A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction

Thromb Haemost. 2014 May 5;111(5):923-32. doi: 10.1160/TH13-08-0672. Epub 2014 Jan 23.

Abstract

A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

Keywords: Inherited / acquired platelet disorders; acquired; gene mutations; platelet pathology / inherited; platelet pharmacology; receptors.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Asparagine / genetics
  • Blood Platelets / physiology*
  • Conserved Sequence / genetics
  • Down-Regulation / genetics
  • Female
  • Genetic Variation
  • HEK293 Cells
  • Hemorrhage / blood
  • Hemorrhage / genetics*
  • Heterozygote
  • Humans
  • Middle Aged
  • Mutation / genetics
  • Platelet Activation / genetics
  • Platelet Function Tests
  • Radioligand Assay
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, Thromboxane A2, Prostaglandin H2 / genetics
  • Receptors, Thromboxane A2, Prostaglandin H2 / metabolism*

Substances

  • Receptors, G-Protein-Coupled
  • Receptors, Thromboxane A2, Prostaglandin H2
  • Asparagine