Chang T. Lim

Anti-peptide 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 platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. Based on the published sequence of the placental TXA2/PGH2 receptor, two decapeptide segments were selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the other 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 chromatography employing each peptide as the immobilized ligand. The combined purification factor for both procedures was approximately 60-fold. By ELISA, both antibodies displayed immunoreactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA2/PGH2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 receptor protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish antibody specificity for the human platelet TXA2/PGH2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation of [3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA2/PGH2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA2/PGH2 receptor.

Thromboxane A2/prostaglandin H2 receptor antagonists

Publisher Summary The chapter presents a study on thromboxane A 2 /prostaglandin H 2 receptor antagonists. TxA 2 , and its precursor PGH 2 , directly interact with membrane-coupled receptors to initiate the process of platelet stimulation. Whether these molecules interact with the same or distinct platelet receptors remains to be resolved; and because of this consideration, they are collectively referred to as TxA 2 /PGH 2 receptors). In addition, studies in vascular smooth muscle have suggested the existence of tissue heterogeneity of TxA 2 /PGH 2 receptors. The chapter describes the characteristics that have served as the minimum criteria for establishing the specificity of a putative TxA 2 /PGH 2 receptor antagonist in platelets and the vasculature. The chapter illustrates a number of compounds of diverse structure and potency, which have been reported to exhibit TXA 2 /PGH 2 receptor antagonist activity in platelets and the vasculature. These compounds include 13-azaprostanoic acid (13-APA); pinane TxA 2 ; derivatives of pinane TxA 2 , and so on. The chapter describes the methods that can be applied to the characterization of platelet TxA 2 /PGH 2 receptors in vitro . These procedures provide a means for the measurement of radioligand interaction with the TxA 2 /PGH 2 platelet receptor. Based on considerations of affinity and specific binding, the most suitable ligand currently available for TxA 2 /PGH 2 receptor binding studies is [ 3 H]SQ29,548.

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets.

A photoactive iodoarylazide derivative (I‐APA‐PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13‐azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor‐binding studies using [3H]U46619, I‐APA‐PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I‐APA‐PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding radio‐iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2‐binding protein in human platelets.