Platelet activation by immobilized monoclonal antibody: Evidence for a CD9 proximal signal

Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the FcγII receptor. We show here that nonstimulatory F(ab')₂ fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')₂ fragments of MoAb 50H.19 by F(ab')₂ fragments of goat antimouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross-linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')₂ fragments of the second antibody readily induce the aggregation of platelets treated with the anti-FcγII receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')₂ fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A₂.