The role of leukocytes in acute ischemic stroke-related thrombosis: a notable but neglected topic

Abstract

Ischemic stroke is one of the most serious diseases today, and only a minority of patients are provided with effective clinical treatment. Importantly, leukocytes have gradually been discovered to play vital roles in stroke thrombosis, including promoting the activation of thrombin and the adhesion and aggregation of platelets. However, they have not received enough attention in the field of acute ischemic stroke. It is possible that we could not only prevent stroke-related thrombosis by inhibiting leukocyte activation, but also target leukocyte components to dissolve thrombi in the cerebral artery. In this review, we expound the mechanisms by which leukocytes are activated and participate in the formation of stroke thrombus, then describe the histopathology of leukocytes in thrombi of stroke patients and the influence of leukocyte composition on vascular recanalization effects and patient prognosis. Finally, we discuss the relevant antithrombotic strategies targeting leukocytes.

Introduction

In the past few decades, ischemic stroke has become one of the most common and serious diseases across the world, featuring extremely high rates of morbidity and mortality [1, 2]. More than nine million people suffer from ischemic stroke each year worldwide, which imposes a huge medical burden on society [3]. Unfortunately, not all stroke patients benefit from the treatment and prevention strategies available today. Neither recombinant tissue plasminogen activator (rt-PA) thrombolysis, which is widely used for treatment of acute ischemic stroke (AIS), nor mechanical thrombectomy, can guarantee the complete recanalization of most occluded vessels [2, 4, 5]. Furthermore, secondary prevention strategies with antiplatelet and anticoagulant therapy are not able to adequately prevent the recurrence of ischemic stroke [2, 4, 5].

Encouragingly, a few glimmers of hope have appeared, as neuroscientists turn their attention to the immune cell components of the stroke thrombus [6–8]. Leukocytes, as the immune cells in the vessels, including neutrophils, monocytes, and lymphocytes, have been found to play a crucial role in thrombosis and in thrombotic inflammation in recent years. In the blood vessels of AIS patients, leukocytes are often activated in advance, which not only damage the vascular endothelial cells through the local inflammatory response, but also trigger blood coagulation cascades and promote platelet activation and aggregation in a variety of ways, leading to rapid thrombosis [7]. Furthermore, leukocytes contribute to the stability and maturation of a stroke thrombus, making it harder to dissolve and remove, which has a huge impact on the clinical outcome of AIS patients [9, 10]. Thus, leukocyte components may be a neglected but promising biological target for stroke treatment and secondary prevention.

In this review, we first expound the mechanisms by which leukocytes are activated and participate in the formation of a stroke thrombus. Then, we discuss the histopathology of leukocytes in thrombi of stroke patients, and the influence of leukocyte composition on vascular recanalization effects and patient prognosis. Finally, we summarize the antithrombotic strategies targeting leukocytes.

How leukocytes promote thrombus formation in stroke

Today, large-artery occlusion in stroke is also considered as a thrombo-inflammatory disease, because leukocytes are recruited to the damaged blood vessels and infiltrate into the brain parenchyma after thromboembolic events, causing a local inflammatory response [11, 12]. Not only that, since the term immune-thrombosis was first proposed in 2013, the interaction between immune cells and thrombosis has rapidly become a research hotspot [6–8]. In fact, innate immunity and thrombosis have always been inseparable to synergistically limit pathogen invasion while maintaining vascular integrity over the course of human evolution [13, 14]. Hence, mechanisms related to immune-thrombosis may also play an essential role in AIS.

Although ischemic stroke is classified into multiple subtypes that encompass distinct etiologic diseases with different pathologic features, peripheral immune cells are frequently found at elevated levels in nearly all subtypes of AIS patients, and considered as an independent predictor of stroke recurrences [15, 16].

First, atherosclerosis, the most prevalent cause of AIS, is often accompanied by basic diseases such as hyperlipidemia and hyperglycemia, which activate bone marrow hematopoietic cells to produce large numbers of leukocytes into the circulating blood [17–19], and inflammatory cells are involved both in the formation and rupture of atherosclerotic plaques [20, 21]. Second, cardiogenic embolus, another important cause of AIS, is accompanied by various infectious or non-infectious heart diseases, of which atrial fibrillation is the most common [22, 23]. They cause a hemodynamic disorder which induces the activation of cardiac endothelial cells via ischemia and hypoxia, which in turn secrete inflammatory factors and chemokines that recruit and activate leukocytes [24–26].

