A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis

We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A2 receptor (TxA2R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA2R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA2R, U46619 did not increase cytosolic free Ca2+ concentration, indicating loss of receptor function. The TxA2R antagonist [3H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA2R. This is the second naturally occurring TxA2R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA2R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA2R function in vivo.