Impaired Platelet Function in a P2Y12-Deficient Patient Caused by a Mutation in the Translation Initiation Codon.

Abstract

Recent studies have demonstrated that ADP plays a critical role in platelet activation. Platelets possess at least two major G protein-coupled ADP receptors that are largely responsible for platelet responses to ADP: P2Y1 and P2Y12. P2Y12 is the therapeutic target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel, and its congenital deficiency results in a bleeding disorder. In this study, we have identified a patient with the congenital P2Y12 deficiency and analyzed the role of P2Y12 in platelet function. The proband (OSP-1) is a 67-year-old Japanese female with a lifelong history of easy bruising. In response to high concentrations of agonists OSP-1's platelets showed a specifically impaired aggregation to ADP stimulation. The impaired platelet aggregation was neither improved even at 100 μM ADP stimulation nor reduced by adding 1 μM AR-C69931MX, a specific P2Y12 antagonist. ADP induced platelet shape change normally and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets, further suggesting the defect in P2Y12 dependent signal. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. HEK293 cells transfected with wild-type P2Y12 construct expressed a 60 kD-protein, while cells transfected with mutant P2Y12 construct failed to express any related protein. These data confirmed that the mutation was responsible for the deficiency in P2Y12 and denied the possibility that the substitution might induce an alternative translation starting at downstream ATGs leading to an expression of shorter form of P2Y12.Platelet spreading onto immobilized fibrinogen was impaired in OSP-1 platelets as well as in control platelets in the presence of 1 μM AR-C69931MX. Under static conditions, activation of integrin αIIbβ3 measured by PAC1-binding in OSP-1 platelets was only 5 to 12 % of controls even at high concentrations of PAR1-TRAP, PAR4-TRAP or U46619. These results suggest that ADP released by Gq-coupled receptors and P2Y12-dependent signal are critical for sustained activation of integrin αIIbβ3. Real time observations of thrombogenesis on a type I collagen-coated surface under a high shear rate (2000 s−1) revealed that thrombi were unstable. Most of the aggregates of OSP-1 platelets were unable to resist against high shear stress and loosely-attached aggregates came off into the blood stream. Thrombus height and volume measured at the plateau phase was 50% of control. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signals are critical for platelet spreading, stable integrin αIIbβ3 activation and, as a consequence, for stabilization of thrombus. These results provide a novel insight for the role of P2Y12 and endogenous ADP released into a limited space between thrombus-forming platelets.