
Endothelial activation and exocytosis of WPBs, central events in DVT initiation, are rapidly (within 1-6 h) induced by stenosis of the IVC [21,28]. In this process, WPBs fuse with the plasma membrane and release their constituents expressing some of them, such as von Willebrand factor (VWF; a coagulation factor present in the WPBs) and P-selectin, on the membrane, thus mediating cell recruitment. This is consistent with increased levels of soluble P-selectin in patients with DVT [29], although soluble P-selectin may originate also from activated platelets. Elevated levels of P-selectin both in the circulation and the venous wall are associated with upregulated DVT in the electrolytic mouse model [30]. Inhibition of Pselectin suppresses DVT and stimulates spontaneous recanalization of the thrombus in an IVC balloon occlusion model in baboons, suggesting its pathophysiological role in venous thrombosis [31].
Hypoxia results in the synthesis of reactive oxygen species (ROS), which activate the endothelium, inducing the expression of adhesion receptors and recruitment and extravasation of leukocytes [32]. ROS may activate endothelial cells also indirectly, for example, via the complement system. Oxidative stress induces complement activation (in particular, by endothelial cells damaged by ROS) and its deposition on the vessel wall leading to progression of inflammation [33,34]. The susceptibility to DVT strongly correlates with complement component C5a (C5a) levels in a mouse model [35]. Mice deficient for C3 or C5 have reduced experimental venous thrombosis, with the lack of C5 not being accompanied by any defects in platelet activation or normal hemostasis [36]. High levels of the C3 are associated with high risk of DVT in humans [37]. Mechanistically, high-molecular-weight multimers of VWF released from WPBs provide a scaffold for complement activation [38]. Complement components bind to VWF strings and become activated through the alternative activation pathway [39]. Thus, in addition to the direct activation of endothelium by hypoxia and ROS, the complement system may represent an additional link between flow restriction and endothelial activation.
In addition to cell recruitment, activated endothelium enhances blood coagulation and exerts suppressed anticoagulant function, thus contributing to thrombus formation [40]. Upregulation of the endothelial surface adhesion receptor ICAM-1 underlies augmented DVT under the endotoxemic conditions [41]. Clinically, endothelial activation has been reported in patients with VTE and thrombosis of superficial veins [42,43].
It has recently become clear that endothelial activation and cell recruitment, the critical events in DVT initiation likely induced by local hypoxia, require an intermediary: MCs. MCs are a part of the innate immune system that largely reside in tissues and are present in the vicinity of blood vessels [44]. These are large cells containing granules enriched with proinflammatory mediators, such as tumor necrosis factor-a and histamine, and antithrombotic factors, such as tissue plasminogen activator (tPA) and heparin. Thus, MCs might be expected to exert opposite effects on thrombosis: its reduction by releasing blood coagulation inhibitors or stimulation by supporting local inflammatory response. The involvement of MCs in DVT is implicitly supported by their accumulation at the site of venous thrombosis [45], but their net functional contribution has long remained obscure.
Using the stenosis DVT model in mice, we have recently demonstrated that two strains of MC-deficient mice are completely protected against DVT [46]. Adoptive transfer of in vitrodifferentiated MCs into MC-deficient animals restored thrombosis, suggesting that it is the lack of MCs that prevents DVT. Thus, the net effect of MC degranulation in this model is the exacerbation of venous thrombosis, suggesting that the activity of MC-originated proinflammatory factors outweighs the activity of antithrombotic factors. Absence of MCs completely prevents thrombosis, suggesting that MCs are absolutely required (although may not be sufficient) for its initiation.
In addition to protection from DVT, MC deficiency is accompanied by reduced cell recruitment at the venous wall after stenosis application [46]. Consequently, the factor or factors released from MCs and responsible for DVT may be involved in endothelial activation. Histamine is one of the most likely candidates produced by MCs. Indeed, local application of histamine accelerated DVT in wild-type mice and induced DVT in MC-deficient mice [46]. The mechanisms of the prothrombotic effect of histamine might involve its ability to induce release of VWF and Pselectin from WPBs [47] and enhance expression of E-selectin and ICAM-1 [48], mediating blood cell recruitment to the vascular wall. Histamine also stimulates the expression of TF, the key initiator of blood coagulation, in different cell types [49,50].
