The Src family kinases and protein kinase C synergize to mediate Gq-dependent platelet activation.
The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y(12) deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G(q). We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G(q) deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G(q)/Ca(2+) signaling. Moreover, A23187-induced thromboxane A(2) synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A(2) production in wild-type and P2Y(12) deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α(IIb)β(3) activation, and aggregation of P2Y(12) deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or LY294002. These data indicate that Ca(2+)/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G(q)-dependent platelet activation.
Saha Cardiovascular Research Center, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, USA.
Binggang
B
Xiang
Guoying
G
Zhang
Lucia
L
Stefanini
Wolfgang
W
Bergmeier
T Kent
TK
Gartner
Sidney W
SW
Whiteheart
Zhenyu
Z
Li
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D018821:CD18 Antigens; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C548577:P2ry12 protein, mouse; D040201:Platelet Membrane Glycoprotein IIb; D058925:Receptors, Purinergic P2Y12; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; D000001:Calcimycin; D005340:Fibrinogen; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D011493:Protein Kinase C; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium