Authors: Atsushi Yamashita (aDepartment of Pathology, Division of Pathophysiology), Toshihiro Gi (aDepartment of Pathology, Division of Pathophysiology), Yuichiro Sato (bDepartment of Pathology, Section of Oncopathology and Morphological Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan)
Categories: VASCULAR BIOLOGY: Edited by Yohei Hisada, amniotic fluid embolism, atherothrombosis, ischemic stroke, pathology, thromboembolism
Source: Current Opinion in Hematology
Histological differences among thrombi in thrombotic diseases
Authors: Atsushi Yamashita, Toshihiro Gi, Yuichiro Sato
Abstract
Purpose of review
This review aims to summarize the histological differences among thrombi in acute myocardial infarction, ischemic stroke, venous thromboembolism, and amniotic fluid embolism, a newly identified thrombosis.
Recent findings
Acute coronary thrombi have a small size, are enriched in platelets and fibrin, and show the presence of fibrin and von Willebrand factor, but not collagen, at plaque rupture sites. Symptomatic deep vein thrombi are large and exhibit various phases of time-dependent histological changes. Cancer-associated venous thromboemboli contain invasive cancer cells that penetrate the vascular walls, and small cancer cell aggregates are observed within the thrombi. The thrombus composition in atherosclerotic and cardioembolic ischemic strokes varies from case to case, while the thrombi in cancer-associated ischemic stroke are rich in platelets and fibrin. A pathological study on amniotic fluid embolism identified uterine vein thrombi and massive platelet-rich microthrombi in the lungs.
Summary
Atherothrombus formation is induced by plaque disruption and may occlude a narrow lumen within a short time. Venous thrombi may grow to a large size in a multistage or chronic manner. Cancer cells can directly contribute to venous thrombus formation. The thrombus formation in amniotic fluid embolism may explain the occurrence of consumptive coagulopathy and cardiopulmonary collapse.
INTRODUCTION
Thrombotic diseases, such as acute myocardial infarction (AMI), ischemic stroke, and venous thromboembolism (VTE), have high morbidity and mortality rates worldwide. It is considered that rapid arterial flow induces platelet-rich thrombus formation, while static venous flow induces erythrocyte- and fibrin-rich thrombus formation. However, histological evaluations of thrombi have mainly been conducted in autopsy studies, and it is problematic to ascertain whether the autopsy findings reflect the findings at disease onset because of the time between disease onset and autopsy, the effects of treatment, and the time between death and tissue fixation. Recently, technological advances in endovascular devices have made it possible to evaluate the thrombus composition at disease onset [1]. To date, pathological and clinicopathological studies have investigated both autopsy specimens and aspirated thrombi [2–9]. Nevertheless, few articles have discussed the thrombus compositions across various thrombotic diseases [10,11]. Although the reports described differences in the proportions of platelets, fibrin, erythrocytes, and leukocytes, the observations were conducted using electron microscopy, which has restricted fields of view on the thrombus surface. Elucidation of other components in thrombi may provide insights into the mechanisms of thrombus formation in thrombotic diseases.
This review focuses on the thrombus sizes and compositions in AMI, ischemic stroke, VTE, and amniotic fluid embolism (AFE), a newly identified thrombosis, mainly based on histological evidence.
