Differential regulation of cyclic adenosine monophosphate by phosphodiesterase 3A discriminates thrombin-induced protease activated receptor 1– and 4–dependent platelet activation

BACKGROUND: Human platelets express 2 thrombin-activated G protein-coupled receptors (GPCRs), protease activated receptor (PAR)1 and PAR4. Previous studies have demonstrated PAR1 activation to be fast on-fast off while PAR4 activation was slower but more sustained. However, how PARs regulate second messengers such as cyclic adenosine monophosphate (cAMP), a negative regulator of platelet activation, remain unknown. OBJECTIVES: This study investigated how PAR1 and PAR4 activation by thrombin regulates cAMP levels and platelet activation. METHODS: . Platelets were stimulated with various thrombin concentrations, and calcium flux and platelet aggregation were measured. PAR regulation of cAMP in megakaryocytes (MKs) was also studied by genetic disruption of PAR1 or PAR4 using CRISPR/Cas9 editing. RESULTS: , cAMP was significantly reduced after thrombin stimulation when platelets or MKs were activated through PAR1, but not PAR4. Calcium flux was highly associated with cAMP level changes. High concentrations of cAMP were associated with low calcium flux with PAR4 stimulation. In contrast, stimulating PAR1 decreased cAMP levels, and this was associated with a more pronounced calcium flux. Phosphodiesterase (PDE)3A is involved in regulating cAMP levels. Blocking PDE3A with a pharmacological inhibitor in platelets or genetic knockout in MKs reversed PAR1-induced decrease of cAMP level, while PAR4 was not affected by PDE3A inhibition. CONCLUSION: The data suggest differences between PAR1 and PAR4 induced thrombin activation are due, in part, to cAMP regulation by PDE3A.