Functional analysis of human thromboxane synthase polymorphic variants.
BACKGROUND
Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), 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 TXA2 generation.


OBJECTIVE
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 TXA2 and fragmentation of PGH2 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 PGH2 (wild-type value: 32 μmol/l PGH2); 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 Vmax/Km ratio was just 27% of the wild-type value.


CONCLUSIONS
The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.
Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Chung-Ying K
CY
Chen
Elizabeth M
EM
Poole
Cornelia M
CM
Ulrich
Richard J
RJ
Kulmacz
Lee-Ho
LH
Wang
D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D044262:Prostaglandin H2 / Q000737:chemistry / Q000378:metabolism; D013929:Thromboxane B2 / Q000096:biosynthesis; D013930:Thromboxane-A Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D050505:Mutant Proteins; D044262:Prostaglandin H2; D013929:Thromboxane B2; D013930:Thromboxane-A Synthase