Now, it has been found that activated leukocytes can contribute to the formation and influence the physicochemical properties of the thrombus in a number of interrelated ways. As shown in Fig. 1, activated leukocytes, especially neutrophils and monocytes/macrophages, not only promote the activation and aggregation of platelets, but also secrete procoagulant substances which trigger the coagulation cascade. Therefore, a better understanding of the mechanisms involved will help us explore new antithrombotic strategies for stroke.

Fig. 1.

The mechanism via which leukocytes promote thrombus formation in stroke. a Four stages of thrombosis: (1) leukocytes flow in the blood vessels, while platelets are activated and adhere to the damaged blood vessel wall; (2) activated leukocytes bind to platelets and simultaneously secrete tissue factors; (3) more leukocytes and platelets aggregate into a primitive thrombus, while fibrin is generated and encapsulates RBCs; (4) complete thrombus including platelet-enriched and RBC-enriched areas. b The binding patterns of platelets and leukocytes. c Structural diagram of NETs. GPIbα, glycoprotein Ibα; PSGL-1, P-selectin glycoprotein ligand 1; NET, neutrophil extracellular traps

How leukocytes promote platelet activation

In traditional views of stroke thrombosis, platelets initially adhere to the extracellular matrix of damaged blood vessels, and recruit and activate more platelets in the bloodstream through autocrine and paracrine mediators, including adenosine diphosphate (ADP), thrombin, epinephrine, and thromboxane A2, forming a growing thrombus [27, 28]. It is now known that leukocytes enhance platelet aggregation while platelets and leukocytes bind into hetero-aggregate formations, also known as platelet-leucocyte aggregates (PLAs) [29–31], which are significantly elevated in the circulating blood of AIS patients [32–36].

The formation of PLAs depends on specific ligands and receptors, while of particular concern is the binding of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and platelet P-selectin (also called CD62P), the pivotal step in the formation of PLAs [37, 38]. In the analysis of genetic polymorphism, PSGL-1 and P-selectin gene variation were significantly associated with PLA formation and the risk of ischemic stroke [32, 39]. Usually, P-selectin is stored in platelet α granules and rapidly transferred to the cell surface upon platelet activation [40, 41]. Meanwhile transmembrane protein PSGL-1 is located in lipid rafts on the top of microvilli, and its intracellular domain connects to leukocyte actin [42]. Through intracellular Src kinase and PI3k signaling, activated PSGL-1 up-regulates the expression of leukocyte integrin, as well as remodeling the cytoskeleton and improving adhesion ability [42–44]. In a carotid photochemical thrombosis model in mice, PSGL-1 deficiency showed protective effects against the prothrombotic effects of the inflammatory cytokine IL-1β [45]. Subsequently, platelets and leukocytes further bind in other specific ways including leukocyte Mac-1 (also called CD11b or integrin α_Mβ₂) and platelet surface glycoprotein GPIbα, and leukocyte CD40 and platelet CD40L, which stabilize the PLA structure, and promote platelet activation and aggregation [46–50]. It has been reported that mice with Mac-1 deficiency or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery injury [51]. Also, the CD40/CD40L ligand system has been considered as the hinge between leukocytes and ischemic stroke [52]. Therefore, activated platelets at the damaged endothelium capture circulating leukocytes which act as the aggregation mediator to accelerate the formation of primary thrombus, especially under arterial high shear blood flow.

