Thromboxane A2 synthase activity in platelet free human monocytes.
Soon after platelets, the highest amounts of thromboxane A2 (TXA2) can be detected in human monocytes activated by serum. Using platelet-free human monocytes, we have shown that foetal calf serum (FCS) induces prostaglandin H synthase (PGH synthase) after 16 h of incubation, as shown by the use of transcriptional inhibitors and Western blotting. The effect of serum can be in part mimicked by recombinant colony stimulating factor-1 (hr CSF-1). It is not known whether the limiting step leading from arachidonate to TXA2 is represented solely by the level of PGH synthase or also by the level of TXA2 synthase. We approached this problem by using a Western blot specific for the enzyme, as well as by using PGH2 as substrate. The results show that TXA2 synthase is constitutively expressed in monocytes, i.e., its levels were high soon after their isolation, and similar to those observed after 24 h of incubation with serum. However TXA2 failed to be synthesized until at least 3 h of incubation, and the pattern of synthesis was dependent on the kinetics of PGH synthase induction. In any condition in which TXA2 synthase was immunodetectable, using PGH2 as substrate a high rate of conversion to TXB2 could be detected. Experiments with actinomycin D and cycloheximide indicate that the half-life of TXA2 synthase was longer than 16 h, therefore much longer than that of PGH synthase, that the gene coding for it is fully active in resting monocytes, and that the conversion of arachidonate to TXA2 induced by serum or CSF-1 is dependent solely on the de novo synthesis of PGH synthase.
Orlandi
M
M
Department of Experimental Biology, University of Bologna, Italy.
Bartolini
G
G
Belletti
B
B
Spisni
E
E
Tomasi
V
V
D001051:Apoenzymes / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D001769:Blood; D003115:Colony-Stimulating Factors; D003513:Cycloheximide; D003609:Dactinomycin; D006207:Half-Life; D006801:Humans; D009000:Monocytes / Q000201:enzymology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D013929:Thromboxane B2 / Q000378:metabolism; D013930:Thromboxane-A Synthase / Q000302:isolation & purification / Q000378:metabolism*
D001051:Apoenzymes; D003115:Colony-Stimulating Factors; D003609:Dactinomycin; D016718:Arachidonic Acid; D013929:Thromboxane B2; D003513:Cycloheximide; D011451:Prostaglandin-Endoperoxide Synthases; D013930:Thromboxane-A Synthase