A novel thromboxane A ₂ 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 (TxA ₂R) 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 TxA ₂R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA ₂R, U46619 did not increase cytosolic free Ca concentration, indicating loss of receptor function. The TxA ₂R 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 TxA ₂R. This is the second naturally occurring TxA ₂R 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 TxA ₂R 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 TxA ₂R function in vivo.