Characterization of a partial prostaglandin endoperoxide a synthase-1 deficiency in a patient with a bleeding disorder

Thromboxane A(2) (TXA(2)), synthesized in platelets, is a powerful aggregat ing agent and vasoconstrictor. To induce platelet aggregation, the platelet s' enzyme, prostaglandin endoperoxide H synthase-1 (PGHS-1), first converts arachidonic acid (AA) into prostaglandin H-2 (PGH(2)). PGH(2) is then conv erted by the enzyme thromboxane synthase into TXA(2). Finally, TXA(2) is se creted and can activate the TXA(2) receptor on the platelet surface, The im portance of TXA(2) in haemostasis has been demonstrated by the presence of a bleeding tendency in patients showing an inherited defect in the TXA(2) p roduction pathway We studied an 18-year-old woman with a lifelong bleeding disorder, moderate thrombocytopenia (55-71 x 10(9)/l) and a prolonged bleed ing time (12.5 min). Her platelets aggregated in the presence of both PGH(2 ) and a stable TXA(2) analogue, but did not aggregate in the presence of AA . The activity of PGHS-1 in platelets, measured using thin-layer chromatogr aphy and radioactive counting of TXA(2) formation from [C-14]-AA, was reduc ed to 13% of the activity measured in control subjects. PGHS-1 protein leve ls, measured using Western blot analysis, were also markedly reduced to 10% of control values. Such levels of PGHS-1 enzyme were too low to sustain pl atelet aggregation in the patient, even if the enzyme was active. The PGHS- 1 protein level was also reduced in the patient's immortalized B lymphocyte s, suggesting a systemic expression defect. Northern blot analysis was then carried out with poly (A)(+) RNA extracted from the patient's immortalized B lymphocytes. PGHS-1 mRNA was detected as a 2.8-kb band in both the patie nt and control. The intensity of the band representing the patient's PGHS-1 mRNA was similar to that of the control subject. The Northern blot result suggests a normal transcriptional rate of the PGHS-1 gene for the patient. Therefore, the defect responsible for the reduced levels of PGHS-1 protein is probably post-transcriptional.