Phosphorylation and activation of Pannexin-1 upon platelet activation and thrombus formation

Objective Pannexin-1 (Panx1) is a single membrane channel, which functions as an ion channel for small molecules. Recently, it was shown that platelet activation induces opening of Panx1 channels, amplifies [Ca2+]int and induces platelet aggregation. Panx1-deficient platelets show defects in hemostasis in in vitro and in vivo. We aim to study Panx1 activation mechanisms in platelets to identify the impact on hemostasis and thrombosis in further detail.

Material and Methods Analysis of activation patterns of Panx1 phosphorylation after platelet activation, Panx1 inhibition by the specific inhibitor Probenecid and ex vivo analysis of thrombus formation on collagen under flow.

Results Western blot analysis demonstrated increased phosphorylation of tyrosine residue 198 (Tyr198) of Panx1 following platelet activation with classical platelet agonists. Tyrosine phosphorylation was shown to be fully dependent on Src - and partially dependent on PKC kinases in human platelets. Moreover, murine Panx1-deficient platelets display reduced Src phosphorylation, which goes ahead with the results in human platelets. Interestingly, ADP does not phosphorylate Panx1 at Tyr198 but at Tyr308; another well-known phosphorylation site of Panx1. Here, Src kinases are only partially involved in phosphorylating Tyr308, which indicates other activation mechanisms of Panx1 channels. Blockage of Panx1 with Probenecid led to reduced [ATP]ex after platelet activation with collagen-related peptide (CRP), Par4 peptide and thromboxane, but not with ADP. Under flow conditions, the inhibition of Panx1 leads to reduced thrombus formation on collagen at low (450 s-1) and moderate (1000 s-1) arterial shear rates ex vivo. Moreover, initial experiments provided evidence that low as well as high agonist activation of platelets lead to extracellular cleavage of Panx1 channels in a time dependent manner, which is known to irreversibly open the channel in inflammatory cells.

Conclusion Panx1 channels function as ATP-release channels in platelets to support arterial thrombus formation. Panx1 activation is regulated by phosphorylation at different tyrosine residues following platelet activation. These results suggest an important role of Panx1 in hemostasis by releasing extracellular ATP to enable cell communication and to support thrombus formation.

Publication History

Article published online:
18 June 2021