THROMBUS SIZES IN ACUTE MYOCARDIAL INFARCTION, ISCHEMIC STROKE, AND VENOUS THROMBOEMBOLISM
Before discussing the thrombus compositions, we will describe the thrombus sizes in AMI, ischemic stroke, and VTE, because little attention is typically paid to the differences in these sizes. Figure 1a shows the mean cross-sectional areas of the thrombi associated with AMI, atherosclerotic or cardioembolic ischemic stroke, deep vein thrombus (DVT), and pulmonary embolism (PE) deduced from autopsy cases. The thrombus areas in DVT and PE are significantly larger than those in AMI, and atherosclerotic and cardioembolic ischemic stroke. In most AMI cases, the coronary artery lumen is narrowed by atherosclerotic lesions, and the lumen can become occluded by small thrombi. The size of coronary thrombi is greatly affected by the degree of plaque rupture [12]. It is difficult to examine the time from plaque disruption to thrombus occlusion in clinical studies. In atherothrombosis models, atherosclerotic intimal lesions induced by balloon injury with or without cholesterol diet express tissue factor (TF) and form thrombi composed of platelets and fibrin, similar to human coronary thrombi [2,12,13]. The atherosclerotic intimal damage and stenosis led to thrombotic occlusion within a few minutes [13], and increased coagulation activity led to thrombotic occlusion within 15 min [14], suggesting that thrombus occlusion occurs within a short period of time. The difference in ischemic stroke may arise because the thrombi formed in the atria are larger than those formed in the cerebral arteries. The sizes of the thrombi in the carotid and cerebral arteries directly affect the infarct area. In Fig. 1a, the thrombus areas in DVT and PE are larger than those in AMI and ischemic stroke. Indeed, the median symptomatic DVT volume measured on magnetic resonance images was as large as 20 ml [15]. Smaller thrombi in atherosclerotic arteries cause symptoms, whereas larger thrombi are required to induce symptoms in veins. The large difference in thrombus sizes between AMI and DVT may have a significant impact on the time from thrombus formation to each disease onset. This difference, along with the difference in their thrombus compositions, should be taken into consideration for prevention strategies.
![FIGURE 1: Thrombus sizes and compositions in acute myocardial infarction, ischemic stroke, and deep vein thrombosis. (a) Mean thrombus histological cross-sectional areas in acute myocardial infarction (AMI; n = 25) [12], atherosclerotic ischemic stroke (IS; n = 6), cardioembolic ischemic stroke (IS; n = 11), deep vein thrombosis (DVT; n = 89), and pulmonary embolism (PE, n = 14). The thrombus area in AMI was adapted from Okuyama et al.[12] with permission. The thrombus areas in others are original data. (b) Median immunopositive areas for glycoprotein (GP) IIb/IIIa, fibrin, and glycophorin A in aspirated thrombi in acute myocardial infarction (AMI; n = 50), atherosclerotic ischemic stroke (IS; n = 11) [42], cardioembolic ischemic stroke (IS; n = 74) [42]; and deep vein thrombosis (DVT; n = 16) [6]. The thrombus composition in AMI is an original data. The thrombus composition in ischemic stroke was adapted from Shimuzu et al.[42] with permission. The thrombus composition in DVT was adapted from Furukoji et al.[6].](cohem-32-146-g002.jpg)
THROMBUS HISTOLOGY IN ACUTE MYOCARDIAL INFARCTION
Coronary atherosclerosis in acute myocardial infarction
Most cases of AMI are triggered by acute morphological changes in coronary atherosclerotic plaque. These morphological changes include plaque rupture and erosion as well as eruption of calcified nodules [16]. Plaque rupture is characterized by disruption of the fibrous cap in thin cap fibroatheroma and thrombus formation [2,16]. Plaque erosion is characterized by superficial erosive injury of smooth muscle cell (SMC)-rich atherosclerotic plaque [2,16]. Calcified nodules are atherosclerotic plaque lesions with nodules of calcification, and their eruption is associated with luminal thrombus formation [16,17]. Thus, the precursor lesions for plaque rupture, plaque erosion, and eruptive calcified nodules are considered to be thin-cap fibroatheroma, SMC-rich plaque, and calcified nodules, respectively.
Pathological characteristics of acute coronary thrombus
Acute coronary thrombi are composed of platelets, fibrin, leukocytes, and erythrocytes. Von Willebrand factor (VWF), which is important for platelet adhesion, is widely distributed in these thrombi [18–20]. In a semiquantitative immunohistochemical analysis of the thrombus components, acute coronary thrombi tended to be dominated by platelets and fibrin, with a few red blood cells. The proportions were unique compared with those in other thrombotic diseases (Fig. 1b). The observed proportions were inconsistent with the findings of another study, in which acute coronary thrombi were mainly composed of fibrin and other components, such as platelets, erythrocytes, cholesterol crystals, and leukocytes [21]. The discrepancy may arise from the different evaluation procedures used, namely assessment of thrombus cross-sections by immunohistochemistry and assessment of the thrombus surface by scanning electron microscopy. The immunopositive areas for platelets, fibrin, and VWF in the thrombi did not change within 12 h of onset [18], suggesting that platelet aggregation and fibrin formation occur simultaneously during thrombus formation, and that VWF is involved in occlusive thrombus formation through platelet recruitment on the thrombus surface [13,22]. A scanning electron microscopy study found that the coronary artery thrombi at <2 h after AMI onset were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, the thrombi at >12 h after AMI onset mainly consisted of platelets at the culprit site, while the red blood cell content increased from a low level at the initiating end to >90% at the proximal end [23]. These observations suggest that thrombus growth to the proximal side occurs after coronary obstruction under the static condition.
