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

Introduction: The interaction of platelet glycoprotein (GP) Ib alpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIb alpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM 17) was identified recently to play an essential role in agonist induced GPIb alpha shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIb alpha shedding. Methods and Results: GPIb alpha was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIb alpha shedding was significantly higher than that in calcium ionophore or alpha-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIb alpha 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 GPIb alpha shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIb alpha 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. Conclusions: These data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIb alpha 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. (C) 2009 Elsevier Ltd. All rights reserved.