Synergistic outside-in regulation of platelet activation by GPIIb/IIIa ligand-induced conformation and oligomerization

Full platelet activation with serotonin secretion and thromboxane A(2) (TxA (2)) formation induced by a low dose of thrombin receptor agonist peptide ( TRAP) or high dose ADP requires platelet aggregation. This requirement can be replaced by pretreatment of platelets with a combination of reagents inc luding: GPIIb/IIIa inhibitors yielding ligand-induced binding sites (LIBS), either arginine-glycine-aspartate-serine (RGDS) peptide or Ro 43-5054, cyt ochalasin to disrupt actin filaments and crosslinking by a GPIIb/IIIa mAb ( p1-62). Crosslinking is required since Fab fragments of p1-62 do not suppor t activation. Engagement of the Fc receptor by the mAb Fc domain is not req uired for p1-62 augmentation, since it is not blocked by the anti-Fc recept or mAb, IV-3. Another GPIIb/IIIa inhibitor, Ro 44-9883, not yielding LIBS e pitopes, serves as a negative control and shows a requirement for LIBS in a ddition to crosslinking. Focal adhesion kinase tyrosine phosphorylation ind uced by TRAP is blocked by these GPIIb/IIIa antagonists, but restored by p1 -62 crosslinking independent of LIBS induction. Tyrosine phosphorylation of a peptide comigrating with p38 MAP kinase is also inhibited by these antag onists and restored by p1-62 crosslinking. However, p38 MAP kinase activati on by low dose TRAP is not affected by these aggregation inhibitors. Tyrosi ne phosphorylation of a 34-kDa phosphoprotein in the absence of aggregation or TxA(2) formation was uniquely augmented by Ro 43-5054 but not Ro 44-988 3 under the above activation conditions. (C) 2001 Elsevier Science Ltd. All rights reserved.