TXA2 is a potent vasoconstrictor and a powerful inducer of platelet aggregation. Despite an intense interest in TXA2 and its involvement in numerous physiological functions, relatively little is known about the enzyme which controls its production (thromboxane synthase). Thromboxane synthase is a microsomal enzyme predominantly found in platelets, lung and brain. The platelet enzyme is thought to play an important role in thrombosis and hemostasis; however, the function of the enzyme in lung and brain and molecular similarities of the enzyme from different tissues is still unknown. Our laboratory has partially purified the enzyme from porcine lung microsomes (17-fold purification) using DEAE-cellulose, Affigel Blue and phenyl-Sepharose chromatography. Further purification of the enzyme will be pursued using hydroxyapatite, gel filtration, affinity and hydrophobic interaction chromatography. The homogeneity of the enzyme will be assessed by analytical gel electrophoresis. Once the enzyme has been significantly purified its pH optimum, pI, molecular weight, temperature stability and kinetic properties will be determined. The enzymatic mechanism of TXA2 production will be investigated. Thromboxane synthase preparations described in the literature not only enzymatically convert PGH2 to TXA2 but also to 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT) and malondialdehyde (MDA). The biochemical significance of these products has yet to be fully investigated and the possible role of HHT in TXA2 production established. The purified enzyme would resolve whether or not TXA2 and HHT are produced by the same enzyme protein and whether different cellular sources of enzyme produce different ratios of TXA2 and HHT. The enzyme will be purified from platelets, lung and brain and the existence of thromboxane synthase isozymes investigated by comparing their immunological, kinetic and chemical properties. Pilot studies have shown that the production of TXB2 from PGH2 is limited. The nature of this limitation must be investigated as it may represent an important step in our understanding the in vivo regulation of TXA2 production. The proposed research will provide basic biochemical information on thromboxane formation and its control. In addition, it will provide information regarding the mechanism of action and active site chemistry of the enzyme, thus aiding the synthetic chemists in the design and production of thromboxane synthase inhibitors.
