Role of CalDAG-GEFI in Calcium-Dependent Thromboxane A2 Generation

The second messenger Ca2+ is essential for various platelet functions, including integrin activation, thromboxane A2 (TxA2) generation and granule release. In previous studies, we have identified Ca2+- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a key molecule regulating Ca2+-dependent activation of Rap1 and integrins in platelets. Here we demonstrate that CalDAG-GEFI/Rap1 signaling is required for Ca2+-dependent ERK 1/2 activation and TxA2 generation in platelets. Impaired TxA2 formation was observed in collagen- and convulxin-activated CalDAG-GEFI deficient platelets, both at high and low doses of agonists. In contrast, no defect in TxA2 generation was observed in mutant platelets stimulated with high doses of PAR4-activating peptide, suggesting that CalDAG-GEFI may be particularly important for TxA2 generation in platelets activated through the major collagen receptor, GPVI. Our data further demonstrate a strong correlation between reduced TxA2 generation and impaired activation of ERK1/2 and Rap1 in mutant platelets. Pretreatment of CalDAG-GEFI−'/− platelets with 2-MesAMP, an inhibitor of P2Y12/Gi signaling, blocked the residual TxA2 production. These data suggest that CalDAG-GEFI synergizes with the Gi signaling pathway to control TxA2 generation. Finally, addition of thromboxane A2 analog, U46619, restored dense granule release in CalDAG-GEFI-deficient platelets stimulated with low doses of convulxin, suggesting that impaired TxA2 generation contributes to the defect in granule release previously described for CalDAG-GEFI−/− platelets. In conclusion, our studies strongly support that CalDAG-GEFI is the elusive Ca2+ sensor that links increases in intracellular Ca2+ to the signaling pathways regulating integrin activation and TxA2 formation in stimulated platelets.