Anti-CD9 monoclonal antibody activates p72syk in human platelets.
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.
Ozaki
Y
Y
Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Japan.
Satoh
K
K
Kuroda
K
K
Qi
R
R
Yatomi
Y
Y
Yanagi
S
S
Sada
K
K
Yamamura
H
H
Yanabu
M
M
Nomura
S
S
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015703:Antigens, CD / Q000276:immunology*; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D004792:Enzyme Precursors / Q000097:blood* / Q000302:isolation & purification; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000276:immunology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D010974:Platelet Aggregation; D011505:Protein-Tyrosine Kinases / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000097:blood / Q000302:isolation & purification; D000072377:Syk Kinase; D060245:Tetraspanin 29; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D060245:Tetraspanin 29; D019000:Phosphotyrosine; D014443:Tyrosine; D011464:Epoprostenol; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); C000606896:SYK protein, human; D000072377:Syk Kinase; D001241:Aspirin