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

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 α_IIbβ₃ 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 α_IIbβ₃. 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 α_IIbβ₃ 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.