Identification and characterization of a novel P2Y₁₂ variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study

Data from the European Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease (MCMDM-1VWD) study, and the Canadian Type 1 VWD Study, suggest locus heterogeneity contributes to the pathogenesis of type 1 VWD, though, as yet, no associated defect in platelet reactivity has been described.^1,2  Given the role of von Willebrand factor (VWF) in primary hemostasis, and the clinical similarities of patients with type 1 VWD and platelet-based bleeding disorders, the bleeding tendency in patients with type 1 VWD may be influenced by defects in the receptor or signaling proteins mediating platelet adhesion and aggregation.

ADP is a feedback mediator of platelet aggregation, which elicits its response through the G protein–coupled receptors, P2Y₁ and P2Y₁₂. Interaction of ADP with P2Y₁ leads to mobilization of intracellular calcium and activation of Rho kinase, resulting in platelet shape change and an initial wave of rapidly reversible aggregation.^3-5  In contrast, interaction with P2Y₁₂ is associated with adenylyl cyclase inhibition and PI3-kinase activation, resulting in sustained aggregation in synergy with the P2Y₁ receptor.⁵  Both receptors are required for a full aggregation response to ADP.⁶ 

We investigated the possible contribution of P2Y₁₂ gene (P2RY12) defects to the bleeding tendency in type 1 VWD patients recruited through the MCMDM-1VWD study.

The phenotypic and genotypic characteristics of the MCMDM-1VWD cohort have been described previously.¹  This study focused on those index cases having normal VWF multimer profiles, usually considered a criterion for type 1 VWD. This study has received local ethics committee approval at each of the centers that participated in the MCMDM-1VWD study, and informed consent was obtained in accordance with the Declaration of Helsinki.

P2RY12 coding sequences were amplified from genomic DNA and sequenced on an ABI 3730 DNA capillary sequencer. The P2RY12 520A>G mutation was sought in other subjects by restriction analysis using BsmI.

Platelet aggregation and ATP secretion were assessed in platelet-rich plasma (PRP) using a dual Chronolog lumiaggregometer (Havertown, PA) as previously described.⁷ 

Ligand binding studies using [³H]2MeS-ADP (3 Ci [111 GBq]/mmol) were performed as previously described.⁸ 

A P2Y₁₂ cDNA clone for the K174E variant was derived by amplification from a hemagglutinin (HA)–tagged P2Y₁₂ construct.⁹  CHO-K1 cells stably expressing either wild-type or K174E variant construct were generated as previously described.⁹  Surface expression and cellular distribution of HA-tagged receptors were assessed in transfected cells by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy as described previously.¹⁰ 

ADP-induced inhibition of forskolin (1 μM)–stimulated cAMP accumulation in CHO cells was performed as previously described.^9,11  Data were expressed as percentage inhibition of forskolin-stimulated adenylyl cyclase.

Cells expressing receptor constructs were harvested and incubated with [³H]2MeS-ADP (3 Ci [111 GBq]/mmol) and binding was determined in the presence of AR-C69931MX (10 μM) as described previously.⁸ 

P2RY12 analysis, in DNA from 92 index cases enrolled in the MCMDM-1VWD study with normal VWF multimers, identified a heterozygous 520A>G transition in one case, P12F13II.1, referred to hereafter as PII.1. The transition, which predicts substitution of lysine 174 by glutamate (K174E) in P2Y₁₂, also occurred in several relatives of PII.1, but not in 80 control subjects from the same center as PII.1, suggesting it was unlikely to represent a polymorphism.

Platelets from 3 carriers of the K174E mutation, PII.1, her mother (PI.1), and her sister (PII.2), changed shape in response to ADP in the concentration range of 3 to 30 μM, indicating normal P2Y₁ receptor function (Figure 1A). However, platelets from all 3 patients exhibited reduced and transient aggregation to a concentration of ADP (10 μM) that induced maximal sustained aggregation of platelets from more than 40 control subjects.⁷  There was also a reduced response to 3 μM ADP, whereas 30 μM ADP induced maximal, sustained aggregation similar to that in controls (Figure 1A). These results indicated a partial defect in the P2Y₁₂ receptor.⁷  This was supported by the further reduction in ADP (10 μM)–induced platelet aggregation caused by the P2Y₁₂ receptor antagonist, ARC-67085 (Figure 1B), and by the reduction in the maximal level of dense granule secretion induced by ADP in platelets from the 2 patients in whom this was measured (Figure 1C). Thus, heterozygous expression of the K174E mutation induces a partial loss in aggregation and secretion to ADP.

