Abstract
A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn-glycero-3-phosphocholine ([ 125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC 50 value of 3.2 ± 1.9 nM as compared to 0.40 ± 0.25 nM for PAF. Specific binding of [ 125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (K d) of 2.4 ± 0.7 nM and a receptor density (B max) of 1.1 ± 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [ 125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr=52,000.
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