Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations.
Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations.
To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution.
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.
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.
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.
The thromboxane A2 (TxA2) receptor (TP receptor) is a member of the prostanoid subfamily of class A G-protein-coupled receptors (GPCRs) that is expressed on platelets, monocytes, macrophages, vascular endothelium, and smooth muscle cells . Activation of the TP receptor by the agonists TxA2, prostaglandin H2 or the isoprostanes results in a variety of responses in vascular tissues, including platelet activation, vasoconstriction, and cellular proliferation . In platelets, activation responses are mediated by the 343-residue TP receptor α-isoform , which couples through Gq family G-proteins to cause phospholipase Cβ-mediated Ca2+ release and protein kinase C activation . The platelet TP receptor also signals through G13 family G-proteins to induce Rho-mediated platelet activation . Thus, the TP receptor is a critical mediator of platelet function in hemostasis, and is an attractive target for antithrombotic drugs .
Critical functional regions within the TP receptor have been investigated with several experimental techniques, including site-directed mutagenesis to generate artificial variant receptors for expression and functional analysis in heterologous cells. These approaches have helped to localize the TP receptor ligand-binding site to extracellular loop (ECL)2 [7, 8] and the Gq coupling site to intracellular loop (ICL)1 and ICL2 [9, 10]. Rare naturally occurring variants in the TP receptor gene (TBXA2R) offer a powerful complementary approach to determine TP receptor structure–function relationships, as the effects of receptor variations can be studied in native cells, such as platelets, from affected individuals. Clinical evaluation of affected individuals also provides insights into the biological consequences of TP receptor dysfunction. To date, only a single quantitative defect causing reduced TP receptor expression (TBXA2R c.167dupG ) and qualitative defects caused by Arg60Leu (R60L) [12, 13] and Asp304Asn (D304N)  TP receptor substitutions have been reported.
We now extend the phenotypic description of thromboxane receptor deficiency (OMIM #614009) by reporting the phenotype of a new index case with a novel Tyr29Cys (W29C) substitution within the TP receptor transmembrane domain (TM)1. We show that the W29C substitution is unique among the recognized variants by causing loss of TP receptor function, both through reduced surface receptor expression and reduced ligand binding.
Control platelets from healthy donors were obtained from the French blood bank institute (EFS), according to the agreement between Paris Descartes University and EFS (C CPSL UNT No. 12/EFS/064). Blood samples were obtained from members of the study kindred after informed signed consent in accordance with the 2008 Declaration of Helsinki. Aggregation in stirred platelet-rich plasma (PRP) was monitored at 37 °C with a four-channel aggregometer (Regulest, Nancy, France) after addition of arachidonic acid (AA; 1.5–2 mm; Helena, Beaumont, TX, USA), U46619 (1–5 μm; Calbiochem, MERK KGaA, Darmstadt, Germany), collagen (1 μg/mL; Horm, Nycomed, Ismaning, Germany), ADP (5–10 μm; Sigma Aldrich, Poole, UK), and ristocetin (1 mg/mL; Sigma Aldrich). To assess platelet secretion, unstirred PRP was first incubated with eptifibatide (4 μg/mL) for 3 min at 37 °C, and then stimulated with the protease-activated receptor 1 agonist peptide thrombin receptor agonist peptide (TRAP) (50 μm; Diagnostica Stago, Theale, UK) or U46619 (20 μm) for 15 min. Platelets were stained with mouse anti-CD62P–phycoerythrin (Immunotech, Marseille, France) and anti-CD63–fluorescein isothiocyanate (FITC) (Immunotech) IgG1 mAbs, or a negative isotype control. mAb binding was determined with a FACScan flow cytometer (Becton Dickinson, Oxford, UK).
The total platelet expression levels of the TP receptor were determined by fixation and permeabilization of platelets in PRP with 4% paraformaldehyde and 0.5% saponin before flow cytometry with the mAbs SC-31260 and SC-18377 (TP receptor extracellular and intracellular regions, respectively; Santa Cruz Technologies, Santa Cruz, CA, USA) and SC-2028 (normal goat IgG isotype control) as goat polyclonal primary antibodies, and FITC–anti-goat IgG as a secondary antibody (Jackson ImmunoResearch, Newmarket, UK). For Western blot analysis, platelet protein extracts prepared in SDS sample buffer were resolved on a 7.5% polyacrylamide gel and visualized with SC-18377, SC-30036 (TP receptor intracellular domain), and anti-goat IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch).
