The mammalian lymphatic system is a one-way transport system of draining fluid and proteins from the interstitialspaces to the blood circulation. The lymphatic circulation is composed of lymphatic vessels, lymph nodes, lymphocytes, and associated lymphoid organs (1). This system is responsible for reabsorbing exudate tissue fluid from the vascular system through lymphatic capillaries. The fluid is then transported back to the bloodstream to replenish blood supply (2, 3). Although blood vesselsand lymphatic vessels differ greatly in structure, they work together to maintain essential functions, including fluid and protein balance in tissues, cellular nutrition, and proper immune function (4). In blood vessels, the clotting mechanism is a protective mechanism for preventing excessive blood loss while maintaining blood flow (5). However, in the lymphatic vasculature, coagulation is a pathological phenomenon that is closely related to but quite different from blood clotting. In 1914, the presence of thrombin, fibrinogen, and other coagulation factors in the lymph was confirmed, indicating that lymph also has the ability to coagulate (6), while the mechanism of coagulation in the lymphatic system has not been thoroughly studied. In this review, we summarize the possible etiologies, processes, and associated diseases of lymphatic coagulation.

The lymphatic system is a unidirectional transit network consisting of a network of vessels and nodes. The lymphatic system is responsible for the reabsorption of plasma components leaking from capillaries and postcapillaryvenules into extracellular spaces (3). When the blood flows through the capillaries, the plasma proteins and part of the liquid are extruded from the vessels into the interstitial fluid because of hydrostatic pressure and osmotic pressure (7, 8). Most of the blood components are reabsorbed by the postcapillaryvenules, while a small portion is absorbed by the lymphatic capillaries to form the lymph (9).

The lymphatic capillaries are composed of monolayers of discontinuous endothelial cells, which having a relatively flat, “oak leaf” shape (10). The basal membrane is discontinuous and unclear, with no smooth muscle medium, thereby allowing greater permeability (11). The specialized discontinuous buttons junctions serve as anchoring sites at the sides of interdigitated flaps of adjacent oak leaf-shapedendothelial cells (12). The abluminal surface of lymphatic capillaries is notably directly connected to the extracellular matrix by anchoring filaments, which help maintain the shape of the lymphatic vessels and the flow of fluid as the pressure in the matrix changes (11, 13, 14). Pre-collecting lymphatic vessels, located in the deep dermis, drain fluid from the lymphatic capillaries (15). The pre-collecting lymphatics drain into the collecting lymphatic vessels, which are covered with a continuous basement membrane and lymphatic muscle cells. In contrast to capillaries, the collecting lymphatic vessels are lined with lymphatic endothelial cells interconnected by zipper-like junctions between the cell (1, 16, 17). The zipper-like junctions were composed of vascular endothelial cadherin and tight junction–associated proteins. Thesecontinuous zipper-like junctions with basement membrane and intraluminal valves prevent leakage or reflux of lymph during its transport (1, 18). The loosely apposed but overlapping borders of the lymphatic capillaries operate as primary valves that provide unidirectional fluid into lymphatics (19). The smooth muscle layer of collecting lymphatic vessels ensures that the phasic contractions propel lymph forward through the network (20). Action potentials in the lymphatic muscle cells elicit phasic contractions of the collecting lymphatic vessels (11). In addition, cyclical compression and expansion by the surrounding tissue significantly contributes to lymph propulsion, allowing lymph to through collecting pre-nodal lymphatic vessels, lymph nodes and collecting post-nodal lymphatic vessels, to replenish the blood cardiocirculatory system (1) (Figure 1A).

The primary function of the lymphatic system is to replenish the vascular system by regulating tissue fluid balance, facilitating interstitial protein transport, and providing essential immune function (21). To accomplish these functions, lymphatics move fluid and other contents from the interstitium to pass across the nodes and enter the great veins (20). Both extrinsic tissue compression and intrinsic contractions of the lymphatic muscle cells provide the lymphatic system with the energy necessary to overcome the opposing pressure gradients and propel lymph along the lymphatic network (22, 23). When lymphatic vessels are damaged or diseased, edema, fibrosis, immune disorders, nutritional failure, and other conditions may occur (3, 23).