The coronary thrombi associated with plaque rupture were fibrin-rich, while those associated with plaque erosion were platelet-rich [2]. Eruptive calcified nodules were surrounded by fibrin with a few interspersed platelet aggregates [17]. These findings suggest different contributions of platelets and coagulation to thrombus formation associated with plaque rupture, plaque erosion, and eruptive calcified nodules [24]. However, owing to the limited number of pathological studies, further research is needed to validate these data.
Initial step of thrombus formation during coronary plaque rupture
Arterial thrombus formation is considered to be initiated by platelet adhesion to the exposed subendothelial matrix via VWF [25,26]. However, the precursor lesion, thin-cap fibrous atheroma, and ruptured plaque are characterized by a paucity of SMCs and fibrous matrix [27]. To reveal the scaffold for platelet adhesion on exposed plaque tissue, we pathologically examined the plaque components and thrombus interface in aspirated acute coronary thrombi with ruptured plaque. A necrotized core and macrophages were frequently observed at the interface between the ruptured plaque and thrombus. Fibrin and VWF were deposited within the plaque and beneath the platelet-rich acute thrombus (Fig. 2 a) [28]. However, no fibrous matrix stained blue by Azan staining was observed at the interface (Fig. 2 a) [28]. TF, a coagulation initiator, was localized in the necrotic core and macrophages, and the macrophages involved in the coronary thrombus were also seen [28,29]. This was the first pathological evidence suggesting that platelet adhesion to fibrin and/or VWF, rather than matrix proteins, is a possible initial step for thrombus formation at ruptured plaque, and that rupture-induced TF exposure in the necrotic core and macrophages initiates the coagulation cascade. An animal model of atherosclerotic plaque rupture has recently been reported, and the mice were susceptible to AMI and ischemic stroke [30]. This model may be useful for elucidating the mechanisms of thrombus formation associated with plaque rupture.
![FIGURE 2: Representative histological images of the interface between ruptured plaque and coronary thrombi and presence of cancer cells in venous thromboembolism. (a) Immunohistochemical staining for CD68 and tissue factor indicates the presence of macrophages and tissue factor expression in the ruptured plaque. The thrombus on the ruptured plaque is rich in platelets (GPIIb/IIIa), and some fibrin formation is visible within the thrombus (arrows). Fibrin deposition is evident around macrophages and cholesterin clefts, and in the necrotic core. In the high-magnification images of the dashed squares (lower panels), fibrin deposits are evident at the interface between the ruptured plaque and thrombus, and no fibrous matrix is observed in the Azan-stained image. The interface is indicated by the dashed line. GPIIb/IIIa, glycoprotein IIb/IIIa; HE, hematoxylin-eosin; P, ruptured plaque; Th, thrombus. (b) Venous thrombus in the inferior vena cava of a patient with pancreatic adenocarcinoma. The dashed line indicates the interface between the thrombus (Th) and the venous wall (V). Cytokeratin (CK)-positive cancer cells show direct invasion into the thrombus from the venous wall, associated with thrombus formation. The lower panels correspond to the high-magnification images in an area of upper panels. (c) Pulmonary thrombi in a patient with gastric adenocarcinoma. Cancer cell clusters are observed in a variety of sizes. There is no direct cancer invasion from the pulmonary artery (PA). The lower panels correspond to the high-magnification images in an area of upper panels. (d) Representative immunofluorescence image of tissue factor (red)-expressing cancer cells (green) in a venous thrombus from a patient with gastric adenocarcinoma. (e) Representative immunofluorescence image of podoplanin (red)-expressing cancer cells (green) in a pulmonary thrombus from a patient with cutaneous squamous cell carcinoma. Figure (a) was adapted from Yamashita et al.[28]. Figures (b–e) are original data.](cohem-32-146-g003.jpg)
![FIGURE 2 (Continued): Representative histological images of the interface between ruptured plaque and coronary thrombi and presence of cancer cells in venous thromboembolism. (a) Immunohistochemical staining for CD68 and tissue factor indicates the presence of macrophages and tissue factor expression in the ruptured plaque. The thrombus on the ruptured plaque is rich in platelets (GPIIb/IIIa), and some fibrin formation is visible within the thrombus (arrows). Fibrin deposition is evident around macrophages and cholesterin clefts, and in the necrotic core. In the high-magnification images of the dashed squares (lower panels), fibrin deposits are evident at the interface between the ruptured plaque and thrombus, and no fibrous matrix is observed in the Azan-stained image. The interface is indicated by the dashed line. GPIIb/IIIa, glycoprotein IIb/IIIa; HE, hematoxylin-eosin; P, ruptured plaque; Th, thrombus. (b) Venous thrombus in the inferior vena cava of a patient with pancreatic adenocarcinoma. The dashed line indicates the interface between the thrombus (Th) and the venous wall (V). Cytokeratin (CK)-positive cancer cells show direct invasion into the thrombus from the venous wall, associated with thrombus formation. The lower panels correspond to the high-magnification images in an area of upper panels. (c) Pulmonary thrombi in a patient with gastric adenocarcinoma. Cancer cell clusters are observed in a variety of sizes. There is no direct cancer invasion from the pulmonary artery (PA). The lower panels correspond to the high-magnification images in an area of upper panels. (d) Representative immunofluorescence image of tissue factor (red)-expressing cancer cells (green) in a venous thrombus from a patient with gastric adenocarcinoma. (e) Representative immunofluorescence image of podoplanin (red)-expressing cancer cells (green) in a pulmonary thrombus from a patient with cutaneous squamous cell carcinoma. Figure (a) was adapted from Yamashita et al.[28]. Figures (b–e) are original data.](cohem-32-146-g004.jpg)
Neutrophil extracellular trap formation in coronary thrombus
Neutrophils are the predominant leukocytes detected in acute coronary thrombi. Other leukocyte subpopulations are present in lower numbers in the following monocytes, T cells, and B cells [31]. A correlation between the myeloperoxidase-immunopositive area and aspirated thrombus area was noted in AMI [3,32]. Neutrophils form extracellular traps designated neutrophil extracellular traps (NETs) that show filamentous and reticulate staining with hematoxylin. The involvement of neutrophils in thrombus formation has attracted much attention, with NETs acting as scaffolds for platelets, fibrin, red blood cells, and activation of blood coagulation factor XII, while histones H3 and H4 promote platelet aggregation [33]. NET formation detected by anti-DNA-histone antibodies was observed in 90% of coronary thrombi, and the extent of NET formation in coronary thrombi was larger than that in DVT [31]. NETs detected by extracellular DNA and antihistone H1 antibodies were frequently observed in fresh (27%) and lytic (80%) thrombi, but never in organized thrombi [34]. Although NETs are thought to be involved in thrombus formation, the presence of the highest frequency of NETs in lytic thrombi suggests that NETs function after, rather than during, thrombus formation. Interestingly, monocytes, eosinophils, and mast cells also form extracellular traps (ETs) in acute coronary thrombi. NETs and monocyte ETs are predominant in coronary atherothrombosis. Furthermore, NETs are dominant in fresh and lytic thrombi, while monocyte ETs are numerous in the organization stage [35].
THROMBUS HISTOLOGY IN ISCHEMIC STROKE
Ischemic stroke can vary in its pathogenesis. It is mainly caused by large artery atherothrombosis and embolism, cardiogenic thromboembolism, or small vessel occlusion, but can have a cryptogenic origin [36]. Arterial conditions with rapid flow are considered to induce platelet-rich thrombus formation, while atrial fibrillation with disturbed flow is considered to induce fibrin-rich thrombus formation. Therefore, large artery atherothrombosis and cardioembolic stroke are mainly treated using antiplatelet agents or anticoagulants, respectively [37]. Cryptogenic strokes, accounting for one-fourth of ischemic stroke cases, are problematic for treatment strategies [38]. Therefore, it is important to clarify the thrombus composition in ischemic stroke cases to achieve secondary prevention.
The mean thrombus area in cardiogenic thromboemboli was approximately three times larger than the atherothrombus area in autopsy cases within 30 days of onset (Fig. 1a). The cardiogenic thromboemboli in the autopsy cases were rich in erythrocytes, while the atherothrombi were rich in fibrin [39]. Recent advances in thrombus aspiration for ischemic stroke have enabled histological analyses of the thrombi and evaluation of the associations between clinical and histological findings. Large studies involving more than 100 cases have consistently shown that cardiogenic emboli contain fewer erythrocytes and noncardiogenic thrombi contain more erythrocytes [40,41]. However, these studies semi-quantified the thrombus compositions using hematoxylin–eosin staining, rather than immunohistochemistry, and the differences between the platelet and fibrin components remain unclear. To identify the immunohistochemical composition of the thrombi in ischemic stroke cases, we assessed 108 aspirated thrombi [42]. No significant differences were noted in fibrin, platelets, erythrocytes, and VWF areas between cardiogenic thromboemboli and large artery atherothrombi (Fig. 1b). These results derived from autopsy and aspiration thrombi were inconsistent. This may be because autopsy thrombi can be affected by the time passed from the onset, and aspiration thrombi cannot be evaluated the entire thrombus. Although the relationship between thrombus composition and etiology remains inconclusive, the histological findings suggest that various degrees of platelet aggregation and blood coagulation reactions occur simultaneously in cardioembolic and large artery atherosclerotic stoke. Meanwhile, the thrombi in cryptogenic stroke consistently show the same components as cardiogenic thromboemboli, suggesting that most cryptogenic stroke cases are cardioembolic [40–42].
Leukocytes and NETs are also observed in ischemic stroke thrombi [43]. The extracellular DNA networks are predominantly observed in platelet-rich areas and in the boundaries between platelet-rich and erythrocyte-rich regions [44]. The association between the degree of NET formation and the etiology of stroke was not as consistent as the associations with other components [43,45]. Older thrombi showed significantly higher amounts of NET formation than fresh thrombi, similar to the case for acute coronary thrombi [34,43]. Accumulating evidence suggests that platelet-rich thrombi are resistant to thrombolysis [46,47]. The platelets, fibrin, VWF, and extracellular DNA form a fibrinolysis-resistant thrombus surface [47]. In addition, recombinant DNase 1 was found to accelerate tissue plasminogen activator-induced thrombolysis of ischemic stroke thrombi, while DNase 1 alone was ineffective [45]. The structural complexity and fibrinolysis resistance may explain why platelet-rich thrombi are resistant to thrombolysis.
Distinctive thrombus composition in cancer-associated ischemic stroke
Ten percent of hospitalized ischemic stroke patients have cancer, and cryptogenic stroke and multiple infarctions are more frequent in these patients compared with noncancer patients [48,49]. As cancer cells can release thrombogenic extracellular vesicles (EVs) and cell-free DNA [50], the thrombus composition in cancer-associated thrombosis may differ from those in other etiologies. An autopsy study found that nonbacterial thrombotic endocarditis was the leading cause of symptomatic ischemic stroke in cancer patients with cerebrovascular disease [51]. The thrombi in patients with active cancer were platelet-rich and erythrocyte-poor, and showed no difference in fibrin formation. This feature was more evident in patients with nonbacterial thrombotic endocarditis [52]. Although further research is needed, the thrombus composition in cancer-associated ischemic stroke may be distinctive, and nonbacterial thrombotic endocarditis may be the cause of thrombus formation in most patients with cryptogenic stroke and cancer. Immunohistochemical assessment of coagulation factors suggested that the extrinsic coagulation pathway was more important than the intrinsic pathway in cancer-associated ischemic stroke [53]. Antiplatelet agents and coagulation inhibitors may be useful for the prevention of cancer-associated ischemic stroke, although the risk of bleeding must be considered.
THROMBUS HISTOLOGY IN VENOUS THROMBOEMBOLISM
All DVTs and pulmonary emboli at vascular wall attachment sites were found to contain fibrin, erythrocytes, platelets, and VWF, and the proportions did not differ among the contents or between DVTs and PE [5]. Aspirated thrombi obtained from the proximal (upper) portion of DVTs were rich in erythrocytes and fibrin with few platelets (Fig. 1b) [6,54]. Platelets were predominantly localized in VWF- and fibrin-rich areas, but not in erythrocyte-rich areas. Factor VIII (FVIII) was closely associated with VWF, platelets, and fibrin [55]. Therefore, the thrombus compositions may differ between the thrombus initiation site and the growing portion, and VWF may recruit platelets and FVIII during venous thrombus formation. Acute DVT within 14 days after onset exhibited histological elements of chronicity, such as organization in 61% and fibrosis in 48% [56]. We pathologically classified the thrombotic areas in terms of fresh, cell lysis, and organizing reaction components. All aspirated thrombi showed a heterogeneous mixture of at least two of the three thrombotic areas. The fresh and organizing reaction components were observed in 83% of the cases [54]. These findings suggest that venous thrombus may grow to a large size in a multistage or chronic manner. This concept is supported by the heterogeneous intensity observed on diffusion-weighted magnetic resonance imaging of symptomatic DVTs. An animal study with in vitro magnetic resonance imaging confirmed that the erythrocyte content affects the signal intensity on diffusion-weighted images [15].
Presence of cancer cells within thrombi in cancer-associated venous thromboembolism
Basic studies have demonstrated that a tumor-bearing state promotes venous thrombus formation and stability via cancer cell-derived EVs, NET formation, platelet increases, or fibrinolysis inhibition [57]. Furthermore, cancer-associated fibroblasts, which are closely located around cancer cells, promote venous thrombus formation via platelet activation by podoplanin on fibroblast-derived EVs [58]. Because fibroblasts are rich in TF, cancer-associated fibroblast-derived EVs may also be involved in the activation of coagulation in cancer-associated thrombosis. However, the pathological findings at thrombus sites in cancer-related thrombosis are unclear. We observed cancer cells within the thrombi, which may have invaded into the vessels from the surrounding tissue (Fig. 2 b) or been incorporated into the thrombi from the venous blood (Fig. 2 c). These cancer cells expressed TF (a coagulation initiator; 76%), podoplanin (a platelet agonist; 44%), tissue-type plasminogen activator (36%), or plasminogen activator inhibitor-1 (20%) in varying proportions. Adenocarcinoma and squamous cell carcinoma exhibited frequent expression of TF (Fig. 2 d) and podoplanin (Fig. 2 e), respectively [59^▪^]. Thus, cancer cells may initiate and promote VTE by destruction of the vessel wall, activation of platelets and coagulation factors, and inhibition of fibrinolysis. The presence of tissue-type plasminogen activator-expressing cancer cells and the increase in TF-expressing macrophages in cancer-associated thrombi may explain the enhanced organizing response in these thrombi [59^▪^].
THROMBUS HISTOLOGY IN AMNIOTIC FLUID EMBOLISM
AFE is a serious obstetric disease in which amniotic fluid flows into the maternal circulation. The mortality rates in AFE range from 13% to 44%, resulting from rapid cardiopulmonary collapse during ongoing labor and massive uterine bleeding caused by coagulation activation and consumptive coagulopathy [60–62]. Regarding its pathogenesis, AFE has been considered to arise when amniotic fluid mechanically obstructs the pulmonary circulation, leading to cardiopulmonary collapse. However, the small amounts of amniotic emboli suggest that mechanical obstruction of the pulmonary circulation is not the main pathophysiology of AFE [63]. Thus, a complex immunological (anaphylactoid) reaction for amniotic antigens is currently thought to be the main underlying mechanism in AFE [64,65]. However, it remains unknown whether amniotic fluid induces thrombus formation and how the coagulation factors are consumed. We observed thrombus formation in the uterine vein and massive thrombus formation with or without amniotic emboli in the small pulmonary arteries and capillaries (Fig. 3 a). The pulmonary thrombi were significantly smaller than the uterine vein thrombus, and were rich in platelets and neutrophils without NET formation (Fig. 3 b). The histological components of the uterine vein thrombus were similar to those observed for a pulmonary embolus [66^▪^]. These histological findings suggest that amniotic fluid induces distinct thrombus formation in the uterine vein and pulmonary vessels, although the exact mechanisms of each thrombus formation remain unclear. It is possible that AFE is a novel thrombotic disease, and that the formation of the pulmonary and uterine thrombi may partly explain the pathophysiology of the sudden cardiorespiratory collapse and hemostatic alteration in AFE (Fig. 3 c).
![FIGURE 3: Representative histological images of pulmonary thrombi in amniotic fluid embolism. (a) Representative histological image of amniotic fluid embolism (hematoxylin–eosin staining). Multifocal small thrombi (asterisks) with or without amniotic fluid materials (arrows) are observed in the small pulmonary arteries. (b) Immunofluorescence images of the pulmonary thrombi. The microthrombi in the small pulmonary arteries are composed of platelets (green) and leukocytes (blue; nuclear staining with 4’,6-diamidino-2-phenylindole [DAPI]) without fibrin formation (red). Aggregated platelets are observed in the alveolar capillary vessels (arrows). (c) Schematic illustration of pulmonary microthrombus and uterine vein thrombus formation in amniotic fluid embolism. Although the pathogenesis remains unknown, amniotic fluid flows into the maternal circulation at the uterus, resulting in amniotic fluid embolism. Amniotic fluid entering the uterine veins forms a thrombus with a similar composition to a conventional deep vein thrombus. In the lungs, microthrombi mainly composed platelets and neutrophils show extensive formation alongside amniotic fluid emboli. NETs form in the uterine vein thrombi, but rarely in the pulmonary microthrombi. Mast cells in the perivascular and interstitial space may increase vascular permeability and affect the hemodynamics. The images are original data.](cohem-32-146-g005.jpg)
![FIGURE 3 (Continued): Representative histological images of pulmonary thrombi in amniotic fluid embolism. (a) Representative histological image of amniotic fluid embolism (hematoxylin–eosin staining). Multifocal small thrombi (asterisks) with or without amniotic fluid materials (arrows) are observed in the small pulmonary arteries. (b) Immunofluorescence images of the pulmonary thrombi. The microthrombi in the small pulmonary arteries are composed of platelets (green) and leukocytes (blue; nuclear staining with 4’,6-diamidino-2-phenylindole [DAPI]) without fibrin formation (red). Aggregated platelets are observed in the alveolar capillary vessels (arrows). (c) Schematic illustration of pulmonary microthrombus and uterine vein thrombus formation in amniotic fluid embolism. Although the pathogenesis remains unknown, amniotic fluid flows into the maternal circulation at the uterus, resulting in amniotic fluid embolism. Amniotic fluid entering the uterine veins forms a thrombus with a similar composition to a conventional deep vein thrombus. In the lungs, microthrombi mainly composed platelets and neutrophils show extensive formation alongside amniotic fluid emboli. NETs form in the uterine vein thrombi, but rarely in the pulmonary microthrombi. Mast cells in the perivascular and interstitial space may increase vascular permeability and affect the hemodynamics. The images are original data.](cohem-32-146-g006.jpg)
CONCLUSION
Acute coronary thrombi, ischemic stroke thrombi, and venous thromboemboli contain varying proportions of platelets, fibrin, and erythrocytes with NET formation. Based on the thrombus sizes and compositions, platelet-fibrin-rich atherothrombus formation is induced by acute plaque disruption and may occlude a narrow lumen within a short time, while venous thrombi rich in fibrin and erythrocytes form and may grow to a large size in a multistage or chronic manner (Fig. 4). Cancer cells in VTE may directly affect thrombus initiation and growth. Although the relationships between thrombus composition and atherosclerotic or cardioembolic stroke remain inconclusive, most cryptogenic stroke cases may be cardioembolic. AFE may be a novel thrombotic disease, and the thrombus formation may partly explain the occurrence of cardiorespiratory collapse and hemostatic alteration. Although NET formation is observed across the thrombotic diseases, further studies are required to clarify whether NET formation is a contributor during or after thrombus formation.
Acknowledgements
We thank Nahoko Udatsu and Kyoko Ohashi for their technical contributions to our research. We also thank Alison Sherwin, PhD, from Edanz ( https://jp.edanz.com ) for editing a draft of this manuscript.
Financial support and sponsorship
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (21K15403, 21K07706, 22K06961, 23K06467), and a Clinical Research Support Grant from University of Miyazaki Hospital.
Conflicts of interest
There are no conflicts of interest.