Prolonged inhibition of protein kinase A results in metalloproteinase-dependent platelet GPIbα shedding

IntroductionThe interaction of platelet glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbα ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbα shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbα shedding.Methods and ResultsGPIbα was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbα shedding was significantly higher than that in calcium ionophore or α-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbα shedding was reversed by PKA activator forskolin and partially inhibited by membrane-permeable calpain inhibitors. Furthermore, the metalloproteinase inhibitor GM6001 or EDTA completely inhibited H89 induced GPIbα shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIbα shedding occurred at 30 minutes after treatment with PKA inhibitor. Platelets prolongedly treated with PKA inhibitor exhibited significant decrease in botrocetin-induced aggregation and shear-induced adhesion on VWF.ConclusionsThese data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIbα ectodomain shedding. This finding has physiological implications for hemostasis and limiting thrombus infinite formation after platelet activation, and it also suggests a novel strategy to develop new drugs for thrombotic diseases.