Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype

Abstract

Objective: Reduced blood flow and tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We, therefore, sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions.

Approach and results: In a cohort of patients with metabolic and peripheral artery disease, platelet activity was enhanced, and inhibition with oral antiplatelet agents was impaired compared with platelets from control subjects, suggesting a difference in platelet phenotype caused by the disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O₂) had increased expression of some proteins that augment platelet activation compared with platelets in normoxic conditions (21% O₂). Using a murine model of critical limb ischemia, platelet activity was increased even 2 weeks postsurgery compared with sham surgery mice. This effect was partly inhibited in platelet-specific ERK5 (extracellular regulated protein kinase 5) knockout mice.

Conclusions: These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses because of changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should, therefore, be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of antiplatelet therapy.

Keywords: hypoxia; inflammation; ischemia; myocardial infarction; peripheral arterial disease.

Figure 1.

Patients with metabolic and vascular disease have dysregulated platelets. A–C, Platelets from healthy patients (4) and healthy controls taking daily 81 mg aspirin (4) were compared with patients with metabolic and vascular comorbidities including diabetes mellitus and PAD (peripheral artery disease) taking platelet inhibitors (8), and patients with diabetes mellitus without PAD taking both platelet aspirin and clopidogrel (4). Platelets were stimulated with (A) a PAR1 (protease-activated receptor-1) agonist TRAP (thrombin receptor–activating peptide), (B) a thromboxane receptor agonist U46619, or (C) a P2Y₁₂ agonist 2-me-ADP for 15 min and activation was assessed by FACS (P-selectin expression, mean±SEM, *P<0.05 healthy vs diabetes mellitus+platelet inhibitors. **P<0.05 healthy vs vascular disease+platelet inhibitor(s). ***P<0.05 healthy vs healthy+aspirin, all by 1-way ANOVA. MFI indicates mean fluorescence intensity.

Figure 2.

Acute hypoxia augments platelet activation. Human platelets were isolated and activation was assessed after 2 h of in vitro normoxia (21% O₂) or hypoxia (5% O₂). Platelets were stimulated with (A) TRAP6 (thrombin receptor–activating peptide-6), (B) U46619, or (C) 2-me-ADP for 15 min and P-selectin expression was determined by FACS (mean±SEM, n=4). *P<0.05 or **P<0.01 normoxia vs hypoxia at each agonist concentration by 1-way ANOVA. D, Human platelets were isolated and activation was assessed after 2 h of normoxia or hypoxia. Platelets were then stimulated with TRAP6 (20 µmol/L), U46619 (20 µmol/L), or 2-me-ADP (10 µmol/L) for 15 min and activation assessed by FACS for GPIIb/IIIA activation (FITC-Fibrinogen binding, mean±SEM, n=3, *P<0.01 by 1-way ANOVA, NS=not significant). E, Human platelet ERK5 (extracellular regulated protein kinase 5) is activated in normoxia or hypoxia (mean pERK5/ERK5±SEM, *P=0.02 by t test, n=5 in each group).

Figure 3.

Murine platelet protein expression changes in vitro after agonist stimulation. A and B, Platelets isolated from mice were stimulated with 0.2 U/mL thrombin, 10 µmol/L U46619, or 10 µmol/L 2-me-ADP for 1 to 3 h. Protein expression was determined by (A) immunoblot (IB) and (B) quantified by densitometry (mean±SEM, *P=0.12 vs 0 for ERK5 (thrombin) and **P<0.05 vs 0 for P70S6K (thrombin) by 1-way ANOVA, n=3). Platelets isolated from mice were incubated for 0 to 6 h under hypoxic conditions (5% O₂) and platelet protein expression was assessed by IB and quantified by densitometry (mean±SEM, *P<0.05 or **P=0.058 vs 0 by 1-way ANOVA, n=4–6). D, Platelets were isolated from wild-type (WT) mice and activation was assessed after 6 h of normoxia or hypoxia and stimulated with thrombin for 15 min. Activation was assessed by P-selectin expression (mean±SEM, n=4, *P<0.05 by 1-way ANOVA). E, Left pneumonectomy (Pnx) or sham surgery mice demonstrate hypoxia by a compensatory increase in circulating blood hemoglobin concentration, P=0.0002 by t test between groups without a change in platelet count (559±44 vs 604±43 between groups, P=0.48 by t test, n=12 in each group). Isolated platelets show platelet ERK5 (extracellular regulated protein kinase 5) activation (mean pERK5/ERK5±SEM, *P=0.009 by t test, n=4 in each group). Mesenteric artery thrombosis was assessed in mice after living for 24 h at ambient oxygen or in hypoxic conditions (10% O₂, mean time to vessel occlusion±SEM, n=5 in each group, *P=0.026 by 1-way ANOVA for normoxia vs 10% O₂). G, Tail bleeding times as an index of hemostasis were calculated as the time in seconds for bleeding to stop after surgical amputation of the tail tip, median value, n=8 to 9, *P=0.021 by Mann–Whitney U test for normoxia vs 10% O₂.

Figure 4.

Human platelet protein expression changes in vitro after agonist stimulation. A, Human platelets were incubated in hypoxic conditions in vitro (5% O₂, Top) or stimulated with TRAP (thrombin receptor–activating peptide-6; 10 µmol/L), U46619 (20 µmol/L), or 2-me-ADP (10 µmol/L). Platelet protein expression was assessed for ERK5 (extracellular regulated protein kinase 5), then quantified by densitometry and reported as mean±SEM *P<0.05 vs 0 by 1-way ANOVA, n=4. B, Human platelets were placed in normoxic or hypoxic conditions for 2 h in the presence or absence of an ERK5 inhibitor (Bix 02189, 0.1–100 nmol/L) for the last 30 min. Platelets were then stimulated with TRAP (10 µmol/L). An ERK5 inhibitor attenuated platelet activation more effectively in hypoxia (% inhibition with ERK5 inhibitor vs no ERK5 inhibitor indicated above each condition). *P=0.040 vs TRAP alone+normoxia or **P<0.0001 vs TRAP alone+hypoxia by 1-way ANOVA, n=4.

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).

Figure 6.

Platelet ERK5 (extracellular regulated protein kinase 5) inhibition improved blood flow in critical limb ischemia. A–C, Wild-type (WT) or ERK5^−/− platelets from mice (A) 3, (B) 7, or (C) 14 d after hindlimb ischemia (HLI) were isolated and stimulated with thrombin for 15 min and activation assessed by FACS. WT platelets had more post-HLI activation compared with ERK5^−/− (P-selectin expression, mean±SEM, n=4, *P<0.01 between groups by 1-way ANOVA). D and E, Thermal Doppler color imaging showed more rapid return of blood flow in ERK5^−/− mouse limbs. D, Representative images, (E) quantification (mean ratio in the ischemic:nonischemic limb±SEM *P<0.001 Sham WT vs WT HLI, **P=0.013 WT HLI vs ERK5 ^−/− HLI, ***P=0.003 WT HLI vs ERK5 ^−/− HLI all by 1-way ANOVA. The group population size is indicated in parentheses.

Figure 7.

Proposed model. ROS indicates reactive oxygen species.