Anti-CD9 Monoclonal Antibody Activates p72 in Human Platelets

Abstract

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab')2 fragments of NNKY 1-19 did not, suggesting that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I2 (PGI2). The activity of p72syk was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72syk-associated tyrosine kinase activity that peaked at 10 s and subsided to the original level 2 min after stimulation. Coinciding with this time course, p60c-src transiently associated with p72syk. In platelets preexposed to GRGDS peptides or PGI2, NNKY 1-19 also increased the p72syk-associated tyrosine kinase activity and led to the association of p60c-src with p72syk. However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation. F(ab')2 fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI2 treatment on intact IgG NNKY 1-19 stimulation. F(ab')2 fragments of another CD9 MoAb, PMA2, had effects on p72syk essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CD9 MoAb to CD9 on the platelet membrane per se induces an increase in the p72syk-associated tyrosine kinase activity but that Fc gamma II receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI2 acts at a step distal to the activation of p72syk and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72syk activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72syk activity induced by anti-CD9 MoAb.

Tetraspanin CD9 induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in human melanoma cells

“…Like other tetraspanin proteins, CD9 has been known to be involved in signal transduction by forming complexes with integrins and other cell surface molecules including tetraspanin members (Maecker et al, 1997;Boucheix and Rubinstein, 2001;Yanez-Mo et al, 2001). In platelets, CD9 was shown to associate with some small GTP-binding proteins (Seehafer and Shaw, 1991) and CD9 ligation with anti-CD9 mAb increased p72syk-associated tyrosine kinase activity, along with cell aggregation (Ozaki et al, 1995). A recent report showed that the CD9 gene transduction downregulates Wnt signaling pathways in fibrosarcoma and lung cancer cells (Huang et al, 2004).…”

Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

“…So far there is no experimental evidence that one of these integrins is involved in CDV-induced cell-cell fusion. It has been reported that several signal transduction pathways are affected by antibodies against CD9 (30,31,47,48), that small G proteins are associated with CD9 (36), and that the binding activity of integrins is regulated by CD9 (23). Therefore, it is possible that antibodies to CD9 might regulate other proteins not identical with the directly associated integrins which may bind CDV or are involved in membrane fusion.…”

Localization of the Transmembrane 4 Superfamily (TM4SF) Member PETA-3 (CD151) in Normal Human Tissues: Comparison with CD9, CD63, and α5β1 Integrin

“…Anti-CD9 and anti-PETA-3 MAbs are able to induce platelet activation and mediator release by crosslinking of Fc ␥ RII, the low-affinity IgG receptor (Roberts et al 1995;Ashman et al 1991;Worthington et al 1990). More recent studies in platelets have demonstrated intrinsic signaling capacity for CD9, with anti-CD9 F(ab Ј ) 2 fragments inducing phosphorylation of the tyrosine kinase syk (Ozaki et al 1995). Signal transduction events have also been demonstrated in B-cells, granulocytes, and monocytes by anti-CD53 MAbs (Olweus et al 1993).…”