Functional analysis of human thromboxane synthase polymorphic variants

摘要

BACKGROUND: Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H 2 into thromboxane H 2 (TXA 2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA 2 generation. OBJECTIVES: The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. METHODS: Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA 2 and fragmentation of PGH 2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. RESULTS: All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 μmol/l PGH 2 (wild-type value: 32 μmol/l PGH 2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose V max/Km ratio was just 27% of the wild-type value. CONCLUSION: The increased Km and decreased V max values observed with L357V suggest that this variant may generate less TXA 2 at the low levels of PGH 2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.