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 membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK 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 contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive 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 phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002109:Caffeic Acids / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D004792:Enzyme Precursors / Q000097:blood*; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007074:Immunoglobulin G / Q000097:blood; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000097:blood; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000172:deficiency / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000097:blood*; D012016:Reference Values; D015398:Signal Transduction; D000072377:Syk Kinase; D013915:Thrombasthenia / Q000097:blood; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000097:blood; D020032:Tyrphostins
D002109:Caffeic Acids; D004792:Enzyme Precursors; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D020032:Tyrphostins; C060152:tyrphostin 47; D019000:Phosphotyrosine; D003094:Collagen; C046617:arginyl-glycyl-aspartyl-serine; D011464:Epoprostenol; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); C000606896:SYK protein, human; D000072377:Syk Kinase; D010738:Type C Phospholipases; D013917:Thrombin