Mechanisms of MC activation by hypoxia remain obscure, but oxidative stress and ROS appear to play a role in this process. ROS can be both the cause and the consequence of MC activation because, on the one hand, inhibition of MC activation downregulates ROS production and, on the other hand, antioxidants prevent MC degranulation [51]. Generation of ROS has a direct impact on DVT in a mouse model. Indeed, H 2 O 2 has been shown to mediate increased susceptibility to DVT in aged mice, whereas mice overexpressing an antioxidant, glutathione peroxidase 1, are protected against the prothrombotic effect of H 2 O 2 [52].
Mastocytosis, which is associated with abnormally high numbers of MCs, is accompanied by bleeding symptoms in a small proportion of patients [53]. The reason for this is unclear but it is possible that when the number of MCs exceeds a certain limit, massive release of antithrombotic factors starts to prevail over the effect of proinflammatory/prothrombotic stimuli. However, the importance of MCs in venous thrombosis is corroborated by a clear link between allergic diseases and VTE [54]. Severity of asthma strongly correlates with the risk of not only DVT but also PE [55]. MCs and histamine are implicated in airway and lung inflammation-related thrombosis induced by diesel exhaust particles and prevention of histamine release has an antithrombotic effect [56]. Thus, targeting MCs by inhibiting their activation and degranulation, as well as further identification of and targeting MC granular constituents exacerbating thrombosis, may represent a fundamentally new approach to fight DVT, although the precise benefit and advantages of this approach in patients are still to be verified.

Stage 3: Blood Cell Recruitment
Leukocytes Restriction of venous blood flow induces rapid leukocyte recruitment. After 1 h of IVC stenosis, leukocytes start to roll along and adhere to the venous endothelium, and after 5-6 h, leukocytes carpet the entire endothelial surface. Neutrophils account for more than 80% of adherent leukocytes and monocytes represent the remainder [28]. Leukocyte recruitment is dependent on P-selectin exposure on the luminal side of the venous endothelium since the number of leukocytes recruited to the venous wall in mice lacking P-selectin on the endothelial surface is reduced by several orders of magnitude. Moreover, these mice are protected against DVT, indicating that leukocyte recruitment is crucial for DVT development in response to blood flow restriction. Although activated platelets also expose P-selectin on their surface, the role of platelet-derived P-selectin in leukocyte recruitment is less prominent [28]. Leukocyte recruitment is also affected by various plasma components. For example, higher plasma levels of low-density lipoproteins (LDL) likely enhance leukocyte accumulation since deficiency in proprotein convertase subtilisin/kexin type 9 (PCSK9), an enzymatically inactive protein that binds the LDL receptor favoring its degradation, significantly reduces leukocyte adhesion and thrombus growth in the stenosis DVT model [57]. Given that leukocyte recruitment to the venous wall is indispensable for DVT, below we discuss the contribution of the main leukocyte subsets recruited, namely neutrophils and monocytes, to the pathogenesis of the disease.

Neutrophils
The involvement of neutrophils in DVT was discovered several decades ago [58]. Recent studies have demonstrated the critical role of neutrophils in the pathophysiology of DVT [28,59,60]. Depletion of neutrophils inhibits venous thrombus formation, indicating that their role cannot be substituted by other leukocytes [28]. This prothrombotic effect of neutrophils, however, is observed only in the stenosis DVT model, whereas in the stasis model, neutropenia does not affect thrombus size in mice and results in development of even larger thrombi in rats [61,62].
Upon recruitment to the venous wall, neutrophils undergo activation and release their nuclear material, forming a web-like extracellular structures designated as neutrophil extracellular traps (NETs). These are composed of DNA, histones, secretory granule constituents, and other components implicated in antimicrobial defense [63]. It has been shown that signals originating from the neutrophil P-selectin glycoprotein ligand-1, a counter-receptor for P-selectin, may trigger the process of NET formation (NETosis) [64]. Under pathogen-free conditions of thrombosis, NETosis may also be induced by high-mobility group box 1 (HMGB1) released by and exposed on the surface of platelets recruited to the venous wall [65,66]. Although monocytes may also be able to form extracellular traps [67], experiments on neutropenic mice have shown that neutrophils are the major source of these traps in venous thrombi [28]. NETs are abundantly present in venous thrombi [59,68], which is in line with increased plasma levels of NETs biomarkers in patients with DVT [69,70]. Prevention of NETosis [71,72] or destruction of NETs by infusion of deoxyribonuclease (DNase) I [28,59] protects mice from thrombus formation in the stenosis DVT model, indicating the crucial role of NETs in the onset of DVT. NET