Atherosclerosis proceeds independently of thrombin-induced platelet activation in ApoE−/− mice

Abstract

Platelet activation has long been postulated to contribute to the development of atherosclerotic plaques, although the mechanism by which this might occur remains unknown. Thrombin is a potent platelet activator and transfusion of thrombin-activated platelets into mice increases plaque formation, suggesting that thrombin-induced platelet activation might contribute to platelet-dependent atherosclerosis. Platelets from protease-activated receptor 4-deficient (Par4−/−) mice fail to respond to thrombin. To determine whether thrombin-activated platelets play a necessary role in a model of atherogenesis, we compared plaque formation and progression in Par4+/+ and Par4−/− mice in the atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) background. Littermate Par4+/+ and Par4−/− mice, all ApoE−/−, were placed on a Western diet (21% fat, 0.15% cholesterol) for 5 or 10 weeks. The percent of aortic lumenal surface covered by plaques in Par4+/+ and Par4−/− mice was not different at either time point (2.2 ± 0.3% vs. 2.5 ± 0.2% and 5.1 ± 0.4% vs. 5.6 ± 0.4% after 5 and 10 weeks, respectively). Further, no differences were detected in the cross-sectional area of plaques measured at the aortic root (1.53 ± 0.17 vs. 1.66 ± 0.16 × 10⁵ μm² and 12.56 ± 1.23 vs. 13.03 ± 0.55 × 10⁵ μm² after 5 and 10 weeks, respectively). These findings indicate that thrombin-mediated platelet activation is not required for the early development of atherosclerotic plaques in the ApoE−/− mouse model and suggest that, if platelet activation is required for plaque formation under these experimental conditions, platelet activators other than thrombin suffice.

Introduction

The pathogenesis of atherosclerosis involves a complex interaction among lipids, plasma proteins and monocytes, vascular endothelial and smooth muscle cells, lymphocytes and platelets (for review see) [1]. Platelets play a central role in thrombus formation in response to erosion or rupture of atherosclerotic plaques, and such thrombi participate in the evolution of complex plaques and cause the major complications of atherosclerosis, myocardial infarction and stroke. Whether platelets make a significant contribution to the genesis of the early atherosclerotic plaque has been less certain. Whilst early work posited a role for platelet-released chemokines and growth factors in recruiting monocyte/macrophages to the vessel wall and promoting smooth muscle cell proliferation to cause plaque formation [2], [3], later work emphasized a key role for adhesion molecules, chemokines and growth factors expressed by activated endothelial cells and monocytes/macrophages themselves in driving these processes [4]. Yet strong evidence has emerged over recent years suggesting that activated platelets do indeed contribute to atherosclerotic plaque progression in well-defined animal models [5], [6], [7], [8]. Despite this, the mechanism(s) by which platelets become activated in the setting of atherogenesis remains unknown. Clues to possible platelet activation mechanisms come from studies demonstrating that tissue factor, the trigger for thrombin generation, is expressed by circulating monocytes in response to inflammatory stimuli [9], [10] and by monocyte-derived macrophages in atheroma [11], [12]. Further, transfusion of thrombin-activated platelets enhances plaque development in atherosclerosis-prone mice [13]. Thrombin is a potent activator of platelets and stimulation of endothelial cells by chemokines released by thrombin-activated platelets and the consequent expression of leukocyte-binding adhesion molecules by endothelial cells provides a potential mechanism by which platelets might promote monocyte recruitment and atheroma formation [13]. Platelet tethering of leukocytes to endothelial cells is well known to enhance leukocyte interactions with the vessel wall [14], and activated platelets and platelet–monocyte aggregates circulate in increased numbers in blood of atherosclerotic patients [15], [16], [17]. Together, these observations provide a possible mechanism by which thrombin-activated platelets might contribute to the formation of atherosclerotic plaques.

Thrombin triggers cellular responses via protease-activated receptors (PARs), and platelets from protease-activated receptor 4-deficient (Par4−/−) mice are unresponsive to thrombin [18]. Thus, such mice provide an opportunity to assess the contribution of thrombin-induced platelet activation in models of disease. To directly assess the importance of thrombin-induced platelet activation in atherogenesis, we examined the effect of Par4-deficiency on atherosclerotic plaque development in ApoE−/− mice. Our results demonstrate that thrombin-mediated platelet activation is not required for the early development of atherosclerotic plaques in the ApoE−/− mouse model and suggest that, if platelet activation contributes to atherogenesis under these experimental conditions, platelet activators other than thrombin are involved.