKGROUND
Platelet adhesion promoted by integrin alpha2beta1 induces integrin alpha(IIb)beta3 activation through the phospholipase C (PLC)-dependent stimulation of the small GTPase Rap1b.


OBJECTIVE
To analyze the mechanism of PLC activation downstream of alpha2beta1 that is required for regulation of Rap1b and alpha(IIb)beta3.


METHODS
Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through alpha2beta1. Tyrosine phosphorylation of PLCgamma2, PLC activation, accumulation of GTP-bound Rap1b and fibrinogen binding were measured and compared.


RESULTS
Integrin alpha2beta1 recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCgamma2, and was suppressed in platelets from PLCgamma2-knockout mice. Moreover, PLCgamma2(-/-) platelets were unable to accumulate active Rap1b and to activate alpha(IIb)beta3 upon adhesion through alpha2beta1. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCgamma2 in adherent platelets, but did not affect its activation, and both Rap1b and alpha(IIb)beta3 stimulation occurred normally. Importantly, alpha(IIb)beta3-induced phosphorylation and activation of PLCgamma2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin alpha2beta1 recruitment triggered the Src kinase-independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCgamma2 phosphorylation. However, when phosphorylation of PLCgamma2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCgamma2 activation, GTP-Rap1b accumulation, and alpha(IIb)beta3 stimulation.


CONCLUSIONS
Src kinases and the Rac GTPases mediate independent pathways for PLCgamma2 activation downstream of alpha2beta1.
Department of Biochemistry, University of Pavia, Pavia, Italy.
G F
GF
Guidetti
B
B
Bernardi
A
A
Consonni
P
P
Rizzo
C
C
Gruppi
C
C
Balduini
M
M
Torti
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002448:Cell Adhesion; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D038982:Integrin alpha2beta1 / Q000502:physiology*; D051379:Mice; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D038982:Integrin alpha2beta1; D047428:Protein Kinase Inhibitors; D014443:Tyrosine; D019061:src-Family Kinases; D051966:Phospholipase C gamma; D020744:rac GTP-Binding Proteins