Aggregation of platelets from PII.1 and PI.1 in response to other agonists was also examined. Platelets from both subjects displayed sustained aggregation, similar to that in controls, in response to high concentrations of the GPVI agonist collagen-related peptide (CRP; 1 μg/mL), collagen (3 μg/mL), PAR-1 (100 μM)– and PAR-4–specific peptides (500 μM), and the thromboxane A₂ analog U46619 (3 μM) (not shown). However, reduced aggregation to lower concentrations of these agonists, consistent with a positive feedback role for ADP, was observed (not shown). Further, a reduction of approximately 50% in platelet ATP secretion was observed in response to a single high concentration of ADP and the PAR-4 peptide, reflecting the critical feedback role of ADP in this response (Figure 1C).

K174 is situated in the second extracellular loop of P2Y₁₂, adjacent to cysteine 175, 1 of 4 cysteine residues in P2Y₁₂ predicted to be exposed on the cell surface and likely to be involved in binding ADP.^12,13  Its substitution by glutamate was therefore likely to inhibit ADP binding. Consistent with this, [³H]2MeS-ADP binding to P2Y₁₂ on platelets from carriers of the K174E mutation was reduced by up to 50%, whereas binding to P2Y₁ was similar to that on control platelets (Figure 1D).

The defect associated with the K174E substitution was further investigated in CHO cells stably expressing HA-tagged wild-type and variant K174E P2Y₁₂ receptors. The wild-type and variant K174E receptors were expressed at similar levels on the surface of CHO cells as assessed by ELISA and immunofluorescence,⁹  indicating that the mutation did not disrupt receptor synthesis or trafficking (not shown). However, the K174E variant demonstrated a marked reduction in its ability to negatively couple to adenylyl cyclase as evidenced by the decrease in both maximal response and rightward shift of the concentration-response curve (Figure 2A). The disruption in ligand binding caused by the K174E substitution in platelets was confirmed in the transfected CHO cells using [³H]2MeS-ADP. Thus, whereas binding to CHO cells expressing the wild-type receptor was saturable and maximal at a ligand concentration of 1 μM (K_d 0.31 ± 0.05 μM), the K174E variant demonstrated approximately 25% of the ligand binding capacity of wild-type P2Y₁₂ at a ligand concentration of up to 10 μM (Figure 2B). Therefore, defective ADP-dependent inhibition of forskolin-stimulated adenylyl cyclase activity in cells expressing the K174E variant occurred as a result of a failure in ligand binding.

The mild bleeding associated with P2Y₁₂ deficiency is thought to be recessively inherited. Whereas this has been true for 6 of the 7 P2Y₁₂-deficient cases reported to date,^14-19  one patient with a hemorrhagic diathesis and a heterozygous P2RY12 mutation (P258T) has been described, whose platelets failed to aggregate in response to low concentrations of ADP (≤ 4 mM), but showed reduced and reversible aggregation at higher concentrations of ADP (20 μM). This is similar to the profile observed in the 3 patients who have been investigated in this study.²⁰ 

In conclusion, the identification of a novel P2Y₁₂ defect and its association with bleeding in a family with type 1 VWD supports a contribution from other loci to the bleeding tendency in patients diagnosed with type 1 VWD, providing further evidence for locus heterogeneity in this disorder.

The contribution of the MCMDM-1VWD study partnership is gratefully acknowledged. We thank Mr John Anson for his contribution to the laboratory analyses.

This work was supported by the British Heart Foundation (BHF; PG/06/038). The MCMDM-1VWD study was supported by the European Community under the Fifth Framework Program (QLG1-CT-2000-00387). S.J.M. holds a BHF Senior Lectureship. S.P.W. holds a BHF Chair.

Contribution: S.P.W. coordinated the study, which was initiated by M.E.D., A.D.M., M.M., J.T.W., I.R.P., and S.P.W.; M.E.D., S.P.W., S.J.M., and B.B.D. wrote the paper, which was read and commented on by all authors; M.E.D., B.B.D., S.J.M., W.A.L., and S.P.W. participated in the data collection and laboratory analyses; and A.C.G., I.R.P., and F.R. initiated and coordinated the MCMDM-1VWD study.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Martina Daly, Academic Unit of Haematology, School of Medicine and Biomedical Sciences, Beech Hill Road, Sheffield, S10 2RX, United Kingdom; e-mail: m.daly@sheffield.ac.uk.