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 αIIbβ3

Abstract

Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin αIIbβ3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack αIIbβ3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% αIIbβ3, functional), and variant type (50% αIIbβ3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional αIIbβ3 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 αIIbβ3 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 αIIbβ3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.

Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP

“…However, cortactin (80 kDa) was still phosphorylated in the early stages after 2-MeS-ADP stimulation whereas the second wave of phosphorylation was inhibited ( Figs 4C and 5C). This observation is in accordance with our previous results: (i) when control platelets were stimulated with thrombin in the presence of RGDS; (ii) when we used platelets from Glanzmann's thrombasthenia patients, indicating that the second wave of phosphorylation of cortactin definitely depends on GPIIb/IIIa engagement (Rosa et al, 1997).…”

Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an αIIbβ3- or CD36-independent mechanism

“…In thrombasthenic platelets, the time-dependent translocation of tyrosinephosphorylated proteins to the cytoskeleton was also observed upon platelet activation. However, lower amounts of most of the proteins were detected as less tyrosine phosphorylation of the proteins occurred in these platelets, as shown by the analysis of the corresponding Triton X-100-soluble protein fractions and as also reported previously by others [36,39]. The presence of the tyrosine kinases Src, Syk and FAK and the lipid and tyrosine kinase PI-3K along with tyrosine-phosphorylated proteins was detected in the actin cytoskeleton from αIIbβ3and CD36deficient platelets ( Figure 7B).…”

A Ser752→Pro substitution in the cytoplasmic domain of β3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the αIIbβ3 integrin and the platelet granule pool of fibrinogen

“…However, macroscopic aggregation did not occur when the patient's platelets were stirred with thrombin (Chen et al, 1992). This was independently confirmed for this patient by Rosa et al (1997), who showed that addition of thrombin in stirred platelet suspensions was not followed by aggregation-dependent tyrosine phosphorylations. Why does aggregation not occur?…”