The P2 Receptors and Congenital Platelet Function Defects

ABSTRACT

Platelets possess three P2 receptors: two (P2Y₁ and P2Y₁₂) are receptors for adenosine diphosphate (ADP), and one (P2X1) is a receptor for adenosine triphosphate (ATP). The P2Y₁ receptor, which is coupled to Gq and phospholipase C-β, is responsible for mobilization of ionized calcium from internal stores and mediates the ADP-induced platelet shape change and initial wave of rapidly reversible aggregation. The other ADP receptor, P2Y₁₂, is negatively coupled to adenylyl cyclase through Gi and mediates a progressive and sustained ADP-induced aggregation not preceded by shape change. In addition, this receptor plays an important role in the potentiation of platelet secretion induced by several platelet agonists. The combined action of P2Y₁ and P2Y₁₂ is necessary for the full platelet aggregation response to ADP. Four patients with severe deficiency of P2Y₁₂ have been described so far. Sequence analysis of the P2Y₁₂ locus of three of these patients revealed homozygous mutations that produced a frame shift mutation and premature truncation of the protein. The fourth patient had an allele with a frame shift mutation and a normal allele, which could be silenced by an additional, as yet unknown, mutation. More recently, we described a patient with a congenital bleeding disorder and a dysfunctional P2Y₁₂. The patient is a compound heterozygote, in whom one allele contained a G to A transition resulting in an Arg²⁵⁶ to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg²⁶⁵ to Trp codon substitution (R265W). The two substitutions are located in TM6 and EL3 of the receptor. Stable Chinese hamster ovaries (CHO) cell lines were established expressing either wild-type P2Y₁₂ and P2Y₁₂ ^R256Q or P2Y₁₂ ^R265W. Neither mutation blocked the ability of the P2Y₁₂ receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1 to 10 μM) greatly inhibited the forskolin-induced increase of cyclic adenosine monophosphate (cAMP) in CHO cells transfected with wild-type P2Y₁₂, whereas CHO cells transfected with either mutant protein were only partially inhibited by ADP. Thus, the molecular basis for the patient’s dysfunctional platelet phenotype is explained by missense mutations and the expression of a dysfunctional P2Y₁₂ receptor. The localization of both mutations in TM6 and EL3 identifies this region of P2Y₁₂ as a structurally and functionally critical region of the receptor.

KEYWORDS

Platelets - P2 receptors - congenital defects - dysfunctional platelets - P2Y₁₂ defects

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