title: Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody.
abstract: NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ib alpha, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-Fc gamma II receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the Fc gamma II receptor. Cross-linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.
authors.affiliation: First Department of Internal Medicine, Kansai Medical University, Osaka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yanabu
authors.forename: Y
authors.initials: Y
authors.lastname: Ozaki
authors.forename: S
authors.initials: S
authors.lastname: Nomura
authors.forename: T
authors.initials: T
authors.lastname: Miyake
authors.forename: Y
authors.initials: Y
authors.lastname: Miyazaki
authors.forename: H
authors.initials: H
authors.lastname: Kagawa
authors.forename: Y
authors.initials: Y
authors.lastname: Yamanaka
authors.forename: N
authors.initials: N
authors.lastname: Asazuma
authors.forename: K
authors.initials: K
authors.lastname: Satoh
authors.forename: S
authors.initials: S
authors.lastname: Kume
authors.forename: Y
authors.initials: Y
authors.lastname: Komiyama
authors.forename: S
authors.initials: S
authors.lastname: Fukuhara
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase