Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin.
We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.
Atkinson
B T
BT
Department of Pharmacology, University of Oxford, UK. ben.atkinson@pharm.ox.ac.uk
Stafford
M J
MJ
Pears
C J
CJ
Watson
S P
SP
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001081:Apyrase / Q000378:metabolism; D018955:CD36 Antigens / Q000378:metabolism*; D003435:Crotalid Venoms / Q000494:pharmacology*; D004357:Drug Synergism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000819:agonists; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D037181:Lectins, C-Type; D051379:Mice; D010712:Phosphatidic Acids / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013928:Thromboxane A2 / Q000494:pharmacology
D018955:CD36 Antigens; D003435:Crotalid Venoms; D007211:Indoles; D037181:Lectins, C-Type; D010712:Phosphatidic Acids; D012701:Serotonin; C028890:convulxin; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D001081:Apyrase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C064758:Ro 31-8220; D007213:Indomethacin