Comp. Biochem. Physiol. Vol. 75B, No. 2, pp. 245 to 253, 1983 0305-0491/83/060245-09503.00/0 Printed in Great Britain © 1983Pergamon Press Ltd COMPARATIVE STUDIES OF PIG PLATELET MEMBRANE PROTEINS Jos~ BUBIS* and XAVIERYSERN Department of Chemistry, Universidad Sim6n Bolivar, Apartado 80659, Caracas 1080A, Venezuela (Received 27 September 1982) Abstract--1. The proteins and glycoproteins of pig platelet membranes have been studied using gel electrophoretic techniques. A nomenclature is suggested from the apparent molecular weights estimated by one-dimensional electrophoresis. Isoelectric focusing showed that the majority of the proteins are in the 4.0-7.0 pH range. 2. Subunits have been inferred from oligoproteins by two-dimensional, reduced nonreduced, electro- phoresis techniques. 3. High resolution two dimensional electrophoresis combining isolectric focusing and sodium dodecyl sulphate allows the observations of 60 polypeptide bands. An identification of some of those bands based on a correlation from reported human blood platelet membrane proteins is presented for comparison. INTRODUCTION The blood platelets are involved in the haemostasis, blocking the loss of blood by clot formation that seals the damaged vessel walls. Under different conditions platelets participate on the development of throm- bosis, obstruction of the free blood circulation avoid- ing blood supply to tissues (Holmsen, 1972). Platelet adhesion and aggregation are central steps in the hae- mostasis and in the thrombosis, in both processes blood platelet membranes play a main role (White & Gerrard, 1978). There is little information about the molecular nature and topology of the platelet mem- brane surface, in particular respect to the protein and glycoprotein complexes present in the membrane, which could act as receptors of the several haemosta- tic stimulus, as transductors that initiate intracellular events, or in the absorption and transport processes. The distinctive characteristics of the membrane sys- tem can be analyzed in a better way in isolated mem- branes from the cellular remainder (Baenziger & Majerus, 1974). As platelets are free cellular blood components they can be separated and their mem- branes isolated in a relatively intact form. Different electrophoretic techniques have been for a long time the best instruments to analyze complex protein mix- tures and several works using this technique have been reported in the study of human blood platelet membrane proteins (Hasitz et al., 1976; Phillips & Poh Agin, 1977a; Clemetson et al., 1979 McGregor et al., 1980). Taylor & Crawford (1976) analyzed the polypeptides from pig membrane platelets by SDS gel electrophoresis at 75.% polyacrylamide and revealed the presence of 1~15 main components within the molecular weight range from 12 to 200 K, six of which were glycoproteins. To be able to carry out studies about the pig platelet membrane topology, it is * Present address: Department of Chemistry, University of California, San Diego, La Jolla, CA 92093, USA. necessary previously a better quantification of their proteins, glycoproteins and polypeptides units. The use of high resolution two-dimensional electro- phoretic techniques to characterize the pig platelet membrane system is being reported in the present work. The use of chemical crosslinkers had been an im- portant tool to predict the molecular association in biological membranes (Peters & Richards, 1977; Ji, 1979; Das & Fox, 1979). Liu et al. (1977) pointed out that pH changes affect significantly the morphology and the physical properties of red blood cell ghosts, and they showed that protein crosslinking induced by pH can be involved in this effect as shown by electro- phoresis. The effect on pig protein platelet membrane electrophoretic patterns at different pH incubations has to be studied in order to characterize the role of pH crosslinking. MATERIALS AND METHODS Reagents Acrylamide, N,N'-methylenebisacrylamide (BIS), N,N, N',N'-tetramethylethylene diamine (TEMED), 2-mercapto- ethanol and Coomassie Blue R250 from Eastman Kodak Co.; Tris (hydroxymethyl) aminomethane or Trizma base (Tris), polyethylene glycol (mol. wt 6000) Dextran (mol. wt 500,000) and N-ethylmaleimide from Sigma Chemical Company; sodium citrate, citric acid, 1-chloro-3-tosyl- amide-7-amino-2-heptanone hydrochloride (TLCK) and L-/-p-tosylamina-2-phenylet hyl chloromethyl ketone (TPCK) from Aldrich Chemical Company; sodium dodecyl sulfate (SDS) from Calbiochem; sodium periodate and ion exchanger V from E. Merck Darmstadt; "Electrophoresis Calibration Kit" for molecular weight determination of high and low molecular weight proteins, "Pharmalyte" ampholytes and Agarose A from Pharmacia Fine Chemi- cals; nonidet P-40 from Imperial Shell; basic fuchsin from Riedel (De Haen Ag Seelze, Hannover). 5-sulpho-salicylic acid and sodium metabisulphite from Hopkin and Williams. Ammonium persulphate from Rhone-Poulenc Labora- tories. 245