In addition, leukocytes also secrete soluble mediators to activate platelets. For example, cathepsin g [53, 54], platelet activating factor (PAF) [55, 56], and major basic protein (MBP) [57, 58] promote platelet activation and aggregation. In an occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, and reduced histologic brain injury [54]. Moreover, inflammatory factors expressed by leukocytes bind to toll-like receptor 2 (TLR2) and TLR4, which are the innate immune receptors expressed on platelets and are related to thrombosis [59, 60]. Neutrophils also secrete vesicles containing arachidonic acid, which are internalized into platelets as the material for the synthesis of thromboxane 2 (TXA2), a paracrine activator of platelets [61]. Meanwhile, proinflammatory leukocytes release inflammatory cytokines, matrix metalloproteinases (MMPs), and reactive oxygen species (ROS), which injure the endothelial cells and cause them to expose platelet-binding sites, such as collagen and von Willebrand factor (VWF) [42, 62].

In turn, platelets release inflammatory cytokines IL-1β, IL-8 [63], and High Mobility Group Box 1 (HMGB1), a damage-related molecular pattern located in the cytoplasm of platelets or the nucleus of karyocytes [64–66], and activate leukocytes into proinflammatory and prothrombogenic phenotypes. Moreover, platelets secrete chemokines such as CXCL4 and CXCL7, which recruit monocytes and neutrophils, respectively, into the thrombus [67–69]. Thus, there is a vicious cycle between leukocytes and platelet activation, accelerating the formation of thrombus.

How leukocytes promote fibrin formation

Blood coagulation is the central process of thrombosis, its end product is thrombin, which can cleavage fibrinogen into fibrin [70]. And activated leukocytes release large amounts of procoagulant mediators, among which tissue factor (TF) is one of the most widely studied. TF, as a trigger of exogenous coagulation, binds to coagulation factors FVIIas, resulting in the activation of thrombin and the formation of fibrin [71, 72]. TF is usually expressed by activated endothelial cells when blood vessels are injured [71, 72]. In fact, macrophages and their microparticles are considered as the main source of TF in atherothrombosis [73, 74]. Interestingly, neutrophils have been determined not to release TF but to mediate the transfer of macrophage-derived TF microparticles [73, 75–77].

Furthermore, neutrophil granzyme effectively assists in blood coagulation [78]. Neutrophil-derived cathepsin g and neutrophil elastase (NE) effect the degradation of natural anticoagulants including heparin cofactor II [79], tissue factor pathway inhibitor (TFPI) [80], and antithrombin III [81]. In addition, neutrophil-derived cathepsin g directly activates coagulation factors FV, FVIII, and FX [82–84]. Leukocytes also provide the cellular surface as sites for the assembly and activation of clotting factors [85, 86]. In summary, activated leukocytes may ultimately increase the fibrin content of the thrombus in ischemic stroke.

In fact, neutrophils and monocytes express protease activated receptors (PARs) [87], which are the specific receptors of thrombin. Once bound, thrombin activates leukocytes into proinflammatory phenotypes [88, 89]. Similarly, a leukocyte–thrombin positive feedback loop forms that continuously amplifies the coagulation response locally.

How neutrophil extracellular traps (NETs) promote thrombosis

In particular, neutrophils, the most abundant leukocytes in human blood, ultimately exhibit a highly thrombogenic cellular form, which is referred to as a NET, and the process by which neutrophils produce NETs is known as NETosis [90–92]. During NETosis, neutrophil chromatin disintegrates, the nuclear membrane dissolves, and ultimately the DNA strands, citrullinated histones and various proteases are released into the extracellular space [93–95]. In fact, it is a death form of neutrophil, but with potent biocidal activities, which can accelerate thrombosis and has a huge impact on the properties of the thrombus in atherothrombosis, venous thrombosis, and even cancer-related thrombosis [96, 97], which are all potential causes of stroke [98, 99].

The mechanism via which NETs promote thrombosis is complicated, but is mainly related to DNA strands and citrullinated histones. The DNA strands bind to FXII and FXI through the negatively charged surface [100–102], then activate the endogenous coagulation cascade and shorten coagulation time [103, 104]. Histones H3 (H3) and H4 promote the activation of prothrombin [105, 106], and regulate the adhesion and aggregation of platelets by activated TLRs [107–109]. Most importantly, NETs can also trap procoagulant particles which contain TF and NE secreted by leukocytes [96], thus becoming the chemical center of thrombosis and the mechanical framework of the thrombus, which makes the thrombus structure more stable.

