Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M_r 21 000 to 29 000, one of which is the M_r 24 000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP₂), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na⁺/H⁺ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca²⁺]_i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP₂-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.