CHEMICAL CROSS-LINKING OF PLECKSTRIN IN HUMAN PLATELETS - EVIDENCE FOR OLIGOMERIZATION OF THE PROTEIN AND ITS DISSOCIATION BY PROTEIN-KINASE-C

The major substrate of protein kinase C (PKC) in platelets is the 40 k Da protein, pleckstrin. Addition of the homobifunctional reagent, bis( sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fractio n or to electropermeabilized platelets resulted in cross-linking of pl eckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting wit h an anti-pleckstrin antibody. Higher levels of cross-linking were obs erved in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of plat elet lysate or lysis of electropermeabilized platelets and, in the cas e of the 90 kDa and 100-120 kDa species, after activation of PKC by ph orbol 12-myristate 13-acetate. Similar experiments with purified pleck strin indicated that the 90 kDa and 100-120 kDa species consist, at le ast in part, of pleckstrin dimers and higher oligomers. After incubati on of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25 % of the protein was present in cross-linked species. The results indi cate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts wit h an unidentified platelet protein of about 28 kDa.