ANTIPEPTIDE ANTIBODIES AGAINST THE HUMAN BLOOD-PLATELET THROMBOXANE-A2 PROSTAGLANDIN-H2 RECEPTOR - PRODUCTION, PURIFICATION AND CHARACTERIZATION/

Two anti-peptide antibodies have been raised against the human blood p latelet thromboxane A2/prostaglandin H-2 (TXA2/PGH2) receptor. Based o n the published sequence of the placental TXA2/PGH2 receptor, two deca peptide segments were Selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the ot her in the C-terminal region of the intracellular domain corresponding to residue 314 through 323. Rabbits were immunized with each peptide, and the antisera were subjected to a two-step purification procedure. The IgG fraction was purified using a DEAE Affi-Gel Blue column, and the peptide-specific IgG was further purified by affinity chromatograp hy employing each peptide as the immobilized ligand. The combined puri fication factor for both procedures was approximately 60-fold. By ELIS A, both antibodies displayed immunoreactivity toward their synthetic a ntigens, solubilized platelet membranes and affinity-purified TXA2/PGH 2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 recepto r protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish anti body specificity for the human platelet TXA2/PGH2 receptor protein. Fu nctional analysis demonstrated that neither antibody interfered with A DP- or U46619-induced platelet aggregation or [H-3]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epi topes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human pl atelet TXA2/PGH2 receptor. These antibodies should prove to be of valu e in the further investigation of the platelet TXA2/PGH2 receptor.