PB0404 - Connexins are post-translationally modified in activated platelets; potential role of calpain

Background: Platelets express several connexins that assemble as hemichannels at the plasma membrane or dock to form gap junctions with adjacent cells facilitating the release or transfer of bioactive molecules. Selective inhibition of connexin 37 (Cx37), Cx40 or Cx62 reduces thrombus formation in vivo and limits platelet activation in vitro. To date, several mechanisms of post-translational modification (PTM) are known to regulate the activity of connexins. The regulation of platelet connexins by PTMs warrants further investigation.

Aims: We aimed to investigate the role of proteolysis-mediated PTM of Cx62 by calpain.

Methods: Proteolytic cleavage of Cx62 was assessed by immunoblot and platelet function was studied by aggregometry and calcium flux assays. We tracked efflux and transfer of calcein dye to evaluate Cx62 activation in single platelets by flow cytometry or within thrombi formed under arteriolar shear rates.

Results: We performed in silico analysis of Cx62 to scan for post-translational modification sites and identified a potential calpain cleavage site within its extracellular face. Immunoblot analysis revealed a cleavage product of Cx62 that appeared following platelet activation, which was blocked by calpeptin. To understand whether Cx62 PTM has functional consequences on hemichannel activities, we designed a mimetic peptide for the predicted cleaved Cx62 N-terminal sequence (62Pept-NT) as a decoy substrate to diminish Cx62 PTM in platelets. Treatment of platelets with 62Pept-NT delayed calcein dye release from activated platelets with no effect on platelet aggregation. Finally, we studied the functional consequence of calpain cleavage of platelet Cx62 within thrombi. Under control conditions we observed real-time transfer of calcein dye between platelets. Indicative of a role for calpain, addition of 62Pept-NT reduced intercellular communication by 30%.

Conclusion(s): Our data demonstrate the presence of a calpain cleavage site on the extracellular face of Cx62 that regulates the opening of hemichannels and gap junctions.