Abstract
The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho–Rho kinase pathway independently of integrin αIIbβ3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50–1000 nM U46619 and/or 10 μM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbβ3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.
Highlights
Thromboxane (TX) A2, or its synthetic and natural analogs have a critical role in platelet activation
Using washed platelets in the presence of the ADP scavenger apyrase and Acetyl Salicylic Acid (ASA) we found that the specific inhibitor PP2 inhibited both α granule secretion (CD62P expression %) and the expression of the active form of the fibrinogen receptor (PAC-1 binding %) in response to low doses of U46619 plus epinephrine (Table 1)
The role of a tyrosine phosphorylation pathways in this response was further investigated examining platelets obtained from patients affected by chronic myeloid leukemia (CML) treated with dasatinib, an Active fibrinogen receptor
Summary
Thromboxane (TX) A2, or its synthetic and natural analogs have a critical role in platelet activation. These compounds, depending on the concentration used and their chemical structure, trigger different signaling pathways downstream the thromboxane-peroxide (TP) receptor that have been the object of several investigations. Stimulation of washed platelets with the thromboxane TXA2 analog U46619 in the nanomolar range of concentrations, in the presence of the ADP scavenger apyrase and under non-aggregating conditions, was shown to induce both Rho kinase-induced phosphorylation of myosin light chain (MLC) and tyrosine phosphorylation signals; to note, these pathways were implicated in triggering of platelet shape change, but not secretion and aggregation (Minuz et al, 2006). Platelets treated with low concentrations of U46619 in the presence of substimulatory doses of epinephrine, that acts through a Gz coupled α2 adrenoreceptor, undergo a full release reaction and platelet aggregation response
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