Photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor

The synthesis, binding and photoincorporation of a thromboxane A2/prostaglandin H2 (TXA2/PGH2) analog (9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-azidophenyl )-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2) [( 125I]PTA-Azido) to washed human platelets was characterized. Kinetic analysis of the binding of [125I]PTA-Azido at 30 degrees C yielded a k1 of 1.83.10(7) M-1.min-1 and k -1 of 0.195 min-1, Kd = k -1/k1 = 11 nM. Incubation of washed human platelets with [125I]PTA-Azido followed by photolysis resulted in the radiolabelling of a number of platelet proteins as assessed by SDS-PAGE autoradiography. The radiolabelling of three of these protein bands could be either uniformly blocked or reduced with a series of structurally dissimilar TXA2/PGH2 receptor antagonists or agonists and corresponded to proteins with a molecular mass of 43, 39 and 27 kDa. In addition, the incorporation of [125I]PTA-Azido into the three proteins was stereoselectively blocked by a pair of optically active stereoisomers that are TXA2/PGH2 receptor antagonists. Two-dimensional gel electrophoresis indicated that the 43 kDa protein possessed a pI value of 5.6 and that the 27 kDa protein exists in at least three isoforms with pI values of 4.9, 5.1 and 5.3. The labelling pattern was not altered by a mixture of proteinase inhibitors. The data suggest that one or more of these specifically radiolabelled proteins may represent the human platelet TXA2/PGH2 receptor.