In a way, NETosis is considerably attributed to the activated platelets in stroke thrombosis [110]. Platelet-derived HMGB1 may induce NETosis through TLR4 [111]. Activated platelets commit neutrophils to NET generation and the event is inhibited in the presence of competitive antagonists of HMGB1 in vitro [112]. In addition, platelets may promote NETosis through P-selectin and integrin α_IIbβ_3, which are found in a thrombin-reduced thrombosis mouse model and in vitro under physiological flow conditions [113, 114].

Furthermore, macrophages, eosinophil granulocytes, and mast cells also release their DNA during thrombosis [115–117], although most extracellular traps originate in neutrophils. Especially in the late stages of thrombotic diseases, these cell-free DNA molecules inhibit the process of fibrinolysis [118, 119]. However, whether they promote thrombosis in the same way as NETs is not yet clear in stroke studies.

Studies of leukocytes in thrombi recovered from stroke patients

Thanks to recent advances in endovascular treatment technology, the thrombi of stroke patients can be obtained by mechanical thrombectomy, which has led to an increase in both clinical and basic research on this topic. This helps us not only to validate the existing theoretical mechanism of stroke thrombosis, but also to accumulate more clinical evidence.

Leukocyte composition in thrombus histopathology

In the present studies, thrombi were collected, and paraffin or frozen sections were prepared to analyze leukocyte counts and distributions by staining for hematoxylin and eosin (H&E), Martius scarlet blue (MSB), and leukocyte-specific markers, including leukocyte universal marker CD45, neutrophil CD66b, neutrophil myeloperoxidase (MPO), NE, monocyte CD68, T cells CD3, B Cells CD20, and NETs marker circulated Histone H 3 (H3cit) [120–123]. Staining for H&E or MSB enables visualization of the general overall structure of the thrombus, and makes the leukocyte nuclei, platelets, and erythrocytes appear in different colors, while immunohistochemical staining for specific receptors was used for more precise quantitative measurements [124]. Here, research findings on the thrombi of stroke patients are summarized in Table 1 [9, 10, 125–138].

Table 1.

Summary of the histopathological studies of leukocyte components in stroke thrombus

Year	Targets	Markers	Sample size	AIS Etiology-related		Prognosis-related	References
2021	Leukocytes	CD45	45	No
	Monocytes/macrophages	CD68				Yes	[137]
	T cells	CD3
2021	Neutrophils	MPO	80	No		–	[124]
	NETs	H3cit		Yes		–
2020	Leukocytes	CD45	71	No		No	[126]
	Neutrophils	Neutrophil elastase/myeloperoxidase		No	No
	NETs	H3Cit		Yes	Yes
	Monocytes/macrophages	CD14		No	No
	T cells	CD20		No	No
	B cells	CD3		No	No
2020	Leukocytes	CD45	188	–	–		[127]
	DNA	Feulgen DNA staining		–	–
2020	Monocytes/macrophages	CD68/KiM1P	85	Yes	–		[128]
2020	Leukocytes	CD45	41	–	Yes		[143]
2019	Leukocytes	MSB	105	No	–		[129]
2019	DNA	H3Cit	78	–	–		[148]
2018	Leukocytes	H&E	37	Yes	Yes		[130]
2018	Leukocytes	H&E	43	No	–		[125]
2018	NETs	H4-Cit3	108	–	Yes		[10]
2018	Leukocytes	MSB	92	–	Yes		[9]
2017	T cells	CD3	187	Yes	–		[131]
	B cells	CD20		No	–
	Monocytes/macrophages	CD68/KiM1P		No	–
2017	Neutrophils	CD66b; neutrophil elastase	68	–	–		[132]
	NETs	H3Cit		Yes	–
2017	Neutrophils	Neutrophil elastase	85	–	Yes		[136]
2016	T cells	CD4	37	–	No		[133]
	Monocytes/macrophages	CD68/KiM1P		–	No
2016	T cells	CD3	54	Yes	–		[134]
2016	Leukocytes	H&E	145	Yes	–		[135]