TP receptor and P2Y12 receptor ligand binding was determined as previously described  in 4% formaldehyde-fixed platelets resuspended in binding buffer (20 mm Hepes and 1 mm MgCl2). After incubation with [3H]SQ29548 (3 Ci mm−1, 0.01–0.1 μm) for 20 min at room temperature, either in the presence or in the absence of unlabeled ligand (10 μm), reactions were terminated with ice-cold binding buffer and rapid filtration through Whatman GF/C glass fiber filters under vacuum. Radioactivity bound to the filters was measured by scintillation counting. Control experiments were performed with the non-specific P2Y purinergic receptor ligand [3H]MeSADP (3 Ci [111 GBq mm]) in the absence or presence of the P2Y12 receptor antagonist AR-C69931MX (10 μm), to determine P2Y12 receptor-specific radioligand binding.
The TBXA2R coding sequence was amplified by PCR from genomic DNA, and was sequenced with an ABI 3100 sequencer (Applied Biosystems, Carlsbad, CA, USA). Sequence variations were identified by comparison with the reference sequence for the TP receptor α-isoform NM_001060.5.
The W29C TP expression construct comprised a DNA3.1 hygromycin vector containing the human TP receptor α-isoform cDNA with an N-terminal FLAG epitope tag mutagenized with a QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Variant and wild-type TP receptor expression constructs were stably transfected into HEK293 cells as described previously . TP receptor expression was assessed by cell surface ELISA and immunofluorescence microscopy with the FLAG M2 mAb (Sigma-Aldrich), as described previously .
Transfected cells cultured on poly(l-lysine)-coated glass coverslips were first incubated with fura-2/AM (3 μm) at 37 °C for 60 min. The cells were then continuously perfused with Locke's solution (154 mm NaCl, 5.6 mm KCl, 1.2 mm MgCl2, 2.2 CaCl2, 5 mm Hepes, and 10 mm glucose, pH 7.4) while fluorescence was measured at 340-nm and 380-nm excitation and 510-nm emission. U46619 (0.001–10 μm) was perfused onto the cell monolayers, and [Ca2+]i was determined from ratiometric data. In order to determine ligand binding, transfected cells were harvested, resuspended in binding buffer (20 mm Hepes and 1 mm MgCl2), and incubated with [3H]SQ29548 (3 Ci mm, 0.01–2 μm) for 20 min at room temperature. Ligand binding was then determined in the absence or presence of unlabeled ligand (10 μm) to determine receptor-specific radioligand binding.
TP receptor and P2Y12 receptor ligand binding was determined as previously described . Reactions were terminated with ice-cold binding buffer and rapid filtration through Whatman GF/C glass fiber filters under vacuum. Radioactivity bound to the filters was subsequently measured by scintillation counting.
The index case (P1) was a 60-year-old male who presented with excessive bleeding 3 days after abdominal surgery and had previously experienced prolonged bleeding after tonsillectomy in childhood. There was no other personal history of bleeding. Plasma coagulation factor and von Willebrand factor activities and platelet count were normal. Template bleeding time, clot retraction and thromboelastometry results were similar to those of healthy controls (data not shown). Platelet function analyzer-100 closure times were minimally prolonged, with CEPI (median, 186 s; reference interval, 80–160; n = 3) and CADP (median, 127 s; reference interval, 59–120; n = 3) cartridges.
Platelet aggregation in response to 1.5 and 2 mm AA was markedly reduced (maximum amplitude [MA] of 6% and 12%, respectively) and transient in P1, whereas these concentrations induced full, sustained aggregation of healthy control platelets (MA of 85% for AA 2 mm; Fig. 1A). There was partial aggregation (MA of 51%), then disaggregation with a high concentration of the TP agonist U46619 (5 μm), and markedly reduced (MA of 6%) and transient aggregation with a low concentration of U46619 (1 μm), as compared with full, sustained aggregation at these agonist concentrations with control platelets (MA of 85% and 86%, respectively; Fig. 1A). Platelet aggregation responses to 1 μg mL−1 collagen, 5–10 μm ADP (Fig. 1A), 20 μm TRAP and 1 mg mL−1 ristocetin (data not shown) were similar in P1 and controls.
Platelets from P1 and from a control showed markedly increased surface binding of anti-CD62P and anti-CD63 in response to 50 μm TRAP as compared with vehicle or isotype control antibody, indicating intact α-granule and dense-granule secretion (Fig. 1B,C). However, platelets from P1 showed no increase in anti-CD62P or anti-CD63 binding in response to 20 μm U46619, whereas this agonist induced a marked increase in binding of both mAbs to control platelets (Fig. 1B,C).
Together, these findings indicated a selective defect in platelet aggregation and secretion responses to TxA2 pathway activation that was likely to be at the TP receptor level rather than a generalized platelet activation defect. To investigate this further, we determined the total TP receptor expression level in permeabilized platelets and other blood cells by flow cytometry with mAbs against the extracellular (SC-32160) and intracellular (SC-18377) regions of the TP receptor. Binding of both mAbs to platelets (Fig. 2A), lymphocytes, monocytes and neutrophils (data not shown) from P1 was similar to that in the control. Western blots of platelet protein extracts probed with SC-18377 and SC-30036 (TP receptor intracellular region) confirmed similar total TP receptor expression in P1 and controls (Fig. 2B).
