Pathophysiology of atherothrombosis: Mechanisms of thrombus formation on disrupted atherosclerotic plaques

Abstract

Atherothrombosis is a leading cause of cardiovascular mortality and morbidity worldwide. The underlying mechanisms of atherothrombosis comprise plaque disruption and subsequent thrombus formation. Arterial thrombi are thought to mainly comprise aggregated platelets as a result of high blood velocity. However, thrombi that develop on disrupted plaques comprise not only aggregated platelets, but also large amounts of fibrin, because plaques contain large amount of tissue factor that activate the coagulation cascade. Since not all thrombi grow large enough to occlude the vascular lumen, the propagation of thrombi is also critical in the onset of adverse vascular events. Various factors such as vascular wall thrombogenicity, local hemorheology, systemic thrombogenicity and fibrinolytic activity modulate thrombus formation and propagation. Although the activation mechanisms of platelets and the coagulation cascade have been intensively investigated, the underlying mechanisms of occlusive thrombus formation on disrupted plaques remain obscure. Pathological findings derived from humans and animal models of human atherothrombosis have uncovered pathophysiological processes during thrombus formation and propagation after plaque disruption, and novel factors have been identified that modulate the activation of platelets and the coagulation cascade. These findings have also provided insights into the development of novel drugs for atherothrombosis.

Keywords: atherothrombosis; blood flow; coagulation factor; platelet; vasoconstriction.

Figure 1

Microphotographs of human coronary plaque rupture and erosion with thrombi. Ruptured plaque comprises large necrotic core and disrupted thin fibrous cap accompanied by thrombus formation. Eroded plaque is fibrous and rich in smooth muscle cells, without visible atheromatous components. Both types of thrombi comprise platelets and fibrin (Ref. 13 with permission).

Figure 2

Localization and activity of tissue factor in human atherosclerotic lesions. (a) Tissue factor is localized in adventitia of nonatherosclerotic artery (infant coronary artery), but is broadly present in atheromatous lesion of coronary artery. (b) Schema of TF localization in atherosclerosis. Tissue factor is localized in SMC and macrophages during early to advanced stages of atherosclerosis. Large amounts of TF are localized in extracellular matrix of advanced lesions. (c) Tissue factor activity is found in all atherosclerotic lesions and is more prominent in fatty streaks and atheroma than in DIT. *P < 0.05, ^† P < 0.001, versus DIT. (Ref. 8 with permission). DIT, diffuse intimal thickening; Mac, macrophage; SMC, smooth muscle cells.

Figure 3

Tissue factor/factor VIIa complex‐dependent coagulation pathway and proteinase‐activated receptors (PAR). Membrane‐ and microparticle (MP)‐associated tissue factor (TF) binding to factor VIIa triggers coagulation pathway, whereas soluble TF with factor VIIa does not. Downstream coagulation factors activate PAR that also play other noncoagulative biological roles (Ref. 47).

Figure 4

Immunohistochemical microphotographs of tissue factor and thrombus in rabbit normal and atherosclerotic femoral arteries. Left and middle columns: Representative immunohistochemical microphotographs of normal femoral artery and of femoral arteries at 3 weeks after balloon injury of conventional (smooth muscle cells (SMC)‐rich neotima) or 0.5% cholesterol diet (Macrophage‐rich neointima). Middle column: Tissue factor is expressed in SMC‐ and macrophage‐rich neointima, and in adventitia. Right column: Thrombus at 15 min after balloon injury on normal artery comprises only small aggregated platelets, whereas that on neointima comprises platelets and fibrin. Thrombus on macrophage‐rich neointima is much larger. Ad, adventitia; HE/VB, hematoxylin and eosin/Victoria blue; I, intima; M, media (Ref. 51 with permission).

