title: Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK.
abstract: Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.
authors.affiliation: Ariad Pharmaceuticals Inc., Cambridge, Massachusetts 02139.
authors.forename: M M
authors.initials: MM
authors.lastname: Huang
authors.forename: L
authors.initials: L
authors.lastname: Lipfert
authors.forename: M
authors.initials: M
authors.lastname: Cunningham
authors.forename: J S
authors.initials: JS
authors.lastname: Brugge
authors.forename: M H
authors.initials: MH
authors.lastname: Ginsberg
authors.forename: S J
authors.initials: SJ
authors.lastname: Shattil
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000199:Actins; D001798:Blood Proteins; D015815:Cell Adhesion Molecules; D016023:Integrins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D015638:Cytochalasin D; D014443:Tyrosine; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human