Plasma membrane phosphorylation by endogenous phosphate donors in human blood platelets. Selectivity of the action of dibutyryl cyclic AMP

Abstract

Incubation of platelet-rich plasma with 32Pi leads to cellular uptake of the isotope and covalent incorporation into several cell constituents. Plasma membranes isolated from intact labelled platelets, delipidated and solubilized in sodium dodecyl sulfate, show, upon gel electorphoretic analysis, three main peaks of radioactivity: two in the molecular weight range 100 000-30 000 and an additional very slow migrating component strong positive by the peirodic acid-Schiff reaction. Treatment of the cells with dibutyryl cyclic AMP under conditions just sufficient to completely inhibit platelet aggregation leads to an increased isotope incorporation. Electrophoretic analysis of membranes isolated from dibutyryl cyclic AMP-treated cell reveals: (a) no change in the general pattern of distribution of the isotope, (b) no difference in the isotope incorporation to the two components of lower mol. wt. and (c) a marked increase (greater than 100%) in isotope incorporation in the slow migrating material as compared to membranes isolated from control cells. This material can be extracted from platelet plasma membranes after treatment of the membranes for 5 h with Triton X-100, at a detergent-to-protein ratio of 7.5. When the membrane material extracted with Triton X-100 is subjected to gel chromatography in Agarose (Biogel A-15m), the phosphorylated material that corresponds to the slow migrating band in polyacrylanide gel electrophoresis is eluted with or very close to the void volume of the column. Isoelectric focussing of this fraction, shows a single radioactive peak corresponding to an isolectric point of 3.78. The isolated component is pronase-sensitive, contains 52% of carbohydrate and 15% sialic acid. Analysis of the stability of the bound phosphate suggests that about 43% of it is bound as acyl-phosphate. The results reported, obtained through an approach that closely resembles physiological conditions are compatible with the participation of this membrane phosphoglycoprotein in the phenomena of platelet aggregation.