A novel thromboxane A 2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis

Andrew D. Mumford, Ban B. Dawood, Martina E. Daly, Sherina L. Murden, Michael D. Williams, Majd B. Protty, Jennifer C. Spalton, Mark Wheatley, Stuart J. Mundell, Steve P. Watson

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)363-369
Number of pages7
JournalBlood
Volume115
Issue number2
DOIs
Publication statusPublished - 14 Jan 2010
Externally publishedYes

Keywords

  • blood platelets
  • cho-k1 cells
  • ligands
  • thromboxane
  • bleeding diathesis
  • agonists
  • hemorrhage
  • blood platelet disorders

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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