Genetic and pharmacological analyses of involvement of Src-family, Syk and Btk tyrosine kinases in platelet shape change. Src-kinases mediate integrin alphaIIb beta3 inside-out signalling during shape change.
Platelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADP- and thromboxane A2-G-protein coupled receptors in human platelets and thromboxane A2 receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin alphaIIb beta3 complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin alphaIIb beta3 exposure during shape change.
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Klinikum der Universität München, Germany.
M
M
Bauer
P
P
Maschberger
L
L
Quek
S J
SJ
Briddon
D
D
Dash
M
M
Weiss
S P
SP
Watson
W
W
Siess
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000201:enzymology; D048429:Cell Size / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000187:drug effects / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018345:Mice, Knockout; D009391:Nephelometry and Turbidimetry; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000187:drug effects / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000187:drug effects / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases