Figure 5.

ERK5 (extracellular regulated protein kinase 5) promotes dysregulated platelet activity in critical limb ischemia. A, Platelets isolated from wild-type (WT) mice (left) or healthy humans (right) were incubated for 2 h under normoxic conditions (21% O₂) or after hypoxia exposure (5% O₂) and then loaded with DCFDA (2’,7’-dichlorodihydrofluorescein diacetate) to indicate reactive oxygen species (ROS) production, quantified by FACS analysis (mean±SD) *P<0.05 vs 21% O₂ by t test, n=3 in each group. B, Platelets isolated from humans with peripheral artery disease (PAD) or from mice after 4 d of unilateral left leg femoral artery ligation (hindlimb ischemia [HLI]) or sham surgery were assessed for ERK5 activation using a phospho-specific antibody (p-ERK5). Actin was used as an additional loading control for ERK5 because ERK5 protein content was increased in mice with HLI. ERK5 activation was quantified by densitometry and reported as mean pERK5/ERK5±SEM. *P=0.025 for control vs PAD, N=3 to 4, and P=0.14 for sham vs HLI mice by t test, N=4. Platelets isolated from mice after 4 d of HLI or sham surgery were assessed for expression of proteins known to affect platelet activation. Protein expression was assessed by immunoblotting (IB), then quantified by densitometry and reported as mean±SEM *P=0.046 vs 0 (ERK5), P=0.034 vs 0 (mTOR) and P=0.022 vs 0 (RAC1) by t test, n=3 to 4 in each group. D, WT mice were subjected to HLI or sham surgery and platelets isolated 7 d later were stimulated with thrombin for 15 min and activation assessed by FACS (P-selectin expression, mean±SEM, n=4 in each group by 1-way ANOVA, *P<0.05 between groups).