We also investigated platelet surface expression of the TP receptor in P1 by measuring binding of the TP receptor antagonist [3H]SQ29548. The maximum binding of [3H]SQ29548 to platelets from P1 was reduced to 54% that of control platelets (Bmax P1 183 d.p.m./4 × 106 platelets ± 20.8 vs. Bmax control 336 d.p.m./4 × 106 platelets ± 14.9; P < 0.05, Mann–Whitney U-test), indicating a partial reduction in platelet surface expression of the TP receptor (Fig. 2C). In addition, the affinity of binding of [3H]SQ29548 to platelets from P1 was lower than that for control platelets (Kd P1 550 nm ± 0.11 vs. Kd control P1 110 nm ± 0.04), suggesting an additional defect in ligand binding. The kinetics of binding of the P2Y12 purinergic receptor antagonist [3H]MeSADP to platelets from P1 were similar to those in controls (Fig. 2D).
In order to identify qualitative defects in the TP receptor, we sequenced the coding sequence of the TP receptor gene TBXA2R. P1 harbored a heterozygous c.87G>C transversion in exon 2 of TBXA2R that predicted a W29C substitution in the TP receptor TM1 (position 1.37, according to the Ballesteros–Weinstein GPCR nomenclature ). This variation was not identified as polymorphic in dbSNP137 (http://www.ncbi.nlm.nih.gov/projects/SNP/). The c.87G>C transversion was also identified in post-mortem material from a second family member (P2; mother of P1) who did not have a history of abnormal bleeding.
In order to investigate further the effects of the W29C substitution, we generated stable HEK293 cell clones expressing either wild-type (WT) or variant W29C TP receptor. We first examined receptor function by measuring changes in [Ca2+]i in response to the TP receptor agonist U44619 (Fig. 3A). HEK293 cells expressing the W29C TP receptor showed a rightwards shift in the dose–response curve for U44619 as compared with WT transfected controls (EC50 61 ± 1.9 nm vs. 0.89 ± 0.21 μm; P < 0.05, Mann–Whitney U-test) and a slight decrease in maximal response, indicating loss of signaling function.
We next examined the expression pattern of the W29C and WT TP receptors in transfected HEK293 cells by immunofluorescence microscopy. Cell surface expression of the W29C TP receptor was lower than that of WT controls (Fig. 3B), with more variant receptor being retained intracellularly. Reduced surface expression of the W29C TP receptor was confirmed by cell surface TP receptor ELISA (surface expression 51% that of WT controls; P > 0.05, Mann–Whitney U-test; Fig. 3C).
In order to investigate the defect in TP receptor function further, we measured [3H]SQ29548 binding to the transfected HEK293 cells. The maximum binding of [3H]SQ29548 to cells expressing the W29C TP receptor was reduced to 57% that of WT cells (Bmax W29C 363 ± 45 d.p.m. mg−1 protein vs. Bmax WT 638 ± 23 d.p.m. mg−1 protein; P < 0.05, Mann–Whitney U-test), confirming reduced surface expression of the W29C TP receptor (Fig. 3D). In common with the ligand-binding experiments with platelets, there was also reduced affinity of binding of [3H]SQ29548 to HEK293 cells expressing the W29C TP receptor as compared with WT controls (Kd W29C 312 ± 27 nm vs. Kd WT 72 ± 19 nm; P < 0.05, Mann–Whitney U-test).
We have reported a heritable platelet function defect characterized by reduced platelet aggregation and secretion responses to high concentrations of AA, the metabolic precursor of TxA2, and U46619, which is a specific TP receptor agonist. By contrast, platelet functional responses were normal with high concentrations of other platelet receptor agonists. This suggested a defect in the platelet TxA2 pathway that was downstream of TxA2 synthesis, but upstream of the signaling effectors of the TP receptor, which are shared with other platelet receptors [4, 5]. In keeping with this phenotype, we identified a novel heterozygous variation in the TP receptor gene TBXA2R, predictive of a W29C substitution in the TP receptor. We demonstrated that platelets from the index case showed a reduction in both TP receptor surface expression and ligand binding. We also showed that the variant W29C TP receptor expressed in HEK293 cells was dysfunctional, and that the platelet phenotype of reduced surface receptor expression and ligand binding was reproduced. The functional defect observed in platelets from the index case was similar to the limited previous reports of heritable TP receptor deficiency [11, 12, 14]. However, the complex effect of the W29C substitution on TP receptor function represents a unique disease mechanism for this disorder.
It is noteworthy that the family index case (P1) showed abnormal bleeding after surgery on two occasions, but did not show lifelong mucocutaneous bleeding, which is a hallmark of other heritable platelet function defects . Moreover, the mother of the index case (P2), who was also heterozygous for the W29C TP receptor substitution, had no abnormal bleeding history. The previously reported kindreds with qualitative (D304N and R60L substitutions) and quantitative (TBXA2R c.167dupG) TP receptor defects both included heterozygous index cases with mild bleeding. However, other heterozygous family members were asymptomatic [11, 14]. Together with our new observations from the W29C TP receptor variant, this suggests that heterozygous TP receptor defects alone are insufficient to cause spontaneous abnormal bleeding. Mild bleeding is a common population symptom that is multifactorial and frequently has no identifiable cause, despite detailed laboratory testing . Therefore, we speculate that the significant bleeding symptoms in the index case in our study, and those in the previously reported families [11, 14], required other, unidentified, hemostatic defects. The mild heritable platelet disorder P2Y12 purinergic receptor deficiency (OMIM #609821) provides support for this hypothesis. In one reported kindred, abnormal bleeding was reported in subjects with a heterozygous defect in the P2Y12 receptor when it was co-inherited with mild von Willebrand disease . However, only subjects with homozygous P2Y12 receptor defects showed abnormal bleeding in most other reported kindreds [22, 23].
We confirmed that the W29C substitution caused TP receptor dysfunction by showing that cytoplasmic Ca2+ responses to U44619 were reduced in HEK293 cells expressing the W29C TP receptor compared to the WT TP receptor. Furthermore, immunofluorescence microscopy and cell surface ELISA identified intracellular retention of the W29C TP receptor and reduced cell surface expression as a plausible mechanism for this defect. Our finding of reduced maximum binding of [3H]SQ29548 to the W29C TP receptor in HEK293 cells confirmed that there was reduced surface expression of the variant receptor. However, we also demonstrated that [3H]SQ29548 bound with lower affinity to the W29C TP receptor on HEK293 cells than to WT receptor controls. This cannot be explained by reduced platelet surface receptor expression but, instead, indicates an additional defect in ligand binding.
We conclude that loss of TP receptor function in platelets from the index case was caused by the W29C substitution, both by reducing trafficking of the variant receptor to the platelet surface, and by reducing ligand binding in the subset of receptors that are correctly expressed. This mechanism is distinct from the previously reported qualitative TP receptor variants, in which there was normal surface expression of variant receptor, but loss of function through markedly impaired ligand binding (D304N ) or loss of Gq coupling (R60L; [12, 13]). It is noteworthy that both the cell surface ELISA and ligand-binding experiments with transfected HEK293 cells indicated that the W29C substitution caused an approximately 50% reduction in surface expression of the variant TP receptor. The ligand-binding experiments in platelets showed a similar 50% reduction in surface expression level. However, this is a greater reduction than expected, because the index case was heterozygous for the W29C substitution, and is expected to express similar quantities of the variant and WT TP receptor. One possible explanation for this discrepancy is that the W29C substitution exerts a dominant negative effect in platelets by also disrupting trafficking of the WT receptor .
The Trp29 residue that is substituted in the index case lies within the TP receptor TM1 (Fig. 3E), at the junction with the N-terminal extracellular region , and is distinct from the TP receptor ligand-binding site, which has been localized to ECL2 [7, 25]. However, substitution of the hydrophobic residue Trp29 with the polar Cys residue is likely to affect the insertion or stability of TM1 in the cell membrane. In other homologous class A GPCRs, TM1 contributes hydrogen bonds to stabilize a critical interhelical association with TM7, which is necessary for receptor structural integrity and helps to define the ligand-binding site in the inactive receptor state [18, 26]. In keeping with this critical role for TM1, variant TP receptors with substituted TM1 residues showed reduced ligand-binding affinity as compared with the WT receptor . Disruption of intrahelical hydrogen bonds in the TP receptor by the D304N substitution in TM7 caused a similar defect in ligand binding, even though this substitution is also remote from the ligand-binding site . Thus, by exerting a wider effect on TP receptor conformation, it is highly plausible that the W29C substitution accounts for the observed defect in ligand binding, and, potentially, receptor mis-trafficking.
This report of a novel W29C TP receptor variant clarifies the phenotypic consequences of heterozygous loss of TP receptor function. It also illustrates a novel pathogenic mechanism in TP receptor deficiency, and highlights the essential structural role of TM1 in TP receptor function.
We acknowledge S. Watson for helpful discussion and review of the manuscript. We would like to thank J. N. Fiessinger for initial patient management, A. Munnich, and V. Remones, F. Grelac and S. Minaee for excellent technical assistance.
This work was funded by the British Heart Foundation (RG/09/007 and PG/06/038) and INSERM. S. J. Mundell is a Senior British Heart Foundation Research Fellow (FS/11/49/28751).