Epinephrine induces intracellular Ca2+ mobilization in thrombin-desensitized platelets: a role for GPIb-IX-V

In this work we have investigated the ability of epinephrine to trigger the release of intracellular Ca2+ in thrombin-desensitized platelets. Addition of thrombin to platelets in the presence of extracellular EGTA caused a rapid and transient release of Ca2+ from intracellular stores and rendered platelets unresponsive to a second addition of the same agonist. Although epinephrine alone had no effect on intracellular Ca2+ mobilization, its addition to thrombin-desensitized platelets was associated to a rapid and evident secondary release of intracellular Ca2+. This effect of epinephrine was not observed when platelets were desensitized with other agonists able to induce phospholipase C activation, including convulxin, U46619, and ADP. Although the platelet receptor for epinephrine is coupled to the Gi family member Gz, no secondary Ca2+ release was seen in thrombin-desensitized platelets upon stimulation of other Gi-coupled receptors, including the P2Y12 receptor and the CXCR4. Addition of hirudin to thrombin-desensitized platelets prevented epinephrine-promoted secondary release of Ca2+, indicating that thrombin, rather than epinephrine itself, is actually responsible for this event as a consequence of thrombin receptors resensitization. Studies with platelets stimulated with specific PAR1- and PAR4- activating peptides proved that neither one of these thrombin receptors were involved in the secondary epinephrine-assisted Ca2+ release. Moreover, we found that thrombin was still able to induce a reduced, but evident release of Ca2+ from internal stores in PAR1- and PAR4-desensitized platelets, which could be followed by a secondary Ca2+ release upon subsequent addition of epinephrine. Importantly, both the primary and the secondary Ca2+ release induced by thrombin and epinephrine in PAR1- and PAR4-desensitized platelets were abrogated upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. These results demonstrate a direct role of thrombin binding to GPIb-IX-V in the mobilization of Ca2+ from intracellular stores, and reveal that epinephrine can restore this process in desensitized platelets, thus prolonging the effect of thrombin stimulation.