Underlying mechanisms of thrombus formation/growth in atherothrombosis and deep vein thrombosis

Abstract

Thrombosis remains a leading cause of death worldwide despite technological advances in prevention, diagnosis, and treatment. The traditional view of arterial thrombus formation is that it is a platelet-dependent process, whereas that of venous thrombus formation is a coagulation-dependent process. Current pathological and basic studies on atherothrombosis and venous thrombosis have revealed the diverse participation of platelet and coagulation activation mechanisms in both thrombus initiation and growth processes during clinical thrombotic events. Atherosclerotic plaque cell-derived tissue factor contributes to fibrin formation and platelet aggregation. The degree of plaque disruption and a blood flow alteration promote atherothrombotic occlusion. While blood stasis/turbulent flow due to luminal stenosis itself initiates venous thrombus formation. The coagulation factor XI-driven propagation phase of blood coagulation plays a major role in venous thrombus growth, but a minor role in hemostasis. These lines of evidence indicate that atherothrombosis onset is affected by the thrombogenic potential of atherosclerotic plaques, the plaque disruption size, and an alteration in blood flow. Upon onset of venous thrombosis, enhancement of the propagation phase of blood coagulation under blood stasis and a hypercoagulable state contribute to large thrombus formation.

Keywords: atherothrombosis; factor XI; thrombus formation; thrombus growth; venous thromboembolism.

Figure 1

Scheme of coagulation factors and endothelium‐dependent anticoagulation and fibrinolysis. Coagulation factors include enzymes (circles) and cofactors (triangles), and are present in blood except for tissue factor (TF). Thrombin (IIa), activated factor XI (FXIa), activated factor IX (FIXa), activated FX (FXa), activated factor VIII (FVIIIa), and activated factor V (FVa) drive the propagation phase of coagulation (red arrows) for thrombin generation. Thrombin contributes to fibrin formation and further platelet aggregation. Activated protein C (aPC) and protein S inactivate FVIIIa and FVa in the presence of thrombin and the thrombomodulin (TM) complex on the endothelium. The endothelium also releases tissue type plasminogen activator (t‐PA) that activates fibrinolysis. The scheme does not show each zymogen, factor XIII, or platelets that provide a lipid membrane for the coagulation reaction.

Figure 2

Constituents of coronary atherothrombus in AMI patients. Double immunofluorescence of an aspirated coronary thrombus. The coronary thrombus is composed of platelets (GPIIb/IIIa), fibrin, and von Willebrand factor (VWF). Adapted from Yamashita et al. with permission.

Figure 3

Tissue factor expression and fibrin deposition in ruptured plaques beneath platelet aggregations in patients with acute myocardial infarction. Tissue factor localizes in the necrotic core and macrophages. The thrombus on the ruptured plaque is rich in platelets, and some fibrin formation is visible in the thrombus. Fibrin formation is present around macrophages and cholesterin clefts, and in the necrotic core. In high magnification images (dashed squares), fibrin formation is present at the interface between the ruptured plaques and thrombus (arrows), and no fibrous matrix is observed (Azan staining). Interfaces are indicated by dashed lines. HE, hematoxylin and eosin; P, ruptured plaque; Th, thrombus. Adapted from Yamashita et al.

Figure 4

Disturbed blow flow induces superficial erosive injury and thrombus formation in SMC‐rich plaques. The SMC‐rich neointima was induced by a balloon catheter injury of the rabbit femoral artery. (a) Rabbit femoral arteries with SMC‐rich neointima were constricted using a vascular occluder (actuating tube) to reduce the blood flow volume to 75%. (b) Immunohistochemistry of VWF, an endothelial marker, and smooth muscle actin, a smooth muscle cell marker. Images show endothelial cell and SMC detachment (arrows). N indicates the SMC‐rich neointima. (c) Longitudinal section of a rabbit femoral artery showing mural thrombus formation (arrows) at the poststenotic portion 30 min after vascular narrowing. (d) Representative histological and immunohistochemical images of occlusive thrombus formation and SMC involvement (arrows) in the thrombus. N indicates the SMC‐rich neointima. Adapted from Sumi, et al. (b, d) with permission and Sawaguchi et al. (a). HE, hematoxylin and eosin; SMC, smooth muscle cell; VWF, von Willebrand factor.

Figure 5

Relationship between intraplaque hemorrhage and coronary thrombosis. (a) Representative in vitro MRI of a nonculprit lesion in an AMI patient, and histological and immunohistochemical images of a high‐signal intensity plaque on a T₁‐weighted image (WI). T₁ high‐intensity portions (asterisks) in the plaque show a low signal intensity on T₂WI and are variably immunopositive for CD68, glycophorin A, fibrin, and matrix metalloproteinase 9 (MMP9). There are tissue factor‐immunopositive foci in the plaque (arrows). The foci are close to the lumen (L). Adapted from Kuroiwa et al. with permission. (b) Bilirubin deposition and expression of ferritin and biliverdin reductase in a coronary ruptured plaque in an AMI patient. Ferritin and biliverdin reductase are predominantly expressed in yellowish (middle row, arrows) and grayish (bottom row, arrows) macrophages. Bilirubin deposition is present in the middle row (arrowhead). AMI, acute myocardial infarction, HE, hematoxylin and eosin; MRI, magnetic resonance imaging. Adapted from Yamashita et al.

