The major platelet integrin αIIbβ3 plays a critical role in thrombus formation and has been a major target for anti-thrombotic therapy. The precise molecular mechanism of activation of the platelet integrin αIIbβ3 is not understood although a critical role for the conserved αIIb-integrin cytoplasmic motif, KxGFFKR, has been identified. We have recently shown, using a synthetic KVGFFKR peptide probe, evidence for a direct and specific interaction between the platelet integrin αIIbβ3 and a protein involved in regulatory volume decrease, ICln. ICln is a volume regulating chloride channel protein that is capable of forming a pore structure within the membrane. Previously we demonstrated by surface plasmon resonance that purified αIIbβ3 binds to His-tagged recombinant ICln (His-ICln). We now show that purified αIIbβ3 binds to His-ICln in protein pulldown assays using a western blot detection system. In addition, His-ICln selectively pulls down endogenous αIIbβ3 from platelet lysates, but in an activation dependent manner. These comparisons were carried out in lysates from resting platelets and platelets activated with U46619 (10μM), TRAP (5μM) and thrombin (0.2U/ml) for 3 minutes at 37°C, unstirred. Reduced integrin precipitation was observed in lysates from platelets treated with acyclovir (100μM), a known pharmacological inhibitor of ICln, consistent with an activation dependent interaction.
As the channel function of ICln is nucleotide sensitive we set out to further identify if ICln regulates the activation of αIIbβ3 or if the integrin contributes to the activation of ICln. Low dose nucleotides were found to effect platelet function. Gel-filtered platelets treated with ATP (2nM) and apyrase (1U/ml) showed an increase in PAC-1 binding that could be inhibited with acyclovir (1mM). U46619 stimulation caused an increase in PAC-1 binding and enhanced platelet aggregation in the presence of ATP/apyrase that could also be inhibited by acyclovir in a dose-dependent manner (10μM–1mM). Thus, activation of ICln by low dose nucleotides can induce αIIbβ3 activation without further stimulus. However maximum αIIbβ3 activation is observed upon stimulation of both αIIbβ3 and ICln. The presence of an ICln inhibitor, acyclovir, interferes with platelet aggregation and integrin activation with and without specific ICln stimulation, suggesting a co-dependence between αIIbβ3 and ICln to obtain maximum platelet activation. Taken together with results of the protein- protein interaction studies this data suggests an activation-dependent, functional interaction between the volume regulating ICln protein and the platelet integrin αIIbβ3.