MECHANISMS OF PLATELET ACTIVATION BY A STIMULATORY ANTIBODY - CROSS-LINKING OF A NOVEL PLATELET RECEPTOR FOR MONOCLONAL-ANTIBODY F11 WITH THE FC-GAMMA-RII RECEPTOR

The mechanisms by which a stimulatory monoclonal antibody (mAb), calle d mAb F11, induces granular secretion and aggregation in human platele ts have been characterized. Fab fragments of mAb F11, as well as an mA b directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, i ndicating that the mAb F11 IgG molecule interacts with the Fc gamma RI I receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot an alysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatogra phy. When added to platelet-rich plasma, the purified proteins dose-de pendently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of I-125 -labelled mAb F11 to intact platelets. The N-terminal 26 amino acid se quences of both the 32 and 35 kDa proteins were identical and containe d a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a sin gle similar to 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glyco sylation. Internal amino acid sequence analysis of the F11 antigen pro vided information concerning 68 amino acids and suggested two consensu s phosphorylation sites for protein kinase C (PKC). The phosphorylatio n by PKC of the isolated F11 antigen was observed following stimulatio n by phorbol 12-myristate 13-acetate. Databank analysis of the N-termi nal and internal amino acid sequences of the F11 antigen indicated tha t the N-terminal sequence exhibited the highest degree of similarity t o the variable region of the alpha-chain of human T-cell receptors (TC R). In contrast, the F11 internal sequences did not exhibit any simila rity to the TCR. Our results demonstrate that the F11 antigen is a nov el platelet membrane surface glycoprotein which becomes cross-linked w ith the Fc gamma RII receptor when platelets are activated by the stim ulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.