Understanding thrombosis: the critical role of oxidative stress

ABSTRACT

Thrombosis, a leading contributor to global health burden, is a complex process involving the interplay of various cell types, including vascular endothelial cells, platelets, and red blood cells. Oxidative stress, characterized by an overproduction of reactive oxygen species (ROS), can significantly impair the function of these cells, thus instigating a cascade of events leading to thrombus formation. In this review, we comprehensively explore the role of oxidative stress within these cells, and its mechanistic contribution to thrombogenesis, and the application of oxidative therapy in inhibiting thrombosis. By dissecting the intricacies of oxidative stress and its impact on thrombosis, we underscore its potential as a viable therapeutic target. Therefore, further research in this direction is warranted to enhance our understanding and management of thrombotic disorders.

KEYWORDS:

• Thrombosis
• oxidative stress
• reactive oxygen species
• endothelial cell
• platelets
• red blood cell
• neutrophils
• neutrophil extracellular traps

1. Introduction

Thrombosis is a common pathological process characterized by abnormal activation of coagulation factors and platelets in the bloodstream, leading to blood coagulation and thrombus formation [1]. Under normal circumstances, blood coagulation is a protective mechanism that prevents bleeding due to injury. However, when blood coagulation becomes excessively active or occurs in inappropriate locations, thrombus formation can occur, leading to the obstruction of blood vessels and resulting in various diseases and can even be life-threatening such as myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism [2]. During the process of thrombus formation, endothelial cells, platelets, and red blood cells (RBCs) play crucial roles [3–6]. Endothelial cells, which line the inner wall of blood vessels, not only contribute to the structural integrity of blood vessels but also produce a range of regulatory factors to maintain vascular function and hemostasis. These regulatory factors include thrombomodulin and vascular regulators such as nitric oxide (NO) and prostacyclin (PGI2). They help maintain the fibrinolytic state of the blood and prevent thrombus formation [7]. However, when endothelial cells are subjected to oxidative stress, their function becomes impaired, leading to an increased tendency of thrombus formation. Oxidative stress-induced damage and inflammatory reactions in endothelial cells disrupt the integrity of the vascular endothelial barrier, dysfunction of vasodilation and contraction, dysregulation of coagulation and fibrinolysis processes and an increased risk of thrombus formation [8]. Platelets also play a crucial role in thrombus formation. They are key players in hemostasis and thrombus formation. When vascular injury occurs, the excessive production of reactive oxygen species (ROS) can cause platelet dysfunction. This leads to abnormal activation and aggregation of platelets, promoting thrombus formation [9]. Additionally, RBCs participate in the process of thrombus formation. RBCs are not only responsible for oxygen transport to tissues and organs but also contribute to the formation and stability of blood clots. However, oxidative stress can cause functional impairment of RBCs, resulting in morphological and functional changes. This further increases the risk of thrombus formation. Oxidative stress plays a significant role in the functional abnormalities of these cell types [10,11]. It is a state characterized by an imbalance between excessive production and inadequate clearance of ROS, leading to disruption of the cellular redox balance. The excessive ROS production damages endothelial cells, platelets, and RBCs, impairing their normal functions and exacerbating the propensity for thrombus formation. Therefore, a comprehensive understanding of the association between oxidative stress and thrombus formation is crucial for unraveling the mechanisms underlying thrombotic diseases and exploring the potential of antioxidant therapy in suppressing thrombus formation.

2. The role of endothelium in thrombosis

2.1. Physiological function of endothelium

The endothelium, a single layer of epithelium covering the surface of the vascular system, plays a crucial role in maintaining the homeostasis of the vascular environment. It serves as a physical barrier between the blood and the vascular wall. Additionally, endothelial cells play a crucial role in preserving blood fluidity by providing a non-adhesive surface that prevents the activation of platelets and the coagulation cascade. Furthermore, these cells possess multiple fibrinolytic and antithrombotic properties on their cell surface, which actively contribute to preventing thrombus formation and ensuring the continuous flow of blood [7,8]. Furthermore, endothelial cells have a variety of functions, including regulation of transport from blood to underlying cells and tissues, permeability, vascular tension, cell adhesion and thrombolysis [7]. In addition, endothelial cells can respond to physical and chemical signals by producing a series of factors that regulate cell adhesion, smooth muscle cell proliferation, angiogenesis and vascular wall inflammation [7]. However, its dysfunction increases the risk of thrombosis.

2.2. Oxidative stress and vascular endothelial dysfunction

Oxidative stress has been extensively studied and has been shown to induce endothelial dysfunction [12]. Specifically, ROS can cause DNA damage, lipid peroxidation, and protein modifications in endothelial cells, leading to structural and functional impairment. This dysfunction can increase the propensity of blood vessels towards thrombus formation, partly due to impaired endothelium-dependent vasodilation, which affects blood flow and may result in the formation of turbulent blood flow within the vessel, a significant factor in thrombus formation [13].

2.2.1. Imbalance of NO

NO is an important bioactive substance synthesized by endothelial cells, which is catalyzed by endothelial NO synthase (eNOS). NO has a variety of physiological functions in vascular endothelium, including regulating vascular tension, inhibiting platelet aggregation, and preventing leukocyte adhesion [14–16]. Importantly, it has a obvious inhibitory effect on thrombosis such as NO can prevent thrombosis by increasing the synthesis of cyclic guanosine monophosphate (cGMP) and inhibiting platelet activation and aggregation [17,18]. It can also directly or indirectly inhibit the activity of several coagulation factors, such as factor Ⅶ a, factor X and factor Ⅱ a, thus inhibiting thrombosis. Under oxidative stress, on the one hand, more oxygen free radicals may be produced due to the increase in the consumption of antioxidants. These free radicals can further occupy the coenzyme position of eNOS and form ‘uncoupled eNOS’, resulting in a decrease in the ability of eNOS to produce NO and more peroxides [19–22]. On the other hand, oxygen free radicals can react directly with NO to form peroxynitrite (ONOO−) [23], which has both oxidizing and nitrifying properties, which can further destroy cell membrane, protein and DNA, and reduce the bioavailability of NO [24], thus contribute to thrombosis.

2.2.2. Coagulation factor

Due to oxidative stress, the expression of some key adhesion molecules such as ICAM-1 (CD54) and VCAM-1 (CD106) on the surface of endothelial cells is up-regulated [25]. These two molecules are members of the superfamily of cell adhesion molecules and are the key structures that mediate the interaction between leukocytes and endothelial cells. Studies have shown that increased expression of ICAM-1 and VCAM-1 can enhance platelet adhesion to endothelium, which may lead to thrombosis [25,26]. ROS enhances coagulation by upregulating tissue factor (TF) expression in endothelial cells [27]. Endothelial cells also express tissue factor pathway inhibitor (TFPI), which is the sole physiological regulator of TF activity, blocking the initiation of the extrinsic coagulation pathway [28], which can be inhibited by oxidative stress, thus leading to a procoagulant effect [29]. Thrombomodulin (TM) is a protein found on endothelial cell membranes. It is a critical mechanism through which the endothelium regulates hemostasis. TM interacts with thrombin, either binding and sequestering it or enhancing its affinity for protein C, an important anticoagulant. This process helps maintain a delicate balance between blood clotting and prevention of excessive clot formation, ensuring proper hemostasis within blood vessels [30,31]. Research has shown that inflammatory stimuli can reduce the levels of TM in endothelial cells [32]. Furthermore, oxidative stress has been demonstrated to activate inflammation in endothelial cells, which promotes thrombus formation [33,34].

2.2.3. Inflammatory response caused by oxidative stress

Oxidative stress can induce changes in the expression of inflammation-related genes in endothelial cells. Among them, nuclear factor-kappa B (NF-κB) is a crucial regulator of inflammatory signaling pathways, and its activation in endothelial cells promotes the expression of various inflammatory factors, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), Monocyte Chemoattractant Protein-1 (MCP-1) [35,36]. The increase in these inflammatory factors leads to the migration of leukocytes to the vascular endothelium and enhances endothelial cell adhesion to platelets, thereby increasing the risk of thrombus formation. Endothelial cells are stimulated with pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1, to up-regulate the production of TF and vWF, while reducing the expression of TM, NO and PGI2 [37]. PGI2 is one of the main mechanisms through which vascular endothelial cells negatively regulate platelets. A decrease in PGI2 indicates impaired anti-thrombotic defense function [38].

3. The role of platelets in thrombosis

3.1. Physiological function of platelets

Platelets are an important cellular component in blood, which is mainly responsible for hemostasis and thrombosis. In cases of vascular damage, platelets are the first to respond, adhering to the injured vascular wall through the binding of platelet glycoproteins (GP) Ia/IIa and vWF and GPVI and collagen [39]. Then, by releasing aggregators such as ADP and thromboxane A2 (TXA2), they promote more platelet aggregation and form primary thrombus [40]. After the aggregation of primary thrombus, the phospholipids and tissue factors on the surface of platelets can effectively catalyze the formation of thrombin, promote the production of fibrin and make the thrombus more stable [1]. Platelets can also release a series of chemical signaling molecules, participating in inflammation and immune responses, such as inducing leukocyte migration and activation.

3.2. Oxidative stress-induced activation, adhesion of platelets in thrombus formation

Platelets contain an efficient antioxidant enzyme system, which includes key components such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione transferase (GST), and glutathione reductase (GSSG-R). Nonetheless, an imbalance between ROS production and the effectiveness of the antioxidant system can contribute to thrombotic disease development by causing high intracellular ROS levels, thereby promoting increased platelet activation [41]. During activation, platelets produce ROS via various intracellular origins including NADPH oxidase, cyclooxygenases, eNOS, xanthine oxidase (XO), and mitochondrial respiratory processes [42,43]. NADPH oxidase, now known to be functionally expressed in platelets as well as in phagocytes, contributes to thrombus formation via superoxide anion production, altering platelet activation and increasing ADP availability, with certain inhibitors shown to reduce such effects, while within the NADPH oxidase family, members NOX1 and NOX2 have been identified as having distinct roles in platelet activation pathways. In addition, ROS can further increase the production of ROS by activating platelet NOX, forming a vicious circle [44]. Increased ROS levels due to oxidative stress can enhance platelet activation signaling pathways. A crucial mechanism involves ROS enhancing the activity of platelet phosphoinositide 3-kinase (PI3 K) and protein kinase C (PKC) signaling pathways, resulting in platelet aggregation, shape change, and granule release [45]. ROS can increase the inflammatory mediators produced by platelets, such as platelet activating factor (PAF) and TXA2, which further promote platelet activation and lead to thrombosis [40]. Furthermore, platelet activation occurs through the intraplatelet ROS-triggered oxidation of arachidonic acid, leading to the generation of isoprostanes [46], which associated with platelet activation. In addition, ROS can enhance platelet adhesion by regulating the expression of platelet adhesion molecules, such as GP IIb/IIIa and P-selectin, thus increasing platelet adhesion to the vascular wall [47,48]. Under the influence of inflammation and oxidative stress, neutrophils can produce structures known as neutrophil extracellular traps (NETs), which are web-like structures composed of DNA, histones, and granule proteins. NETs have the ability to capture and eliminate microorganisms, but they are also associated with thrombosis [11,49]. The DNA and histones on NETs can interact with platelets and coagulation factors, activating platelets and promoting blood coagulation, while simultaneously inhibiting the activation of plasminogen, thereby reducing the clot-dissolving capacity of the blood [50]. The impaired and activated endothelial barrier may lead to the exposure or release of prothrombotic proteins like collagen, tissue factor, and von Willebrand Factor [51,52], as well as chemotactic proteins, such as cytokines and adhesion molecules [53], into the bloodstream. These factors promote additional coagulation, platelet aggregation, and recruitment of leukocytes. ROS can indirectly increase platelet reactivity by inhibiting endogenous mechanisms responsible for platelet inhibition. For instance, ROS can impair the NO produced by endothelial cells, which normally exerts an anti-platelet aggregating effect [54]. In addition, ROS can affect the calcium signal in platelets, which is an important process of platelet activation [11].

4. The role of erythrocyte in thrombosis

4.1. Physiological function of RBCs

RBCs, also known as erythrocytes, are one of the most common cell types in our body, and their primary physiological role is to transport oxygen and carbon dioxide.

Transporting Oxygen: Hemoglobin within RBCs can bind with oxygen, forming oxyhemoglobin. As RBCs pass through the lungs, they pick up oxygen and transport it to various parts of the body. This is a crucial step for tissues and organs to undergo oxidative reactions and obtain energy. Transporting Carbon Dioxide: When the body’s tissues and organs undergo oxidative reactions to produce energy, carbon dioxide is generated. Carbon dioxide is transported back to the lungs through hemoglobin in RBCs and then exhaled through respiration [55,56]. In addition, erythrocytes regulate the balance of acid-base metabolism through carbon dioxide metabolism [57,58] In addition to transporting oxygen and carbon dioxide, RBCs also serve several other important functions. Regulating Blood Viscosity and Flow: The shape, quantity, and deformability of RBCs influence the viscosity and flow of blood, affecting the smooth circulation through microcapillaries and the workload of the heart’s pumping [6,57]. These functions collectively play a critical role in maintaining the body’s normal physiological functions.

4.2. The relationship between RBCs and thrombus

Traditionally, RBCs were considered to play a bystander role in hemostasis and thrombosis. However, recent studies reveal that RBCs have significant impacts on these processes and various other important functions [6,59–61]. The earliest clinical studies demonstrate that transfusing RBCs significantly improved bleeding times in thrombocytopenic patients, despite their low platelet counts, indicating a potential role for RBCs in blood coagulation [62]. Consequently, RBC transfusions have been utilized to address various bleeding disorders, regardless of whether platelet levels are normal or low. Therefore, RBCs play an important role in thrombosis.

RBCs primarily affect blood viscosity, which increases with hematocrit and can lead to a prothrombotic state by slowing blood flow [63]. At low shear rates, erythrocytes can form ‘rouleaux’ structures, increasing viscosity [64], especially in lower shear-rate vessels like the lower limbs, predisposing them to venous thrombosis [65]. High hematocrit levels can cause platelets to accumulate near vessel walls, increasing their interaction with the endothelium [66]. Reduced local viscosity due to increased hematocrit can also decrease NO release, potentially leading to a prothrombotic state [67]. Furthermore, Blood viscosity increases non-linearly with hematocrit, influencing thrombosis through slowing down blood flow, a prothrombotic factor within Virchow’s triad. This increase in blood viscosity can also promote platelet margination and interaction with blood vessel walls [61]. However, the correlation between high hematocrit levels and thrombosis is not absolute. A study have shown that extremely high hematocrit mice also displayed a tendency to bleed, while animals with lower hematocrit levels were not distinguishable from controls in thrombosis models [68]. The relationship between RBC content and thrombosis may be complex and warrants further study.

Besides, RBCs’ deformability can also affect thrombosis. Increase of RBC membrane stiffness can cause hemolysis [69]. Hemolysis leads to the release of harmful hemoglobin and heme [70]. Studies have shown that free hemoglobin and heme are also beneficial to production of ROS and activation of macrophages and endothelial cells [71], leading to the formation of thrombus [70,72]. In addition, rigid RBCs have a higher thrombogenic potential as they have difficulty navigating the microvasculature and promote platelet margination.

4.3. The role of oxidative stress of erythrocytes in thrombus

As mentioned above, there is a notable correlation between RBCs and thrombus formation. However, it is important to highlight that dysfunction in the functionality of RBCs can contribute to the propensity for clotting. Oxidative stress is a common factor that plays into this dynamic [73]. First of all, ROS can activate calcium ion channels, leading to damage of the RBC membrane and reduced cell deformability, and inducing cell lysis [74], increase of viscosity [60]. Furthermore, the influx of calcium ions can result in the exposure of phosphatidylserine on the RBC membrane. Phosphatidylserine, under normal conditions, is located on the inner side of the cell membrane, but when it flips to the outside, it serves as a key signal for the initiation of coagulation and promotes the activation of prothrombin, contributing significantly to thrombus formation [75,76]. In addition, oxidative stress can oxidize divalent iron ions to hemoglobin of trivalent iron ions, which are reported to contribute to erythrocyte lysis and thrombosis [10]. In addition, free hemoglobin and heme can further lead to inflammation, vasoconstriction and increased permeability through the toll-like receptor signal pathway [77–79]. Nevertheless, haeme released from damaged cells can also activate platelets through its binding to glycoprotein-1b alpha (GPIbα) receptors on platelet surfaces [80]. Red blood cells are rich in iron. The increase of oxidative stress can oxidize Fe²⁺ to Fe³⁺, which will further cause erythrocyte lysis, oxidative stress and thrombosis [10]. Moreover, Heme released from lytic erythrocytes can induce the formation of NET and the activation and injury of endothelium [81]. In conclusion, it is crucial to emphasize the significant role of oxidative stress on RBCs in promoting thrombosis.

