The P2 receptors and congenital platelet function defects.
Platelets possess three P2 receptors: two (P2Y (1) and P2Y (12)) are receptors for adenosine diphosphate (ADP), and one (P2X1) is a receptor for adenosine triphosphate (ATP). The P2Y (1) receptor, which is coupled to Gq and phospholipase C-beta, 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, P2Y (12), 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 P2Y (1) and P2Y (12) is necessary for the full platelet aggregation response to ADP. Four patients with severe deficiency of P2Y (12) have been described so far. Sequence analysis of the P2Y (12) 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 P2Y (12). The patient is a compound heterozygote, in whom one allele contained a G to A transition resulting in an Arg (256) to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg (265) 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 P2Y (12) and P2Y (12)(R256Q) or P2Y (12)(R265W). Neither mutation blocked the ability of the P2Y (12) receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1 to 10 muM) greatly inhibited the forskolin-induced increase of cyclic adenosine monophosphate (cAMP) in CHO cells transfected with wild-type P2Y (12), 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 P2Y (12) receptor. The localization of both mutations in TM6 and EL3 identifies this region of P2Y (12) as a structurally and functionally critical region of the receptor.
Cattaneo
Marco
M
Unit of Hematology and Thrombosis, Ospedale San Paolo, Department of Medicine, Surgery and Dentistry, DMCO University of Milano, Via di Rudini 8, 20142 Milan, Italy. marco.cattaneo@unimi.it
D000244:Adenosine Diphosphate / Q000494:pharmacology / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D001791:Blood Platelet Disorders / Q000097:blood / Q000151:congenital* / Q000188:drug therapy / Q000235:genetics; D016466:CHO Cells; D018389:Codon, Nonsense; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology; D016368:Frameshift Mutation; D020868:Gene Silencing; D005838:Genotype; D006801:Humans; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D020125:Mutation, Missense; D010974:Platelet Aggregation / Q000187:drug effects; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D014162:Transfection
D018389:Codon, Nonsense; D008565:Membrane Proteins; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; C546384:P2ry1 protein, rat; C553058:P2ry12 protein, rat; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP