Mechanisms involved in platelet activation induced by a monoclonal antibodye anti glycoprotein IIb–IIIa: Inositol phosphate production is not the primary event

Abstract

The mechanisnms involved in platele aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (Ins P) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [ 32 P]- Ins(1,4,5)P 3 and [ 32 P]- Ins(1,4,5)P 4 , was observed between 20 and 90 s. [ 32 P]- Ins(1,3,4)P 3 was aslo produced with a manimumafter 90 s. Addition of ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and of the cycloxygenase inhibitor aspirin together with P256 almost totally abolished InsP P formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab′) 2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable Ins Ps. To characterize further P-256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P265-F(ab′) 2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular P-Tyr. The data indicate that the binding of P256 to HPIIb,-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab′) 2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab′) 2 Fc respectively allows the activation of all platelet activation systems.