Leukocytes can be observed in almost all sections of stroke thrombi, suggesting that leukocytes are commonly involved in the thrombosis of various stroke subtypes. Leukocytes constitute up to 4% of all cells by number, and are distributed in 0.5–20% of the area of the thrombus [9, 10, 125–138]. Moreover, the proportions of neutrophils, macrophages, and lymphocytes are quite different in a stroke thrombus. Neutrophils account for the majority of leukocytes in stroke thrombi, followed by macrophages and T cells, with almost no B cells [120]. This difference is partly attributed to their proportion in circulating blood in the physiological state, but is also related to the activation levels of different types of leukocyte in ischemic stroke. Usually, as the time of thrombus formation increases, the number of neutrophils in the thrombus also gradually increases [121, 133]. In addition, in a stasis-induced deep vein thrombosis mouse model, the number of macrophages in the thrombus also increases continuously and exceeds the neutrophils by day 7 [139]. This suggests that leukocytes continue to infiltrate into the thrombus after its formation and may modulate thrombus maturation.

In a study on the detailed spatial structure of a stroke thrombus, the thrombus was found to be mainly composed of two types of structure; platelet-rich regions and red blood cell (RBC)-rich regions, although they are often interlaced and the outer layer of a thrombus is usually a dense structure of platelets [128, 134, 140]. A recent study involving 188 thrombi of stroke patients found that leukocytes, especially neutrophils and macrophages, were mainly detected in the platelet-rich regions and the interfaces of RBC regions and platelet regions, but rarely in the RBC-rich regions [128]. This may be due to the close interaction between platelets and leukocytes. In fact, it differs in the affinities between platelets and different leukocytes, which depend on the type and number of platelet-binding receptors they express on the surface. Neutrophils and monocyte/macrophages bind most easily to platelets, while T cells do not bind so easily [141, 142]. In fact, if monocytes and T cells are found in the sections, they are often randomly distributed throughout the whole thrombus [128, 134, 135]. As for NETs, these are more commonly located in the outer layer of the thrombus or in the platelet aggregation area of the thrombus core [10, 127]. Regrettably, most analyses of the thrombi of stroke patients are limited to observing the density and distribution of leukocytes under the microscope, with little protein-level validation and exploration.

Relationship between leukocyte features of the thrombus and etiological subtypes of ischemic stroke

The Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification is the most commonly used international classification of etiological subtypes of ischemic stroke based on the origin of the thrombus, and denotes five subtypes: (1) large-artery atherosclerosis (LAA), (2) cardioembolism (CE), (3) small-vessel occlusion (SAA), (4) stroke of other determined etiology, and (5) embolic stroke of undetermined source (ESUS) [98]. A large number of studies have investigated the relationship between leukocyte composition and TOAST classification (Table 1). In most studies that have accumulated hundreds of thrombi of stroke patients, the contents of leukocytes, including neutrophils, NETs, and macrophages, were higher in CE thrombi than in LAA thrombi [125, 129, 132, 143–145], although different results have also been reported [130, 131]. The difference in leukocyte distribution may contribute to the different pathophysiological environments of thrombosis, inferring that more leukocytes and inflammation are involved in the formation of cardiogenic emboli.

In addition, a study of 54 thrombi of stroke patients found that there are more T cells in LAA thrombi then in CE thrombi [135]. Since T cells are activated in arteriosclerosis plaques, they may be mixed into thrombi in ischemic stroke. Furthermore, the similarity of leukocyte features in thrombi between CE and ESUS seems to indicate that most ESUS thrombi may have underlying cardiac causes [129–132, 135, 136, 145]. Clinically, 70% of ESUS patients can also be examined for atrial fibrillation and other cardiogenic diseases within 1 week of hospitalization [146]. However, because no substantial thrombus can be removed from SAA patients and because of the scarcity of ESUS thrombi, no related clinical data have ever been published.

Furthermore, leukocytes in stroke thrombi and myocardial infarction thrombi were also compared. Compared with coronary thrombi, stroke thrombi have a similar number of neutrophils, but a more pervasive distribution of NETs and greater counts of T cells, B cells, monocytes, and eosinophils [127, 147, 148], which indicate that the leukocytes have higher activity in the thrombotic process of stroke then in myocardial infarction.

