Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: evidence that phosphorylation of cortactin and a 64-kD protein is dependent on thrombin activation and integrin alphaIIb beta3.
Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin alphaIIb beta3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack alphaIIb beta3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% alphaIIb beta3, functional), and variant type (50% alphaIIb beta3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional alphaIIb beta3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by alphaIIb beta3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of alphaIIb beta3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.
Rosa
J P
JP
INSERM Unité 348, IFR Circulation-Lariboisière and IVS, Hôpital Lariboisière, Paris, France.
Artçanuthurry
V
V
Grelac
F
F
Maclouf
J
J
Caen
J P
JP
Lévy-Toledano
S
S
D001792:Blood Platelets / Q000378:metabolism*; D051356:Cortactin; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D013915:Thrombasthenia / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology*
C495868:CTTN protein, human; D051356:Cortactin; D008840:Microfilament Proteins; D009842:Oligopeptides; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; C046617:arginyl-glycyl-aspartyl-serine; D013917:Thrombin