Bleeding Tendency and Impaired Platelet Function In a Patient Carrying a Heterozygous Mutation In Thromboxane A2 Receptor

Abstract 2524

Characterization of inherited platelet function disorders has revealed critical molecules and receptors for hemostasis and thrombosis. Thromboxane A2 (TXA2) is one of the major platelet agonists, and thromboxane A2 receptors (TP) are seven transmembrane small G protein coupled receptor. There are two isoforms, α and β, which differ only their C-terminus. TPα is mainly expressed in platelet. In general, platelet receptor abnormalities such as Glanzmann thrombasthenia and P2Y₁₂ deficiency are inherited in an autosomal recessive manner. In other words, a subject carrying a heterozygous mutation does not show any platelet functional abnormality. In this study, we examined a patient with bleeding tendency and platelet dysfunction, and demonstrated that the patient is heterozygous for a novel mutation in TP, G insertion at nt.167-8.

The proband (OSP-2) was a 7-year-old Japanese girl, and had repeated nasal bleeding and mild purpura from 3 years old. The nasal bleeding was sometimes severe and continued for 1 hour to be stopped. She was born from non-consanguineous parents. She was referred to Osaka University Hospital because of suspected platelet dysfunction. There were no abnormalities in blood cell counts and coagulation. Bleeding time was slightly prolonged (7.5 minutes) and ADP-induced platelet aggregation was impaired. U46619 (2.5 mM)-induced platelet aggregation was remarkably impaired in the proband. The impaired platelet aggregation was still observed even at high concentration of U46619 (10 mM). Essentially the same platelet functional abnormality was observed in her father. Informed consent was obtained from the patient and her family members. We firstly confirmed that there is no abnormality in the expression of platelet glycoproteins such as α_IIbβ₃, GPIb-IX, GPVI and GPIa. Nucleotide sequence analysis of TP cDNA obtained from the proband revealed a novel mutation, G insertion between nt.167 and nt.168 leading to a frame shift. No other mutation was detected in the coding region of TP. Sequencing of genomic DNA showed that the mutation was heterozygous in her father as well as the proband. Thus, the abnormal allele was inherited from her father. The wild-type TP expression construct or the mutant construct in pcDNA3.1 vector indicated that the expression of the mutant TP was markedly impaired. Consistent with molecular genetic analysis, immnoblotting with polyclonal antibody against cytoplasmic domain demonstrated that the amount of TP in platelets from the proband and her father were approximately half of controls.

P-selectin expression as well as PAC-1 binding on platelets obtained from the proband was markedly impaired when stimulated with U46619 but not with 0.5 U/ml thrombin or 20 μM ADP. Essentially the same defects were obtained in platelets from her father. To evaluate more precisely the dynamic changes in the α_IIbβ₃ activation, we performed initial velocity analysis for PAC-1 binding. In brief, FITC-PAC-1 was added to the activated platelets at the indicated time points after stimulation and incubated for only 30 s. Interestingly, we detected PAC-1 binding about 36∼42% of control on platelets from the proband and her father at 5 sec after stimulation with 5μM U46619. However, at 1 min and 5 min PAC1 binding velocity rapidly decreased, suggesting that the sustained α_IIbβ₃ activation rather than initiation of α_IIbβ₃ activation was impaired. Consistent with these data, Rap1 activation at 10 sec, 1 min and 5 min after stimulation of U46610 were 50%, 17%, and 11% of control, respectively. We have shown that the P2Y₁₂ plays a critical role in the sustained α_IIbβ₃ activation (T Kamae, et al. J Thromb Haemost 2006; 4:1379–87) From these data, we assume that the impaired α_IIbβ₃ activation may be due to a reduction in ADP release from the proband. Indeed, small amount of exogenous ADP (0.5μM) significantly improved the impaired α_IIbβ₃ activation induced by U46619. Our data demonstrate that the impaired platelet function to U46619 observed in the patient with heterozygous mutation in TP is, at least in part, due to a decrease in granular secretion, especially ADP.