Figure 5

Plaque hypoxia and thrombogenicity in rabbit atherosclerotic lesion. (a) Representative histological and immunohistochemical images of rabbit atherosclerotic lesion show close proximity of pimonidazole (hypoxic marker) and tissue factor expression. (b) Effects of hypoxia on TF gene and protein expression in cultured macrophages. Tissue factor (TF) messenger RNA (mRNA) and protein levels in cultured THP‐1 macrophages are significantly increased after 6 h under 1% O₂ (hypoxia), and suppressed by inhibitors of either HIF‐1 (dimethyl‐bisphenol A, 100 μmol/L) or nuclear factor‐kappa B (NF‐kB) (Bay 11–7085, 20 μmol/L). *P < 0.0001 versus normoxia (21% O₂), and HIF‐1 and NF‐kB inhibitors. (c) Effects of hypoxia on TF and PAI‐1 protein expression in cultured atheromatous lesions. Protein levels of TF and PAI‐1 in cultured atheromatous lesions from rabbits are significantly increased at 6 h under hypoxic, versus normoxic conditions. *P < 0.0001 versus atheromatous lesions cultured under normoxia (21% O₂), or with HIF‐1 or NF‐kB inhibitor (Ref. 62 with permission).

Figure 6

18F‐FDG‐PET imaging and radioactivity accumulation in rabbit arteries. (a) Coronal image shows more 18F‐FDG accumulation in atherosclerotic (arrows) than normal (arrowheads) arteries. (b) Autoradiographic and histologic findings of atherosclerotic arteries with thrombus. Radioactivity has accumulated in sections of atherosclerotic arteries. Thrombus (arrows) consists of platelets and fibrin. (c) Correlations between 18F‐FDG uptake and vascular (a) and thrombus (b) components in arterial sections. Uptake of 18F‐FDG positively correlates with immunopositive areas for pimonidazole (hypoxia), macrophages, tissue factor and thrombus size. ARG, autoradiography; FDG, fluorodeoxyglucose; PET, positron emission tomography; SUV, standardized uptake value; UB, urinary bladder (Ref. 74, 77 with permission).

Figure 7

Computational flow simulation and microphotographs of erosive injury of rabbit stenotic femoral artery with SMC‐rich plaque. (a) Rabbit femoral arteries at 3 weeks after balloon injury were constricted using a vascular occluder (actuating tube) to reduce blood flow volume to 75%. (b) Representative computational reconstructed image and flow simulation in Reynolds‐Averaged Navier‐Stokes model. Red and blue mesh indicates high and low wall pressure, respectively. Flow velocity in this model increases at stenosis and decreases at post‐stenotic portion, resulting in disrupted flow. (c, d) Distribution of wall shear stress (WSS) and turbulence kinetic energy (TKE) of 3D‐image in reconstructed artery. Magnitude of WSS is increased at stenotic portion. Magnitude of TKE is broadly and heterogeneously increased in this model and is maximal at stenotic portion. (e, f) Representative microphotographs of erosive injury and thrombus formation. Neointimal endothelial cells and SMC are broadly detached at stenotic and post‐stenotic portions 15 min after vascular stenosis (e), and large mural thrombi formed 60 min after vascular stenosis (f). (Ref. 88, 89, 90 with permission).

Figure 8

Vasoconstriction induced by 5‐HT in rabbit femoral arteries. (a) Rabbit femoral artery without (i) and with (ii) smooth muscle cells (SMC)‐rich neointima. Neointima was induced by balloon catheter injury 3 weeks previously. Vasocontraction induced by 5‐HT is significantly more augmented in arteries with, than without (normal) SMC‐rich neointima. Femoral arteries without (●) or with (■) SMC‐rich neointima after endothelial denudation (iii). *P < 0.01 versus normal artery. Concentration‐response curves for 5‐HT of separated neointima with endothelial denudation (b) and separated media (c). Femoral artery gently separated into neointima and media. Cumulative amounts of 5‐HT induces proportional contraction in neointima and media. Sarpogrelate (selective 5‐HT_2A receptor antagonist) and fasudil (specific Rho‐kinase inhibitor), significantly inhibit both contractile responses. (Neointima (■), media (□), with 1.0 μM sarpogrelate (○) or 3.0 μM fasudil (▲) *P < 0.01 versus neointima or media (Ref. 95 with permission).

Figure 9

Activation of platelets and coagulation pathway at site of disrupted atherosclerotic plaque. 5‐HT, 5‐hydroxytryptamine; ADAMTS‐13, a disintegrin and metalloprotease with a thrombospondin type 1 motif 13; ADP, adenosine diphosphate; CLEC‐2, c‐type lectin‐like receptor 2; CRP, c‐reactive protein; NTPDase‐1, ecto‐nucleoside triphosphate diphosphohydrolase‐1; Mac, macrophage; SMC, smooth muscle cell; TF, tissue factor; TXA₂, thromboxane A₂; VWF, von Willebrand factor.