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 aggregating agent and vasoconstrictor. To induce platelet aggregation, the platelets' enzyme, prostaglandin endoperoxide H synthase-1 (PGHS-1), first converts arachidonic acid (AA) into prostaglandin H-2 (PGH(2)). PGH(2) is then converted by the enzyme thromboxane synthase into TXA(2). Finally, TXA(2) is secreted and can activate the TXA(2) receptor on the platelet surface, The importance 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) production pathway We studied an 18-year-old woman with a lifelong bleeding disorder, moderate thrombocytopenia (55-71 x 10(9)/l) and a prolonged bleeding 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 chromatography and radioactive counting of TXA(2) formation from [C-14]-AA, was reduced to 13% of the activity measured in control subjects. PGHS-1 protein levels, 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 platelet aggregation in the patient, even if the enzyme was active. The PGHS-1 protein level was also reduced in the patient's immortalized B lymphocytes, 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 patient 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.