The apoptosis or senescence of red blood cells gives rise to small extracellular membrane structures known as microvesicles (MVs) or microparticles (MPs). MVs serve as a mode of intercellular communication and are involved in numerous physiological and pathological regulatory mechanisms, such as acting as pathogenic factors in various thrombotic and hemostatic disorders. Studies indicate that elevated levels of MVs can promote the generation of thrombin, enhance the expression of phosphatidylserine [82], and reduce clotting time [83]. Furthermore, red cell-derived MVs initiate the coagulation pathway through Factor XII, bypassing the need for tissue factor [84]. Additionally, excessive ROS-induced exposure of phosphatidylserine in red blood cells may accelerate MPs production, thereby promoting prothrombotic events [73]. Oxidative stress and intercellular crosstalk in endothelial cells, platelets and erythrocytes play an important role in promoting thrombosis, as shown by Figure 1.

Figure 1. Oxidative stress of endothelial cells, platelets and erythrocytes and their crosstalk promote thrombosis.

5. The role of neutrophils in thrombosis

5.1. Physiological role of neutrophils

Neutrophils are the most abundant white blood cells, accounting for 50% of all circulating leukocytes, accounting for 70% of all circulating leukocytes [85]. Neutrophils are essential for innate immunity and resistance to pathogens, and act as the first line of defense to control infection and help clear cell fragments. It can be induced to express genes encoding key inflammatory mediators, including complement components, Fc receptors, chemokine and cytokine [86]. In addition, recent evidence suggests that neutrophils can also produce anti-inflammatory molecules and factors that promote inflammatory regression [87]. The decrease of neutrophils in the blood leads to severe immune deficiency in humans. Therefore, neutrophils play an important role in immune homeostasis.

5.2. The role of neutrophils in thrombosis

In the past, it is believed that platelets have hemostatic effect at the injured site after endothelial injury. A study has found that neutrophils bind to activated endothelial cells through the interaction of leukocyte functional antigen-1 (ICAM-1), and immediately adhere to the damaged blood vessels, and this process precedes platelets [88], which indicates neutrophils also play a vital role in the process of thrombosis and has garnered increasing attention in thrombosis research. Neutrophils contribute to the pathogenesis of thrombosis by the extrusion of web-like structures known as NETs, which consist of decondensed chromatin and antimicrobial proteins [89]. These NETs provide a scaffold that supports platelet adhesion and activation, leading to subsequent platelet aggregation. Additionally, NETs serve as a platform for the assembly of clotting factors, which furthers the coagulation cascade [90]. The interplay between activated neutrophils and platelets, along with the pro-inflammatory milieu they create, not only perpetuates vascular inflammation but also enhances the collaborative interactions among various cell types within the blood, including endothelial cells and monocytes [49,91]. This multifaceted role of neutrophils, through the release of NETs, the promotion of platelet activity, and the intensification of inflammatory reactions, collectively augments the propensity for thrombus formation. Therefore, NETs are the central mechanism by which neutrophils contribute to thrombus formation.

5.2.1. The contribution of NETs to thrombus formation

NETs are structures released by neutrophils in response to certain stimuli, comprising intracellular DNA, histones, and various enzymes. Initially perceived as a defensive mechanism for trapping and killing pathogens [90], recent studies have elucidated a significant role for NETs in the formation of thrombi [90,92]. Interaction between NETs and platelets is pivotal in thrombus formation, where platelets are captured by NETs, laying the foundation for thrombus development. Specifically, DNA within NETs binds to platelets [93], promoting platelet activation, aggregation, and thrombosis [49]. This binding is attenuated following treatment with deoxyribonuclease (DNase). Moreover, histones in NETs can activate platelets via toll-like receptor 2 (TLR2) and TLR4 receptors [93,94], and citrullinated histone (citH3) in NETs interacts with von vWF, further promoting the formation of thrombi rich in erythrocytes and platelets [95]. Studies indicate that platelets, activated by their interaction with NETs, bind to neutrophils through GPIb, thereby enhancing NETs release and perpetuating a vicious cycle [96,97].

