Src family tyrosine kinases activate thrombin-induced non-capacitative cation entry in human platelets.
Platelet activation is critically regulated by an increase in intracellular calcium concentration ([Ca2+](i)). Although Ca2+ release from intracellular Ca2+ stores and subsequent store-operated Ca2+ entry are often thought to be the major contributors to increases in [Ca2+](i) evoked by most agonists, high concentrations of thrombin activate a Ca2+ entry pathway that is independent of Ca2+ store depletion (known as 'non-capacitative cation entry'-NCCE). The channel that conducts NCCE has not previously been clearly identified, and the mechanisms that regulate its activation are also unknown. Here we have investigated NCCE using fura-2-loaded human platelets. To investigate NCCE independently of other Ca2+ signaling pathways, the intracellular Ca2+ stores were first rapidly depleted in the absence of extracellular Ca2+. Sr2+ was then added to monitor maximal store-operated cation influx. Thrombin was then added to stimulate NCCE. Flufenamic acid, which inhibits Ca2+ entry through most TRPC isoforms, but potentiates entry through TRPC6, was found to block store-operated cation entry. In contrast, thrombin-induced NCCE was increased, suggesting the possible involvement of TRPC6. Since TRPC6 is regulated by Src family tyrosine kinases in some cells, we investigated the possible role of this kinase family in NCCE. PP2, a Src family tyrosine kinase inhibitor, completely abolished thrombin-induced NCCE. Furthermore, NCCE was enhanced by phenylarsine oxide and could be directly induced by vanadyl hydroperoxide, both tyrosine phosphatase inhibitors. These data indicate that Src family tyrosine kinase activation is a required step in NCCE activation. In conclusion NCCE may be an important regulator of platelet activation when local thrombin concentrations are high.
Harper
Matthew T
MT
Department of Physiology, Development & Neuroscience, University of Cambridge, Cambridge CB23EG, UK. m.harper@bris.ac.uk
Sage
Stewart O
SO
D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D015220:Calcium Channels / Q000097:blood; D002413:Cations, Divalent / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects; D050052:TRPC Cation Channels / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D013917:Thrombin / Q000378:metabolism*; D019061:src-Family Kinases / Q000097:blood*
D015220:Calcium Channels; D002413:Cations, Divalent; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D019061:src-Family Kinases; D013917:Thrombin; D002118:Calcium