Activation of human platelets by a stimulatory monoclonal antibody.
The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.
Kornecki
E
E
Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn 11203.
Walkowiak
B
B
Naik
U P
UP
Ehrlich
Y H
YH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D015539:Platelet Activation / Q000276:immunology*; D010974:Platelet Aggregation / Q000276:immunology; D010980:Platelet Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D012016:Reference Values
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate