Platelet dysfunction associated with the novel Trp29Cys thromboxane A2 receptor variant
Platelet dysfunction associated with the novel Trp29Cys thromboxane A2 receptor variant
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DOI：
10.1111/jth.12117
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发表时间：
2013-03-01
影响因子：
10.4
通讯作者：
Gaussem, P.
中科院分区：
文献类型：
--
作者：
Mumford, A. D.;Nisar, S.;Gaussem, P.
背景影响血栓素 A2 受体（TP 受体）结构的遗传变异为了解这一关键血小板和血管受体的功能提供了见解，但在未经选择的人群中非常罕见。目的 确定 TP 受体 Trp29Cys (W29C) 替代的功能后果。患者/方法 我们对指示病例 (P1) 进行了详细的表型分析，该病例具有血小板聚集和对 TP 受体途径激活剂的分泌反应减少以及杂合 TP 受体 W29C 替代的情况。对异源细胞中表达的变体 W29C TP 受体进行了分析。结果 P1 期血小板中的总 TP 受体表达与对照相似，但最大结合力降低，与 TP 受体拮抗剂 [3H]SQ29548 的结合亲和力降低。用 W29C TP 受体 cDNA 转染的 HEK293 细胞显示出与野生型 (WT) 对照相似的总 TP 受体表达。然而，与 WT 对照相比，TP 受体激动剂 U46619 在诱导表达 W29C TP 受体的 HEK293 细胞中胞质游离 Ca2+ 升高方面的作用较弱，表明受体功能降低。免疫荧光显微镜和细胞表面 ELISA 显示 HEK293 细胞中 W29C TP 受体的细胞内滞留和细胞表面表达减少。与血小板表型一致，与WT对照相比，[ 3 H]SQ29548与W29C TP受体的最大结合力和结合亲和力均降低。结论 这些发现扩展了非常罕见的 TP 受体缺陷性疾病的表型描述，并表明 W29C 取代通过减少表面受体表达和破坏配体结合来降低 TP 受体功能。
Background Genetic variations that affect the structure of the thromboxaneA2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. Objectives To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. Patients/Methods We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Results Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [3H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca2+ in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [3H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. Conclusion These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.