To the Editor:
In the March 1 issue, Yanabu et al1 reported on a platelet-activating antiglycoprotein Ib monoclonal antibody (MoAb) NNKY5-5. The intact antibody, but not Fab-fragments, caused activation, and (Fab′)2 -fragments could not be prepared. Inhibition of the platelet FcγII-receptor by the antibody IV.3 did not prevent platelet activation by NNKY5-5. From this the investigators concluded that the action of the antibody on platelets was FcγII-receptor-independent, and hence due to signal transduction via glycoprotein Ib.
We2 previously analyzed MoAb-mediated platelet activation, where we could show that the mechanism of activation clearly depends on the antibody subtype. Activating IgG1 -antibodies can do so by cross-linking the FcγII-receptor, Fab- or (Fab′)2 -fragments are inactive and inhibition is obtained by blocking FcγRII. Activating IgG2 -antibodies, on the other hand, essentially cause platelet activation via the complement system, where again Fab- or (Fab′)2 -fragments are inactive. However, FcγRII-inhibition has no effect but leupeptin-treatment or complement inactivation prevents platelet activation. High concentrations of antibody may result in cell lysis and washed platelets are not sensitive or less sensitive.
In their report, Yanabu et al1 mention that only a minimal aggregation response to NKKY5-5 was obtained in washed platelets in contrast to what was seen in platelet-rich plasma. Furthermore, NKKY5-5 is an IgG2b -antibody. Both findings may be an indication that complement activation through the Fc-part of the antibody could have been involved in the platelet aggregation response. This possibility was not really excluded.
Only very few MoAbs are able to activate platelets in an Fc-independent manner,3-5 and they indeed represent the most interesting ones for further investigation of receptor involvement in signal transduction. However, a fully detailed characterization of all possible mechanisms of activation is necessary.
Response
We thank Deckmyn et al for their interest in our study on platelet activation induced by an anti-GPIb monoclonal antibody (MoAb), NNKY5-5.1-1 They have raised a possibility that complement activation through the Fc-part of the antibody is involved in platelet aggregation induced by NNKY5-5. Although their points are interesting and deserve attention, we have several lines of evidence to suggest that complement activation cannot totally explain the entire picture of platelet activation induced by NNKY5-5. Several MoAbs belonging to the IgG2 -subclass activate the complement system.1-2 Platelet aggregation induced by antibodies with complement-activating property is extensive and accompanied with cell lysis in platelet-rich plasma.1-3,1-4 NNKY5-5, even at high concentrations, showed no sign of cell lysis when added to platelet-rich plasma. In washed cells it induced weak and irreversible aggregation. Although Fab′ fragments of NNKY5-5 failed to induce platelet aggregation, they could induce tyrosine phosphorylation and activation of Syk, a tyrosine kinase, and potentiated platelet aggregation induced by other agonists. These findings clearly indicate that NNKY5-5, independent of the Fc portion, elicit certain intracellular activation signals by interacting with GPIb. We have no direct evidence to exclude the possibility that complement activation partly contributed to platelet activation observed with platelet-rich plasma, as suggested by Deckmyn et al. We hope to address this in the near future. However, our main finding with NNKY5-5, an anti-GPIb MoAb, which we described in our previous report in Blood,1-1 is that an antibody that interacts with GPIb can mediate platelet activation signals, independent of Fc receptors or Fc portion of the antibody.
