Abstract
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.
Highlights
Which actin filaments had been removed by DNase I- The results demonstratethat themembrane glycoproteinsare induced depolymerization and high-speed centrifuga- attached to actin-binding protein, which serves as a linkage tion
Since earlier studies have shown tein links actin filaments oftheplateletmembrane that theCa2+-dependentprotease is activated during platelet skeleton to three plasma membrane glycoproteins andactivation [6] and that actin-binding protein is one of the that filaments are released from their attachmenstite major substrates [7], experiments were performed to when actin-binding proteinis hydrolyzed by the Ca2+- determine whether actin filaments were released from their dependenpt rotease platelet activation
Isolation and Analysis of Actin Filaments andAssociatedProteins- aureusProteinA was washed thwe times by centrifugation and Platelets were lysed by the addition of an equal volume of buffer resuspended in a mixture (1:l)of normal Tyrode's and TritonX-100 containing 2% Triton X-100 (Sigma), and 100 mM Tris-HC1, pH 7.4, lysis buffer
Summary
Plateletscontain large which is polymerized into amounts filaments of in actin,about 40% of unstimulated platelets. Isolation and Analysis of Actin Filaments andAssociatedProteins- aureusProteinA was washed thwe times by centrifugation and Platelets were lysed by the addition of an equal volume of buffer resuspended in a mixture (1:l)of normal Tyrode's and TritonX-100 containing 2% Triton X-100 (Sigma), and 100 mM Tris-HC1, pH 7.4, lysis buffer. Sepharose 6MB (Sigma), and antibodies against actin-binding protein Protease-Ca'+-dependent protease was purified from fresh human were further purified on Affi-Gel 10(Bio-Rad),to which actin-binding platelet concentrates [7] and dialyzed into a buffer containing 5 mM protein, purified to homogeneity as previously described [7], hadbeen EDTA, 100 mM potassium chloride, 5 mM dithiothreitol, 0.02%. The final concentration of the previously.' Purified protein was electrophoresed through an SDS- purified Ca2+-dependentprotease was 13 pg/ml, as determined by the polyacrylamide gel containing 5% acrylamide. The nitrocellulose paper was incubated with 1251-ProteinA (Sigma),which was radiolabeled by the
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