Influence of leukocyte composition in thrombi on the therapeutic effect and prognosis of stroke

Considering the current treatment strategy, the curative effects on AIS are most dependent on the sensitivity to rt-PA and the physical properties of the thrombus, which may be influenced by leukocyte composition.

In a study of 92 patients who were treated with bridging therapy, there was a significant correlation between higher content of neutrophils and NETs in the thrombus and lower reactivity to rt-PA [9]. And thrombi with fewer leukocytes have a greater reduction in volume with a more favorable recanalization after a thrombolytic procedure [149]. This suggested that leukocytes reduce the thrombolysis effects of intravenous rt-PA, perhaps by promoting the production of fibrin, more importantly, the activation of platelets and NETs.

Because of their impacts on the physical properties of thrombi, leukocytes extend the time of thrombectomy and increase the procedure time and the number of passes required for mechanical recanalization, and are associated with a lower vascular recanalization rate [126, 138, 150]. Thus, it is believed that leukocytes stabilize the structure of the thrombus. Especially, NETs, as the physical skeleton of the thrombus, enhance resistance to both mechanical and enzymatic destruction. Furthermore, the older the thrombus, the greater the degree of thrombus adhesion to the vessel wall, making thrombus aspiration more difficult [133]. With continuous infiltration of leukocytes into a thrombus, the total content of leukocytes in the thrombus increases over time. Thus, more leukocytes in a thrombus represent a deeper vascular wall adhesion. In addition, the presence of neutrophils in a thrombus increases the risk of thrombus fragmentation, with the proposed thrombolytic capacity of NE, which is awaiting further exploration and explanation [137].

From the available evidence, leukocytes in blood clots actually determine the clinical outcome of stroke patients. A large amount of clinical data have shown that high leukocyte levels in a thrombus are associated with higher baseline scores on the National Institutes of Health Stroke Scale (NIHSS), which is a comprehensive measure of patient symptoms that is positively correlated with stroke severity, less cerebral reperfusion, and higher long-term modified Rankin scale (mRS) scores which represents poor functional recovery [137, 138, 144]. Furthermore, the NET content of a thrombus is also associated with the severity of ischemic stroke, and studies of more than 100 thrombi of stroke patients showed an independent association between NETs and the incidence of complications such as atrial fibrillation and all-cause mortality during one-year follow-up of ischemic stroke [143]. Overall, a high leukocyte content in the thrombus represents a reduction in the therapeutic efficacy and a poor prognosis of stroke.

Antithrombotic strategies for leukocytes

Targeting leukocyte composition may help prevent thrombosis, and more importantly, help with thrombolysis in patients with AIS. Currently, rt-PA is the only FDA drug recommended for intravenous thrombolysis, which promotes the conversion of plasminogen and the fibrinolysis of thrombi. However, the rate of arterial recanalization after intravenous injection of rt-PA is relatively low, at about 20–46%[151], mainly because rt-PA breaks down only part of the fibrin chain crosslinking in the thrombus, and has little effect on the platelet shell and leukocyte components. At present, targeting NETs holds the promise of solving the rt-PA resistance problem. Recently, the levels of DNase-1, which is the natural dissolver of NETs, in the blood of stroke patients have been found to be lower than in healthy people [152], and in a mouse photothrombotic stroke model, administration of DNase-1, which promotes NET lysis, without t-PA, recanalized the occluded vessel thus improving photothrombotic stroke outcome [153]. It is expected that the clinical use of DNase-1 will overcome the problem of rt-PA resistance.

More importantly, modulating leukocyte activity can prevent the recurrence of thrombi. Because of the powerful thrombus-promoting effect of leukocytes, we can try to inhibit leukocyte activation in general, or by targeting the precise cell receptor or signal pathway, block specific functions of leukocytes. The selective suppression of PAD4, which is the necessary enzyme of NETosis, prevented thrombosis in a mouse DVT model [154, 155]. Anti-inflammatory drugs such as roflumilast can lessen the activation of platelet surface markers, neutrophil adhesiveness and the release of NETs [156], showing antithrombotic activities. Moreover, antibodies to inflammatory factors, such as anti-IL-1, can improve atherosclerosis, protect endothelial cells, and prevent thrombosis [157]. Furthermore, the interference of PLAs with platelet-leukocyte binding targets has also received considerable attention, including P-selectin, PSGL-1, and CD40L inhibitors, which are also being tested to prevent thrombosis in rodent models of arteriosclerosis and in venous models [41, 158, 159].

