title: Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase.
abstract: The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3- kinase (PI 3-kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3-kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3-kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3-kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3- kinase.
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Tamaho, Nakakoma, Yamanashi, Japan.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wu
authors.forename: Naoki
authors.initials: N
authors.lastname: Asazuma
authors.forename: Kaneo
authors.initials: K
authors.lastname: Satoh
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yatomi
authors.forename: Toshiro
authors.initials: T
authors.lastname: Takafuta
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ozaki
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003435:Crotalid Venoms / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011233:Precipitin Tests; D025941:Protein Interaction Mapping; D021122:Protein Subunits; D021381:Protein Transport / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011940:Receptor Aggregation; D015398:Signal Transduction; D000077191:Wortmannin; D019061:src-Family Kinases / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000730:Androstadienes; D003435:Crotalid Venoms; D015638:Cytochalasin D; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D016392:Proto-Oncogene Proteins pp60(c-src); D000077191:Wortmannin; D019061:src-Family Kinases; D014841:von Willebrand Factor; C030961:botrocetin; C060902:lyn protein-tyrosine kinase