Platelet Inhibitors Reduce Rupture in a Mouse Model of Established Abdominal Aortic Aneurysm

Abstract

Objective: Rupture of abdominal aortic aneurysms causes a high morbidity and mortality in the elderly population. Platelet-rich thrombi form on the surface of aneurysms and may contribute to disease progression. In this study, we used a pharmacological approach to examine a role of platelets in established aneurysms induced by angiotensin II infusion into hypercholesterolemic mice.

Approach and results: Administration of the platelet inhibitors aspirin or clopidogrel bisulfate to established abdominal aortic aneurysms dramatically reduced rupture. These platelet inhibitors reduced abdominal aortic platelet and macrophage recruitment resulting in decreased active matrix metalloproteinase-2 and matrix metalloproteinase-9. Platelet inhibitors also resulted in reduced plasma concentrations of platelet factor 4, cytokines, and components of the plasminogen activation system in mice. To determine the validity of these findings in human subjects, a cohort of aneurysm patients were retrospectively analyzed using developed and validated algorithms in the electronic medical record database at Vanderbilt University. Similar to mice, administration of aspirin or P2Y12 inhibitors was associated with reduced death among patients with abdominal aortic aneurysm.

Conclusions: These results suggest that platelets contribute to abdominal aortic aneurysm progression and rupture.

Keywords: angiotensin II; aortic aneurysm, abdominal; aspirin; blood platelets; clopidogrel; mice.

Figure 1. Platelet inhibition was protective against rupture of established AAAs in mice

Ldlr^−/− mice were fed a HFD and infused with AngII for 28 days. Mice were then stratified based on in vivo suprarenal abdominal aortic diamters into 4 equal-sized groups and then placed on placebo (n = 14) or ASA (n = 16) and placebo (n = 15) or clopidogrel bisulfate (n = 15) and infused for an additional 42 days. Ultrasonically measured maximal luminal diameters of in vivo suprarenal aortas were measured at days 0, 28, 49, and 70 (A, D luminal diameters over time; B, E luminal diameters at day 70). Survival curves were also determined in these groups between days 28 and 70 (C, F). Circles represent group means ± SEM (A, D). Circles represent individual mice, diamonds represent means ± SEM (B, E). *P < 0.001 treatment groups versus controls. Data was analyzed with a Repeated Measures ANOVA, a Mann-Whitney Rank Sum with Dunn's post hoc, or a Kaplan Meier estimator.

Figure 2. Platelet inhibition decreased platelet and macrophage accumulation and MMP activity in mouse abdominal aortas

Ldlr^−/− mice underwent interventional therapy with placebo or ASA and placebo or clopidogrel bisulfate. Platelets (5 days before sacrifice) and macrophages (24 hours before sacrifice) were labelled with anti-GPIX conjugated 700nm fluorophore (red) or MMP 680-sense fluorophore (red) and dextran-coated nanoparticles conjugated to DyLight 800 fluorophore (green), respectively (treatments), or IgG placebo controls (controls). (A) Representative platelet, macrophage, merged, and grayscale images, (B) representative MMP, macrophage, merged, and grayscale images, and (C) subsequent quantification. Histobars represent means ± SEM of 4-8 mice. The abdominal aorta within the dotted yellow lines were analyzed for total fluorescent signal in panel C. *P < 0.001 placebo versus treatment groups. Data were analyzed with a One Way ANOVA on Ranks with Dunn's post hoc.

Figure 2. Platelet inhibition decreased platelet and macrophage accumulation and MMP activity in mouse abdominal aortas

Ldlr^−/− mice underwent interventional therapy with placebo or ASA and placebo or clopidogrel bisulfate. Platelets (5 days before sacrifice) and macrophages (24 hours before sacrifice) were labelled with anti-GPIX conjugated 700nm fluorophore (red) or MMP 680-sense fluorophore (red) and dextran-coated nanoparticles conjugated to DyLight 800 fluorophore (green), respectively (treatments), or IgG placebo controls (controls). (A) Representative platelet, macrophage, merged, and grayscale images, (B) representative MMP, macrophage, merged, and grayscale images, and (C) subsequent quantification. Histobars represent means ± SEM of 4-8 mice. The abdominal aorta within the dotted yellow lines were analyzed for total fluorescent signal in panel C. *P < 0.001 placebo versus treatment groups. Data were analyzed with a One Way ANOVA on Ranks with Dunn's post hoc.

Figure 3. Active MMP-2 and MMP-9 was decreased in the abdominal aorta of mice receiving platelet inhibitors

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total different pools analyzed n = 4). Pooled lysates (1 μg total protein/well) were analyzed by MMP-2 (A) and 9 (B) Biotrak Activity Assay ELISAs. Protein (20 μg/lane) from clopidogrel bisulfate (C) and ASA (D) administered mice was also examined using gelatin zymography. Histobars represent means ± SEM of n = 4 pooled aortas. *P < 0.001 clopidogrel bisulfate and ASA total MMP-2, active MMP-2, and active MMP-9 versus placebo controls. Data were analyzed with a One Way ANOVA with a Holm-Sidak Post Hoc.

Figure 4. The plasminogen activation system was decreased in plasma and abdominal aorta of mice receiving platelet inhibitors

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total n = 4). Plasma (n = 14-16 each treatment group) was analyzed on total or active (A) PAI-1, (C) tPA, or (E) uPA ELISA plates. Protein (1 μg protein/well) was run on total or active (B) PAI-1, (D) tPA, or (F) uPA ELISA plates. Histobars represent means ± SEM of n = 14-16 plasma samples (A, C, and E) or n = 4 pooled aortas (n = B, D, or F). *P < 0.001 clopidogrel and ASA total and active PAI-1, uPA, and tPA versus placebo controls. Data were analyzed with a Two Way ANOVA with a Holm-Sidak Post Hoc.

Figure 5. Platelet inhibition reduced plasma and tissue cytokines in established AAAs in mice

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total n = 4). Plasma (n = 14-16 each treatment group) or protein (1μg protein/well) was run on a PF4 ELISA (A). Plasma samples (n = 10 each treatment group) were analyzed on a chemokine/cytokine luminex array and (B) G-CSF, (C) IFN-γ, (D) RANTES, (E) IL-1α, or (F) IL-1β were quantitated. Histobars represent means ± SEM of n = 10 plasma samples (A-F) or n = 4 pooled aortas (A). *P < 0.001 clopidogrel and ASA versus placebo controls. Data were analyzed with a One Way ANOVA on Ranks with Dunn's Post Hoc (B-F) or Two Way ANOVA with a Holm-Sidak Post Hoc (A).

Figure 6. The effects of ASA and P2Y₁₂ inhibitors on AA rupture in a human cohort

Kaplan-Meier plot of adverse event-free rates stratified by (A) ASA and (B) platelet inhibitors and adjusted for age, sex, race, BMI, smoking, diabetes, CKD, dialysis, heart failure, and atrial fibrillation. *P < 0.001 drug therapy versus control. Data was evaluated with the proportional hazards test (PHtest) within the parameters of a kaplan-meier estimator in STATA and were found to be satisfied. All statistical tests assumed two-tailed distributions.