Figure 5

Relationship between intraplaque hemorrhage and coronary thrombosis. (a) Representative in vitro MRI of a nonculprit lesion in an AMI patient, and histological and immunohistochemical images of a high‐signal intensity plaque on a T₁‐weighted image (WI). T₁ high‐intensity portions (asterisks) in the plaque show a low signal intensity on T₂WI and are variably immunopositive for CD68, glycophorin A, fibrin, and matrix metalloproteinase 9 (MMP9). There are tissue factor‐immunopositive foci in the plaque (arrows). The foci are close to the lumen (L). Adapted from Kuroiwa et al. with permission. (b) Bilirubin deposition and expression of ferritin and biliverdin reductase in a coronary ruptured plaque in an AMI patient. Ferritin and biliverdin reductase are predominantly expressed in yellowish (middle row, arrows) and grayish (bottom row, arrows) macrophages. Bilirubin deposition is present in the middle row (arrowhead). AMI, acute myocardial infarction, HE, hematoxylin and eosin; MRI, magnetic resonance imaging. Adapted from Yamashita et al.

Figure 6

Constituents of deep venous thrombus. Representative light and immunohistochemical microphotographs of an aspirated deep vein thrombosis. The thrombus is stained with hematoxylin and eosin (HE) and antibodies against glycophorin A, integrin α2bβ3 (a platelet protein), and fibrin. The thrombus is rich in glycophorin A and fibrin. Adapted from Furukoji et al.

Figure 7

Heterogenous components of DVT: The possibility of multistep growth phases. (a) An aspirated DVT at 33 days after onset shows various phases of reactions, fresh components without a degenerative change, a cell lytic change, macrophage infiltration and an organizing reaction (hematoxylin and eosin staining). (b) Representative magnetic resonance images of DVT patients. High‐signal intensity and high‐to‐iso signal intensity lesions on T₁‐weighted images (WI) occupied bilateral femoral to popliteal veins (arrows), indicating DVT. Merged diffusion weighted image (DWI) and T₁WI‐localized heterogeneous high‐to‐iso signal intensity lesions in deep veins on DWI. Areas of DVT with a high signal intensity on DWI have a low apparent diffusion coefficient (ADC) and high‐to‐iso signal intensity on T₂WI. ADC indicating the diffusion capacity of a water molecule and a low value reflecting restriction of its diffusion capacity. (c) DWI contrast‐to‐noise ratio in proximal and distal portions of DVT. (d) ADC of coagulated blood measured by in vitro MRI. Human venous blood containing sodium citrate was separated into WB (whole blood), PRP (platelet‐rich plasma), and ERB (erythrocyte‐rich blood) 1 and 2. Blood was coagulated using human placental tissue factor and a CaCl₂ solution. ADC values are associated with the erythrocyte content in clotting blood. n = 9 in each. Adapted from Gi et al. (b–d). DVT, deep vein thrombosis.

Figure 8

FVIII and FXI contribute to venous thrombus growth. (a) Recombinant FVIII (rFVIII) administration promotes venous thrombus formation. Fluorescence images at 15 min after rabbit jugular vein endothelial denudation and at 15 s after indocyanine green (ICG) infusion with saline (control, left) or 100 IU/kg rFVIII (right). Histological images show mural (control) or occlusive (rFVIII infusion) thrombus formation at 1 h after endothelial denudation. (b) Venous thrombus growth was monitored over time by ICG administration after endothelial denudation by rFVIII infusion. The ratio of the ICG fluorescence intensity to the background positively correlated to the thrombus size (r = 0.84). When the average fluorescence intensity of ICG exceeded three‐fold of the background, argatroban (thrombin inhibitor) or an anti‐tissue factor (TF), anti‐FXI, or anti‐VWF antibody was infused. The graph shows the difference in the fluorescence intensity of ICG before each inhibitor administration or at 1 h after endothelial denudation (n = 4 in each). (c) Venous thrombus components in rabbits and effect of the anti‐FXI antibody (XI‐5108, 3 mg/kg) on venous thrombus formation. Rabbit jugular vein thrombus is composed of erythrocytes, platelets (GPIIb/IIIa), and fibrin. Venous thrombus weight at 4 h after vessel ligation or endothelial denudation in jugular veins. Adapted from Takahashi et al. and Sugita et al. with permission. HE, hematoxylin and eosin.

Figure 9

Possible difference in mechanisms between hemostasis and venous thrombosis. (a) Blood coagulation in hemostasis. Vascular wall damage induces exposure of vascular wall‐derived tissue factor (TF) and initiates the coagulation cascade. FIX is predominantly activated by the FVIIa/TF complex, and activated FIX (FIXa) and activated FVIII (FVIIIa) are required for hemostatic thrombus formation. Activated FXI (FXIa) plays a minor role in hemostasis. FXII is not essential for hemostasis. Activated protein C (aPC) and protein S inactivate FVIIIa and FVa in the presence of the thrombin (IIa) and thrombomodulin (TM) complex on the endothelium. (b) Blood coagulation in venous thrombosis. Blood flow restriction or stasis induces leukocyte accumulation on a dysfunctional endothelium and/or detachment of the endothelium. Leukocyte‐ and/or vascular cell‐derived TF initiates the coagulation reaction. FXI activated by thrombin enhances further thrombin generation via FIX and FX activation. The propagation phase and blood flow alteration promote thrombus formation. This process is augmented by diminished activity of aPC and protein S. The contribution of FXII to thrombus growth is under debate. The schemes do not show each zymogen, factor XIII, or platelets that provide a lipid membrane for the coagulation reaction.