Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes.
Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.
D000199:Actins / Q000097:blood* / Q000302:isolation & purification; D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D008840:Microfilament Proteins; D008970:Molecular Weight
D000199:Actins; D001798:Blood Proteins; D002352:Carrier Proteins; D018260:Gelsolin; D006023:Glycoproteins; D008840:Microfilament Proteins; C016953:brevin