Figure 3. Secondary agonists augment ASK1 activation.

Western blot analysis of human platelets pretreated with aspirin (1mM) and apyrase (1U/mL) before being stimulated with various doses of thrombin (A) or collagen (B) for 1 and 3 min respectively. (Ai & Bi) Images of representative immunoblots of phosphorylation of ASK1 and p38 using phospho-specific antibodies. Blots were reprobed with anti-ASK1 and anti-p38 antibodies to ensure equal protein loading. (Aii & Bii) Quantification of band intensities of ASK1 from (Ai & Bi). ****P<0.0001, two-way ANOVA. (C) Intracellular Ca²⁺ rise was assessed by flow cytometry in washed human platelets pretreated with aspirin (1mM) and/or apyrase (1U/mL) before being stimulated with thrombin. (Ci) Representative tracings of Ca²⁺ rise in Fluo-4-loaded human platelets when stimulated with 0.1 U/mL thrombin. Baseline measurements were recorded before agonist addition at 60 s. Vertical dotted line denotes point of agonist addition. (Cii) Maximum fold change was assessed from 0–120 s. *P<0.05, one-way ANOVA with Dunnett’s multiple comparisons test.