It can be expected that targeting leukocytes to prevent stroke thrombosis will become an important part of clinical decision-making. How to inhibit leukocyte activity at the risk of thrombosis in stroke is a general principle for targeting leukocytes, but the use of different biomarkers at different disease stages may selectively increase the efficacy of drugs and reduce side effects.

Conclusion and perspective

In conclusion, leukocyte components (e.g., neutrophils, monocyte/macrophages, NETs) promote the activation of coagulation cascades and platelets. Positive feedback between thrombosis and immune cell activation usually forms in AIS. In addition, leukocytes have been shown to be associated with the etiologic subtype of stroke in the analysis of thrombus sections from stroke patients. In patients with cardiogenic stroke, more leukocytes are involved in the formation of thrombi, which is also related to reduced thrombolytic results and poorer clinical outcomes. Therefore, we may be able to target leukocytes to accelerate the dissolution of thrombi and prevent stroke recurrence.

We admit that many of these mechanisms remain to be witnessed in experimental studies of stroke thrombosis, mainly due to the limits of stroke experimental models, including the endovascular suture model, endothelin model, electrocoagulation model, photo-induced and chemical-induced model, and the autologous embolus model [160, 161]. Among them, only the photo-induced and chemical-induced models that relied on photosensitive substances or ferric chloride could induce the complete process of stroke-related thrombosis. The endovascular suture model, which is the most widely used, as well as the endothelin model and the autologous embolus model do not represent the process of intravascular thrombosis. However, stroke is a collective concept centered on vascular-neurological disorders, involving not only in situ atherosclerosis, but also cardiogenic thrombosis which is similar to venous thrombosis, as well as thrombosis related to cancer, immune diseases, and coagulation abnormality-related thrombosis [146, 162, 163]. Therefore, various disease thrombosis models resemble stroke to some extent. Moreover, the roles of leukocytes have been shown in the thrombi of stroke patients.

More importantly, leukocytes may be involved in the long-term regression of the thrombus [164–166]. Leukocytes express plasminogen receptors, including enolase, annexin II and histone H2b, which bind to plasminogen and promote its activation [167]. At the same time, macrophages modulate thrombus and coagulation factor clearance through phagocytosis [168]. Through TLR4 signaling, fibrin and its degradation products activate the scavenger receptor phagocytosis of macrophages, contributing to the clearance of platelets, RBCs and fibrin. In vitro, significant amounts of RBC and NET phagocytosis products were found in macrophages of mature thrombi [169].

Thus, excessive suppression of leukocytes may lead to poor regression. In a rat model of DVT, 2–7 days of neutrophil reduction resulted in larger thrombus residues, accompanied by an increase in thrombotic fibrosis, and a decrease in u-PA and MMP9 [170]. In addition, monocyte chemotactic factor (MCP-1) knock-out mice showed a thrombolytic disorder in a DVT mouse model [171, 172]. These studies suggest that we may be able in the future to promote thrombus elimination by regulating leukocyte activity and phenotype, which will be very meaningful for the removal of unremoved or undissolved thrombi in stroke patients.

It is worth noting that the regulation of leukocytes may significantly affect the body’s immune function and increase the risk of infection, which is important in stroke patients during the recovery period. Therefore, a delicate balance of leukocyte function should be achieved, which requires precise control of disease development.

Author contributions

RB and SC wrote and revised the manuscript. SC and QP helped with the literature search and correction of the manuscript. BH and HJ provided the conception and design of the review, and directed the writing of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2018YFC1312200 to Bo Hu), the National Natural Science Foundation of China (Grants: 81820108010 to Bo Hu, and 81671147 to Huijuan Jin) and Major Refractory Diseases Pilot Project of Clinical Collaboration with Chinese and Western Medicine (SATCM-20180339 to Bo Hu).

Data availability

No data have been generated in the completion of this review.

Declarations

Conflict of interest

The authors declare no conflicts of interest.