PB0403 - Real-time assessment of inter-platelet communication and coordination of calcium signals by connexin gap junctions

Background: Following vascular injury, platelets rapidly aggregate to prevent blood loss and promote wound repair. Mechanisms regulating inter-platelet communication and coordination of these responses are incompletely understood. Platelets express several members of the connexin gap junction family that form intercellular channels. Selective inhibitory peptides of these channels reduce thrombus formation in vivo, but inter-cellular signals conveyed by platelet connexins remain unclear.

Aims: To evaluate the role of connexins in coordination of agonist-evoked platelet calcium signalling.

Methods: Calcein diffusion between platelets was employed as a marker of connexin activation during thrombus formation assays. Briefly, 10% of platelets were co-stained with calcein and CD61 antibodies, allowing monitoring of calcein diffusion beyond these ‘donor’ platelets. For calcium imaging, platelets were spread on fibrinogen and TRAP6-evoked calcium transients were monitored by confocal microscopy.

Results: Under arteriolar shear we formed thrombi and tracked movement of calcein dye from donor platelets into the cytosol of adjacent cells. To evaluate the role for connexins in this process, we used the pan-connexin inhibitor carbenoxolone or the connexin 37 (Cx37)-specific peptide inhibitor 37,43Gap27. In the presence of carbenoxolone calcein diffusion was reduced by 60%, whilst 37,43Gap27 led to a 30% reduction. We therefore investigated whether multiple connexin isoforms contributed to inter-cellular communication using a peptide cocktail to target the major platelet connexins (Cx37, Cx40 and Cx62). This approach reduced dye transfer to the same extent as carbenoxolone. Finally, we studied TRAP6-evoked calcium signals in immobilised platelets and calculated the time interval between calcium spikes in adjacent cells (lag time). Incubating of platelets with carbenoxolone reduced coordination of calcium signals and increased lag time.

Conclusion(s): Connexins facilitate coordination of inter-platelet calcium signals, which may involve diffusion of cytosolic calcium or IP3. It is interesting to speculate that connexins may relay signals between the core and shell to regulate thrombus formation.