Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation.
A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose-dependently induces platelet aggregation and serotonin release in an Fc-independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross-linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA-13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.
De Reys
S
S
Center for Molecular and Vascular Biology, Faculty of Medicine, University of Leuven, Belgium.
Hoylaerts
M F
MF
De Ley
M
M
Vermylen
J
J
Deckmyn
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments / Q000502:physiology; D007141:Immunoglobulin Fc Fragments / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D015539:Platelet Activation; D010974:Platelet Aggregation; D012701:Serotonin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D008565:Membrane Proteins; D012701:Serotonin; D002118:Calcium