TYROSINE PHOSPHORYLATION AND P72(SYK) ACTIVATION BY AN ANTI-GLYCOPROTEIN IB MONOCLONAL-ANTIBODY

NNKY5-5, an IgG monoclonal antibody directed against the von Willebran d factor-binding domain of glycoprotein (GP) Ib alpha, induced weak bu t irreversible aggregation (or association) of platelets in citrate-an ticoagulated platelet-rich plasma. This phenomenon was defined as smal l aggregate formation (SAF). Platelets in hirudin-anticoagulated plasm a or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of ade nosine diphosphate or platelet-activating factor. NNKY5-5 did not indu ce granule release or intracellular Ca2+ mobilization. However, NNKY5- 5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72(syk). An anti-fc gamma II receptor antibody ha d no effect on SAF, suggesting that NNKY5-5 activated platelets by int eracting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induc e SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72(syk), suggesting th at such activation was independent of the Fc gamma II receptor. Cross- linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondar y antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but h ad no effect on 64-kD protein tyrosine phosphorylation or p72(syk) act ivations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72(syk) ac tivation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibit or, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72(syk) activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interacti on between fibrinogen and GPIIb/IIIa through a mechanism associated wi th p72(syk) activation and tyrosine phosphorylation of a 64-kD protein . (C) 1997 by The American Society of Hematology.