NETs also enhance venous thrombosis by activating the coagulation cascade. In addition to inducing platelet aggregation, histones in NETs also promote thrombin generation [98]. Studies demonstrate that histones induce platelets to release inorganic polyphosphates, exposing membrane-bound phosphatidylserine and activated Factor V, thereby enhancing the activity of the prothrombinase complex [94]. Furthermore, histones facilitate the exposure of phosphatidylserine on erythrocytes, promoting the assembly of the prothrombinase complex and fibrin formation [76]. Additionally, thrombin production can be attenuated by DNase treatment, confirming the contribution of DNA in NETs to thrombin generation [98]. NETs activate Factor XII [96], and studies have shown that FXII promotes the generation of neutrophil NETs via uPAR-mediated phosphorylation of AKT2 at S474 [99], creating a feedback loop that exacerbates thrombus formation. Additionally, NETs are decorated with TF and protein disulfide isomerase, facilitating the activation of the extrinsic coagulation pathway [100,101]. NETs also contain serine proteases, which lead to the degradation and inactivation of TFPI, thus promoting thrombin and fibrin clot production [102].

Furthermore, NETs regulate other blood cells, playing a significant role in thrombus formation. In terms of their effect on erythrocytes, the histones in NETs promote red blood cell aggregation, fragility, and rigidity, potentially leading to hemolysis and the release of detrimental factors such as hemoglobin [103]. Moreover, after incubation with primary endothelial cells or endothelial cell lines, histones upregulate functional transferrin, while downregulating thrombomodulin expression [104,105]. In addition, the activation of P-selectin glycoprotein ligand-1 (PSGL-1) and C-X-C chemokine receptor type 2 (CXCR2) on moving neutrophils sends out coordinated signals leading to β2 integrin-dependent neutrophil attachment to the endothelium, resulting in immobilization [106]. In addition, the interaction between neutrophils and platelets and the PSGL-1 and CXCR2 signals of neutrophils contribute to the formation of NET [107,108]. NETs adhere to the vascular wall, disrupting blood flow and promoting endothelial damage. Additionally, NETs provide a binding site for various coagulation components, including platelets, leukocytes, erythrocytes, and soluble clotting factors. This facilitates intercellular interactions, further promoting the formation of thrombi [91,95,109]. Consequently, Thus, neutrophils, especially through the formation of NETs, play a pivotal role in thrombus formation that cannot be overlooked. NETs may offer new targets for the development of drugs to treat deep vein thrombosis.

5.3. ROS and NETs in thrombosis

Most processes leading to NETs release are initiated by ROS, produced by the NOX2 enzyme of phagocytes [110,111]. Various NOX2 stimulators, including proinflammatory cytokines, LPS, TLR agonists, and agents like PMA, are known to induce NETs formation [11]. In addition, neutrophil NOX can also promote platelet-neutrophil interaction, which indirectly promotes the formation of NET [112]. ROS initiates NETs formation by moving neutrophil elastase (NE) and myeloperoxidase (MPO) from cytoplasmic granules to the nucleus. This action leads to chromatin decondensation, with NE cutting nucleosomal histones and MPO further aiding this process, thus triggering NETs formation [113]. ROS also promotes chromatin decondensation via citrullination dependent on PAD4 [114]. Furthermore, ROS induce the activation of MAPK, which are involved in the formation of the net-like structures [115]. Additionally, ROS can induce neutrophil apoptosis, thereby promoting the release of NETs and contributing to thrombus formation [116]. NLRP3 inflammasome which can be promoted by ROS plays a role in promoting NET formation. It is involved in the process of NETs formation under sterile conditions and contributes to both nuclear envelope and plasma membrane rupture in neutrophils, facilitating the release of NETs. This indicates that NLRP3 is a key factor in the formation of NETs, especially in non-infectious conditions and in the context of deep vein thrombosis [117]. In summary, NETs play a central role in the contribution of neutrophils to thrombus formation, with ROS being a key factor in promoting NET generation. Therefore, inhibiting ROS can be beneficial in reducing NETs formation by neutrophils. The role of neutrophils in thrombosis is shown in Figure 2.

Figure 2. The role of neutrophil oxidative stress in thrombus formation.

6. Strategies for the treatment of oxidative stress in thrombosis

Oxidative stress is an important factor in thrombosis. Studies of antioxidant treatments have shown great promise in pre-clinical studies, but have so far been pretty disappointing in applied to the patients. Therefore, it is necessary to explore research targeting ROS in the inhibition of thrombus formation. Angiotensin-converting enzyme (ACE) inhibitors prevent the conversion of Angiotensin I to Angiotensin II, which, in turn, promotes endothelial dysfunction by facilitating the recruitment of white blood cells and the production of ROS. This leads to increased LDL oxidation and the degradation of NO [118]. Statins have anti-inflammatory and antioxidant effects [119,120]. Research indicates that statins can interfere with the migration and proliferation of white blood cells, as well as disrupt the interaction between white blood cells and endothelial cells [121], which are risk factors for thrombosis. Furthermore, activation of Rho family members is a major source of ROS production in the vascular system. Statins can inhibit the activation of Rho and Rac, thus reducing endothelial cell activation while increasing eNOS expression and endothelial NO production, which contributes to the prevention of thrombus formation [122,123]. Research has shown that vitamin E has antioxidant effect and demonstrates multifaceted antithrombotic effects both in vitro and in vivo. These effects involve decreasing the expression and release of adhesion molecules on endothelial cells, thereby impeding interactions between white blood cells and endothelial cells [11]. In addition, in healthy volunteers, 600 mg daily vitamin E significantly inhibited collagen-induced platelet activation and H₂O₂ production [124] and induced an anti-adhesive effect in platelets [125]. Vitamin C has also been shown to inhibit platelet oxidative stress, thereby inhibiting platelet activation [126]. Furthermore, some antioxidants such as catalase [46], N-acetylcysteine (NAC) [127], polyphenols [126] have been reported to weaken ROS-mediated platelet activation in thrombosis. In terms of antioxidant genes, knockout of GPX3 promotes platelet-dependent thrombosis in mice [128], while overexpression of GPX1 inhibits platelet overactivation and reduces thrombosis [129]. Oxidative stress of red blood cells is an important factor causing thrombosis. HO-1 is an important antioxidant gene, which can increase the size of thrombus after thrombus formation in HO-1 knockout mice [130]. In addition, the decreased activity of HO-1 increases the risk of recurrent venous thromboembolism [131]. Moreover, many dietary therapies also have antioxidant effects, such as tocopherol in beer has been reported to reduce ROS and inhibit platelet activation to prevent arteriovenous thrombosis in mice [132]. The concentration reached after moderate wine intake activates platelet eNOS, which passivates the inflammatory pathway associated with p38MAPK, thus inhibiting the activity of NADPH oxidase and production of ROS and ultimately inhibiting platelet function [133]. Olive oil has been reported to reduce the activity of NOX2 in platelets [134]. Nattokinase, a serine protease found in traditional Japanese food Natto, produces significant anti-inflammatory activity in RAW264.7 macrophages by inhibiting the activation of TLR4 and NOX2 induced by lipopolysaccharide, thus inhibiting the corresponding ROS production, MAPK activation and NF-κB, which alleviated inflammation-induced thrombosis [135]. Selenium can regulate apoptosis and necrosis in carp neutrophils through the ROS/MAPK pathway, thereby mitigating the inhibitory effect of TBBPA on NET release [116]. Hesperetin can inhibit NETs formation in a ROS/autophagy-dependent manner [136]. Therefore, it is effective to develop a treatment scheme targeting oxidative stress for the intervention of thrombus.

7. Conclusion

In conclusion, a comprehensive understanding of oxidative stress and its effect on various cellular components offers valuable insights into the pathogenesis of thrombosis. Future research is warranted to further explore the potential of antioxidant therapy in the management of thrombosis, which could mark a significant step forward in our fight against this global health challenge.

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