Author Institution: Department of Chemistry, Texas A\M Department of Natural Sciences, Fayetteville State University, Fayetteville, NC 28301; School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO, 64110-2499

A potential limitation of anti-thrombotic therapies directed at platelet GPIIb/IIIa is immune mediated thrombocytopenia. Reagents that mimic the behavior of patient antibodies would provide a valuable tool for studies directed at understanding the basis of the immune mechanism involved in GPIIb/IIIa antagonist induced thrombocytopenia. Such reagents would bind epitopes that are exposed when the conformation of the receptor is modified in response to inhibitor binding. We describe the production and characterization of monoclonal antibodies that were raised against platelet GPIIb/IIIa bound to a potent antagonist, XP280. These antibodies have high affinity and specificity for XP280 bound GPIIb/IIIa using either purified protein or human platelets. We have demonstrated that the antibodies recognize a conformationally altered form of the receptor, that both subunits are required for binding, and that the antagonist itself does not form part of the binding epitope. Competition experiments indicate that multiple drug-dependent epitopes are exposed on the receptor in response to antagonist binding. The antibodies bind with high specificity to some but not all GP IIb/IIIa/antagonist complexes indicating that different conformational epitopes are exposed when GP IIb/IIIa is bound to different antagonists.

Abstract: The heating of samples sealed in special heatable glass cells [1] allows samples with high boiling points to be studied as vapors by Raman spectroscopy. The investigation of the potential energy surfaces (PESs) of non-rigid molecules in our laboratory [2,3] is typically complemented by the Raman spectra of the vapors as these nicely provide data not available from infrared and ultraviolet absorption and fluorescence measurements. Examples of molecules recently investigated are 2cyclohexenone, tetralin (TET), 1,4-benzodioxan (14BZD), and 3-methylindole, which are shown below in that order.

3-Methylindole (3MI), which serves as a structural model for the tryptophan side chain in proteins, has been investigated using vapor phase Raman spectroscopy. The vapor phase spectrum of 3MI identifies the Raman signature of the indolyl moiety free of intermolecular interaction and extends previously reported solution Raman studies of 3MI and related tryptophan derivatives. The Raman spectrum of 3MI vapor is also complemented here with newly obtained vapor phase infrared data and ab initio calculations to refine and extend previous vibrational assignments. The present results provide an improved basis for assessing the dependence of the indolyl Raman signature on the local environment of the tryptophan side chain of proteins. The principal conclusions of this work are the following. (i) The vapor phase 3MI molecule exhibits Raman bands at 3506, 1585, 1409, 1349/1341 (Fermi doublet) and 881cm−1, which differ greatly from their counterparts in the Raman spectrum of 3MI liquid and thus serve as spectral markers of the indolyl ring environment. (ii) The Fermi doublet relative intensity ratio (I1/I2, where I1 and I2 are, respectively, the Raman intensities of the higher and lower wavenumber components of the doublet) is highly sensitive to the state of 3MI condensation, consistent with the previously reported sensitivity of I1/I2 to solvent polarity. The maximum value of the intensity ratio (I1/I2=3.0) is observed for 3MI vapor, while the minimum value (I1/I2=0.43) is observed for 3MI in CHCl3 solution. Implications of the present results for Raman analysis of hydrogen bonding states, hydrophilic interactions and hydrophobic interactions of tryptophan residues in proteins are considered.

The most effective way to obtain high quality vapor-phase Raman spectra is to heat the samples to increase their vapor pressure. Many samples can be heated to 350 °C and higher without decomposition. We have designed a simple Raman cell to allow these high temperature studies to be carried out. The high-temperature Raman spectra of nine molecules will be presented and discussed. Most of these are non-rigid molecules containing aromatic rings for which vibrational potential energy surfaces have been determined from their spectra. Two molecules (p-cresol and 3-methylindole) are model compounds for amino acids and their vapor-phase spectra are characteristic of environments with no hydrogen bonding.