Thrombin and other soluble agonists promote platelet activation by binding to G-protein-coupled receptors, which typically recruit phospholipase C and trigger cytosolic Ca2+ increase and protein kinase C stimulation. Platelets also express several tyrosine kinases, including different members of the Src family (mainly Src and Lyn) and of the focal adhesion family (such as FAK and Pyk2), which are known to be activated upon stimulation with thrombin. Both Src family kinases (SFKs) and Pyk2 are required for thrombin-induced aggregation and thromboxane A2 production, and both of them have been shown to be regulated by intracellular Ca2+ (Xiang et al, J Biol Chem 2012; Canobbio et al, Blood 2013). However, the hierarchy of protein tyrosine kinases activation, as well as the molecular mechanism linking agonist-induced increase of cytosolic Ca2+ to protein tyrosine phosphorylation is poorly understood. In this study, we investigated the interplay between intracellular Ca2+, Pyk2 and SFKs in the regulation of protein tyrosine phosphorylation in thrombin-stimulated platelets.
In the presence of the ADP scavenger apyrase, thrombin caused the time-dependent tyrosine phosphorylation of several proteins in aspirin-treated human, as well as mouse platelets. This effect was severely reduced by the intracellular Ca2+ chelator BAPTA-AM and by the SFKs inhibitor PP2, and was totally suppressed only when BAPTA-AM and PP2 were used in combination, indicating that cytosolic Ca2+ and SFKs activity cooperate in the regulation of the whole process of protein tyrosine phosphorylation elicited by thrombin. Analysis of SFKs activity in human and mouse platelets with an anti-pY416Src antibody, that detects the autophosphorylation site in the catalytic domain, revealed that cytosolic Ca2+ is upstream SFKs activation upon thrombin stimulation. Accordingly, SFKs phosphorylation in the catalytic domain was also directly promoted by the Ca2+ ionophore A23187. Immunoblotting analysis on high resolution gels allowed to distinguish between Src and Lyn, and revealed that PP2 prevented phosphorylation of both kinases, but BAPTA-AM only inhibited Src , but not Lyn phosphorylation caused by thrombin. The Ca2+ ionophore A23187 also triggered the phosphorylation and activation of the Ca2+-dependent focal adhesion kinase Pyk2, and stimulation with thrombin promoted Pyk2 activation by a Ca2+-dependent mechanism. However, thrombin- or A23187-induced activation of Pyk2 was also reduced by the SFKs inhibitor PP2, and both PP2 and BAPTA-AM were required to completely suppress Pyk2 activation. In platelets from Pyk2 knockout mice, stimulation of SFKs by thrombin was strongly reduced, as it was the overall pattern of protein tyrosine phosphorylation. Importantly, also SFKs activation caused by Ca2+ionophore was significantly impaired in Pyk2-deficient platelets. Finally, in the absence of Pyk2, thrombin-induced aggregation was reduced to a level comparable to that of wild type platelets treated with PP2, although inhibition of SFKs by PP2 further reduced aggregation of Pyk2-deficient platelets.
These results indicate that in thrombin-stimulated platelets intracellular Ca2+ increase promotes Pyk2 stimulation, which initiates SFKs activation and establishes a positive loop which reinforces the Pyk2/SFKs axis to allow the subsequent massive tyrosine phosphorylation of multiple platelet substrates.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.