Tryosine phosphorylation and p72(syk) activation by an anti-glycoprotein Ib monoclonal antibody

Abstract

NNKY5-5, an IgG monoclonal antibody directed against the von Willabrand factor-binding domain of glycoprotein (GP) Ibα, 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 plateletactivating 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-FcyII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein lb. 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 p72(syk) suggesting that such activation was independent of the FcγII 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- 5induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72(sky) activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72(syk) activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72(sky) activation. Our findings suggest that binding of NNKY5-5 to GPlb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72(syk) activation and tyrosine phosphorylation of a 64-kD protein.