Abstract/Summary

Introduction: Connexins (Cxs), a large family of proteins, are expressed in numerous mammalian cells. Cxs oligomerise into hexameric hemichannels in the plasma membrane. The hemichannels on adjacent cells can dock together to form gap junctions (GJs), which regulate the direct trafficking of molecules (up to approximately 1 kDa) from one cell to another, and serve to allow coordinated responses between cells in tissues. Cx37 and Cx40 are expressed in human platelets and their selective inhibition in human platelets or deletion in transgenic mice attenuates platelet function. Notable levels of Cx62 transcripts have been reported in megakaryocytes, although nothing is known of the protein expression or function of this relatively recently discovered Cx in any tissue. The objective of this study was to determine the role of Cx62 in human platelets. Results: The expression of Cx62 in human and mouse (Cx57, mouse homologue) platelets was confirmed by western blotting and immunocytochemistry. A novel mimetic peptide that targets the second extracellular loop of Cx62 was designed in this study. The newly designed Cx62 mimetic peptide, 62Gap27, reduced hemichannel permeability and GJ-mediated intercellular communication. Several features of agonist-induced platelet activation, such as aggregation, degranulation, fibrinogen binding to integrin αIIbβ3, Ca2+ mobilisation and integrin αIIbβ3 outside-in signalling (which controls clot retraction and spreading), were inhibited by 62Gap27. No effects were observed after treatment with the scrambled peptide control. The selectivity of 62Gap27 for Cx62 was confirmed using Cx57 knockout mice. Additionally, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Thrombus formation (in vitro and in vivo) and tail bleeding were inhibited substantially by 62Gap27, suggesting that Cx62 regulates thrombosis and haemostasis, respectively. III Experiments performed to explore the effects of Cx62 inhibition on platelet signalling revealed that treatment with 62Gap27 can negatively-regulate tyrosine phosphorylation of multiple glycoprotein VI (GPVI) signalling mediators, including Syk, LAT phospholipase Cγ2; it also negatively regulates PKC substrate phosphorylation. In addition, treatment of resting and activated platelets with 62Gap27 increased protein kinase A (PKA) activation, as observed via vasodilator-stimulated phosphoprotein VASP S157 phosphorylation. This activation was induced in a cyclic adenosine monophosphate (cAMP) -independent manner. Conclusion: Cx62 and Cx57 are expressed in human and mouse platelets, respectively, where they play a fundamental role in platelet function, thrombus formation, and thereby haemostasis and thrombosis. Future work is required to determine the protein interactions of Cx62 and the mechanisms that allow platelets to control Cx function. Furthermore, the nature of the molecules that traffic through Cx62 hemichannels in isolated platelets or GJs within a thrombus also requires investigation.