Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets.
Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G(q), G(i), and G(z). Although activated by several platelet agonists, including thrombin and thromboxane A(2), the contribution of the G(12/13) signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G(12/13) activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 microm), selectively activates the G(12/13) signaling cascade resulting in platelet shape change without stimulating the G(q) or G(i) signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160(ROCK) inhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G(12/13) signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective G(i) or G(z) signaling, via selective stimulation of the P2Y(12) receptor or alpha(2A)-adrenergic receptor, respectively. Similar data were obtained when using low dose (10 nm), a thromboxane A(2) mimetic, to activate G(12/13) in the presence of G(i) signaling. These results suggest that selective activation of G(12/13) causes platelet GPIIb/IIIa activation when combined with G(i) signaling. Unlike either G(12/13) or G(i) activation alone, co-activation of both G(12/13) and G(i) resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G(12/13) and G(i)-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160(ROCK), PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of G(q) and G(i) pathways, but not by G(12/13) and G(i) pathways. These data demonstrate that co-stimulation of G(12/13) and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI 3-kinase is an important signaling molecule downstream of G(q) but not downstream of G(12/13) pathway.
Dorsam
Robert T
RT
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Kim
Soochong
S
Jin
Jianguo
J
Kunapuli
Satya P
SP
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048728:MAP Kinase Kinase Kinase 1; D008954:Models, Biological; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000378:metabolism; D054460:rho-Associated Kinases
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D004533:Egtazic Acid; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D004837:Epinephrine