Abstract
Abstract 1132
The protein family of pannexins (Panx) contains three members (Panx1, Panx2 and Panx3) that are highly evolutionarily conserved from worms to mammals, and that form hemichannels involved in the transport of ions like Ca2+ and small molecules such as ATP into and out of cells. Panx1 is ubiquitously expressed in human tissues, and has been shown to mediate ATP release in erythrocytes, taste buds, airway epithelia and astrocytes. In platelets, ATP serves as an important paracrine and autocrine signaling molecule, binding to P2X1 purinergic receptors on the platelet surface, regulating shape change, and amplifying platelet responses to agonists such as collagen. Based on the role of Panx and the significance of ATP release in platelets function, we investigate the expression and function of Panx1 in human platelets.
Because the role of Panx1 in platelet function has not been studied previously, we sought to investigate the expression and function of Panx1 in human platelets.
Materials and methods.
Washed human platelets, isolated from ACD anticoagulated whole blood, were resuspended in Tyrode's saline solution-HEPES to a final concentration of 2.5 × 108/mL, stimulated with collagen and collagen related peptide (CRP), and their activation and secretion measured by whole-blood lumi-aggregometry in the presence or absence of Panx1-specific channel blockers: carbenoxolone (Cbnx) 100 μM; probenecid (Pbn) 100 μM and mefloquine (Mfq) 10 μM. Expression of Panx1 was determined by western blot, flow cytometry and fluorescence microscopy using a specific rabbit polyclonal anti-Panx 1 antibody. Panx hemichannel function was assessed by sulforhodamine B dye uptake taken up by ADP or collagen-stimulated platelet in the presence or absence of known Panx1 channel blockers.
Results.
Western blots of platelet lysates with rabbit anti-Panx1 antibody revealed a 47 kDa band corresponding to the known molecular weight of the Panx1 monomer. Flow cytometry performed on permeabilized platelets showed a significant shift in fluorescence intensity in platelets incubated with the anti-Panx1 antibody. Strong, specific staining was also observed by fluorescent microscopy of permeabilized platelets incubated with the anti-Panx1 antibody. Both platelet shape change and ATP release induced by CRP (0.5μg/ml) or collagen (1.0 μg/ml) were inhibited more than 50 % by Cbnx and Pbn; however, the most significant effect was obtained with platelets exposed to mefloquine 10 μM (see table):
Platelet stimulation with Collagen or ADP induced an increase in sulforhodamine B uptake which could be significantly inhibited by preincubation with the Panx1 channel blocker probenecid.
Conclusions.
Our results demonstrated that human platelets express Panx1 Taken together, these data demonstrate that Panx1 is expressed by human platelets, evidenced by the presence of the protein by western blot, flow cytometry and immunofluorescence microscopy. From a functional standpoint, the significant effect of carbenoxolone, probenecid and especially mefloquine on collagen and CRP-induced shape change and ATP release, suggest that in human platelets Panx1 is involved in ATP release. In other cell types Panx1 channels can be opened by activation of P2 receptors; however, further studies are required to elucidate a possible association and functional interaction of Panx1 with P2X1 in platelets. Since purinergic signaling is a fundamental mechanism in platelet activation, inhibition of Panx1 hemichannels could have therapeutic potential when dampening platelet activation is desired.
Disclosures:
No relevant conflicts of interest to declare.
Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets
