Figure 1.

Sustained Syk phosphorylation in thrombin-stimulated platelet is integrin-dependent. Washed platelets at 5 × 10⁸/mL were stimulated with Horm collagen (30 µg/mL), thrombin (1 U/mL), TRAP (10 µM) or fibrinogen (200 µg/mL) in the presence or absence of eptifibatide (9 µM). Activation was stopped at stated time after addition of the agonist. (A) Representative blot of Syk tyrosine phosphorylation from 6 donors. (Ai) Whole cell lysates were immunoprecipitated with anti-Syk mAb and probed with anti-phospho-tyrosine mAb 4G10; (Aii) Percentage of Syk tyrosine phosphorylation, based on Western blots, from 6 donors (mean ± s.d.); (Aiii) Aggregation trace of platelets stimulated with thrombin (1 U/mL), representative of 3 donors, and (Aiv) graph showing aggregation as % light transmission (mean ± s.d.) after 50 min of agonist stimulation. (B) Representative Western blot (Bi), and calculated percentage of Syk (Bii), PLCγ2 (Biii) and LAT (Biv) phosphorylation in response to thrombin (Thromb) from 5 donors (mean ± s.d.). Horm collagen (Col) was used as positive control. (C) Representative blot (Ci); mean ± s.d. of Syk (Cii), PLCγ2 (Ciii) and LAT (Civ) phosphorylation in response to collagen (Col), TRAP or fibrinogen (Fgn) from 3 donors. * p < 0.05, ** p < 0.005, one-tailed paired Student’s t-test. Where stated, proteins were immunoprecipitated (IP).