Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.
Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.
Oda
A
A
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02215.
Druker
B J
BJ
Smith
M
M
Salzman
E W
EW
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D003902:Detergents; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D017830:Octoxynol; D009842:Oligopeptides / Q000494:pharmacology; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010766:Phosphorylation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011092:Polyethylene Glycols; D011108:Polymers; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology
D000199:Actins; D003902:Detergents; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011108:Polymers; D011450:Prostaglandin Endoperoxides, Synthetic; D011092:Polyethylene Glycols; D017830:Octoxynol; C059453:RGES peptide; C046617:arginyl-glycyl-aspartyl-serine; D015688:Oncogene Protein pp60(v-src)