Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event.
The mechanisms involved in platelet 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 (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP 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 InsPs. To characterize further P256-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 P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-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 and Fc respectively allows the activation of all platelet activation systems.
Bachelot
C
C
U 150 INSERM, Hôpital Lariboisière, Paris, France.
Sulpice
J C
JC
Giraud
F
F
Rendu
F
F
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000096:biosynthesis*; D007700:Kinetics; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010725:Phosphocreatine / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; C039077:platelet protein P47; D010725:Phosphocreatine; D003402:Creatine Kinase; D001241:Aspirin