CYCLIC AMP-INSENSITIVE ACTIVATION OF C-SRC AND SYK PROTEIN-TYROSINE KINASES THROUGH PLATELET MEMBRANE GLYCOPROTEIN-VI

Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membr ane protein, whose deficiency results in selective impairment in colla gen-induced platelet aggregation, Our group previously reported a huma n polyclonal antibody (anti-p62 IgG) that induces activation of normal , but not of GPVI-deficient, platelets in an Fc-independent manner. Th e F(ab')(2) fragments of this antibody (F(ab')(2)-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevente d even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhi bitor tyrphostin A47 completely abolished F(ab')(2)-anti-p62-induced p latelet aggregation in parallel with dose-dependent inhibition of prot ein-tyrosine phosphorylation, indicating an essential requirement of P TK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')(2)-anti-p62 in a way insensitive to elevation of cAMP, In cont rast, in the presence of prostacyclin, F(ab')(2)-anti-p62 did not stim ulate tyrosine phosphorylation of the focal adhesion kinase, cAMP-inse nsitive activation of c-Src and Syk was also observed in collagen-but not thrombin-stimulated platelets, Moreover, either F(ab')(2)-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of p hospholipase C-gamma 2. These results indicate that the receptor-media ted activation of several PTKs in platelets is regulated through a cAM P-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-ins ensitive activation of c-Src and Syk accompanied by tyrosine phosphory lation of numerous substrates including phospholipase C-gamma 2 in a m anner similar to collagen stimulation.