Biochemicd Pharmacology, Vol. 35, No. 4. pp. 621-624. 1986. OOM2952/86 $3.00 + 0.00 Printed in Great Britain. @ 1986 Pergamon Press Ltd. zyxwvutsrq AUTOCOID BINDING TO SERUM PROTEINS INTERACTION OF PLATELET ACTIVATING FACTOR (PAF) WITH HUMAN SERUM ALPHA-l-ACID GLYCOPROTEIN (AAG) PATRICK J. MCNAMARA,~~ KENNETH R. BROUWER~ and MARK N. GILLESPIES tDivision of Pharmaceutics and Pharmaceutical Analysis, and *Division of Pharmacology and Toxicology, College of Pharmacy, University of Kentucky, Lexington, KY 40536, U.S.A. (Received 14 February 1985; accepted 19 July 1985) Abstract-Platelet activating factor (PAF; I-alkyl-2-acetyl-sn-glycero-3-phosphochoiine) is a potent bioactive phospholipid released from platelets, neutrophils, basophils and macrophages that has been proposed as a mediator of anaphylaxis, acute lung injury and other disorders. Specific factors which stabilize PAF and/or regulate PAF activity in body fluids are largely unknown. As part of a general autocoid-serum protein binding screen, the platelet activating factor-alpha-l-acid glycoprotein (PAF- AAG) interaction was indirectly characterized by examining the ability of PAF to displace propranolol from AAG. Both PAF and its deacetylated metabolite (lyso-PAF), at 2OpM, doubled the fraction unbound of propranolol(0.4 PM) from purified human AAG (20 @I). None of the other autocoids that were studied (epinephrine, serotonin, spermidine, putrescine, leu-enkephalin or phenethylamine) exhibited any propranolol displacement activity. Scatchard analysis indicated that PAF competitively displaced propranolol from AAG, causing the apparent affinity constant for propranolol-AAG to decrease from 1.8 x lo5 M to 6.9 x IO4 M. PAF behaved qualitatively like chlorpromazine (a docu- mented inhibitor of propranolol binding to AAG), but PAF was less effective at displacing propranolol. The apparent binding to AAG may help stabilize and transport extracellular PAF. Furthermore, the interaction of PAF and AAG suggests that serum AAG, which fluctuates in a number of diseases, may function to regulate PAF activity during acute and chronic disease states. Platelet activating factor (PAF) is a potent bioactive phospholipid found in platelets, neutrophils, baso- phils and macrophages [l-5]. PAF can provoke platelet and neutrophil activation, hypotension and bronchoconstriction [l, 5-71. It has also been impli- cated in the pathogenesis of hyperactive airway dis- ease, acute lung injury and other disorders [.S,81. PAF can be readily metabolized to inactive Iyso- PAF by acetylhydrolases located in both intracellular and extracellular (i.e. vascular) spaces [9, lo]. Those factors which stabilize PAF and/or regulate PAF activity in body fluids are largely unknown. The importance of alpha-l-acid glycoprotein (AAG) in the binding of cationic drugs is widely accepted [ll-131; however, the biological role for AAG has not been established. It would appear likely that, as with serum albumin, AAG may func- tion to transport endogenous substances (e.g. endogenous amines) in serum and extravascular spaces. Serum AAG concentrations have been shown to increase or decrease (2- to 3-fold) in a variety of diseases including inffammation, myo- cardial infarction, renal and pulmonary disease, and altered physiological conditions, such as surgery, obesity and smoking [ 14-191. Neither the underlying causes nor the consequences of these fluctuations have been identified; however, an interaction of AAG with circulating and extravascular autocoids remains an interesting possibility. During the course of a screening program for’ autocoid interactions with serum proteins, an inter- * Correspondence. action between AAG and PAF was observed. The purpose of this study is to characterize the interaction of PAF with AAG, specifically as it relates to the displacement of propranolol from its AAG binding site. METHODS PAF; l-alkyl-2-acetyl-sn-glycero-3-phosphochol- ine was obtained from Calbiochem-Behring (La Jolla, CA) and chlorpromazine was obtained from Smith, Kline & French Laboratories (Philadelphia, PA). Human AAG, lyso-PAF, d,Z-propranolol, epi- nephrine, serotonin, spermidine, putrescine, phen- ethylamine, imipramine and leu-enkephalin were obtained from the Sigma Chemical Co. (St. Louis, MO). Radiolabeled propranolol (d,f-[4-3H]propran- 0101) was obtained from the Amersham Co. (Arling- ton Heights, IL). Radiochemical purity was greater than 98% when acquired and was verified (via TLC) periodically and following dialysis to ensure stability. A stock solution containing 20 PM AAG was made up in a phosphate buffer (0.13 M, pH 7.4). Aliquots of this protein stock solution were used to make working solutions containing 50,uM PAF, lyso- PAF, chlorpromazine, imipramine, epinephrine, serotonin, spermidine, putrescine, phenethylamine or leu-enkephalin. These protein solutions were then used to deter- mine the binding of propranolol in a previously described 118,191 equilibrium dialysis method. Pro- tein solutions were dialyzed at 37” across a semi- permeable ~12,00~14,~0 mol. wt. cut-off) cellulose 621