The normal blood coagulation process consists of three steps: vascular response, the initiation of platelet plugs, and the formation of fibrin clots (24). Platelet adhesion and aggregation at the site of vascular injury are necessary to stop bleeding (25). When blood vessels are injured, reactive vasoconstriction occurs first, reducing blood flow to the damaged area. Subcutaneous collagen components are also exposed, triggering platelet accumulation and activation (26). Exposureofbloodtotissuefactor (TF) is the major physiological initiatorof blood coagulation. It triggers the production of thrombin, which not only converts fibrinogen to fibrin but also activates platelets (27). Activated platelets secrete aggregatory mediators including thromboxane A2 and adenosine diphosphate. These mediators may provide ligands for platelet adhesion, diffusion, and activation when exposed to circulating blood (28, 29). Platelet adhesion and aggregation at the site of injury result in the formation of a primary “platelet embolism” and the binding of coagulation factors during the clotting cascade leads to the formation of a fibrin mesh. This mesh encapsulates and enhances the thrombus formation, prevents bleeding at the wound site, and promotes healing (30).

Coagulation includes both endogenous and exogenous pathways. Each pathway is initiated by different molecules, and both pathways converge into a common pathway that involves coagulation factors II, V, and X, leading to the formation of fibrin (31). Coagulation reactions start with exposure of TF-bearing cells to blood and continues mainly with activated platelets (32). After vascular endothelial injury, in the presence of calcium, factor IXa binds to factor VIIIa and activates factor X. Factor VIIa comes into contact with the exposed or expressed TF to form vitamin K-dependent enzymecomplexes (28). The factor VIIa-TF complexes can also activate factor X directly or indirectly by activating factor IX, but its efficiency is much lower in the absence of factor VIIa. Activated factor X stimulates a small amount of prothrombin to form thrombin (30). At the initial stage of thrombin production, activation of platelets, factor V, and factor VIII trigger a very small amount of thrombin to cause a burst of thrombin production (32). Experiments have revealed that a large number of prothrombin-activated products were detected at a bleeding time of about 4 minutes (33).

Endothelial cells produce anticoagulants and procoagulant molecules to form structures that rapidly activate platelets and clot blood (34). Von Willebrand factor (vWF) is well-known for its crucial roles in hemostasis; it is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs) (35, 36). Platelet adhesion is mediated by vWF, which acts as a bridge between exposed subendothelial collagen and platelet receptors (32). The immobilized vWF attaches platelets to the injured area by binding to the glycoproteinIb-IX complex on the platelet surface (37, 38). This process ultimately leads to a stable platelet adhesion through interaction with the platelet collagen receptors glycoprotein VI and glycoprotein Ia-IIaintegrin (39). Hemostasis is a balancing process of anticoagulant and procoagulant factors. On the one hand, quiescent endothelial cells express thromboregulatory protein (TM), tissue plasminogen activator (tPA), tissue factor pathway inhibitor (TFPI), and heparin sulfate. On the other hand, activated endothelial cells express TF, thrombin receptor, vWF, and plasminogen activator inhibitor-1 (PAI-1) to promote hemostasis (40, 41).

An imbalance consisting of an excessive concentration of antithrombin in the lymph and a low concentration ofthrombokinase (activated coagulation factor X) greatly reduces the possibility of lymphatic thrombosis. The lack of anionic phospholipids on the cell surface and low concentrations of thrombinand TFPI also offset the production of fibrin in the lymph (42–44). Immunoelectron microscopy has illustrated that coagulation factor X is attached to the cell surfaces of lymphocytes under normal conditions (45, 46). Lymphatic thrombosis can be triggered by the lymphatic stream in contact with necrosis cells or by infection of the tissues in the neighborhood of the lymphatic vessels. Under these conditions accompanied by a low lymph flow or a hypercoagulable state, the release of coagulation factor X by the disintegration of the lymphatic endothelium significantly furnishes favorable conditions for thrombosis within the lymphatic vessels (42, 47). When necrotic cells in contact with the lymph stream, thromboplastin (i.e., tissue factor) is released and enters the lymph to activate factor VII (46, 48). When free factor VIIa binds to thromboplastin in the context of lymphatic endothelial cells membrane surface, its proteolytic activity is enhanced substantially (48). The factor VIIa- thromboplastin complexes activate factor X released by the injured lymphatic endothelium. Activated factor X stimulates to production of prothrombinand formations of thrombin, which facilitates the conversion of a large amount of fibrinogen to fibrin. Eventually, this process leads to the formation of a compact mass of fibrin containing lymphoid cells (43, 46, 49, 50) (Figure 1B). The formation of lymphatic thrombosis are supported by the release of thromboplastin substances from the injured lymphatic endothelium and the chronic obstruction of lymph flow in the presence of a hypercoagulable milieu, thus mirroring the Virchow's triad (hypercoagulability, stasis and endothelial injury) (5, 51). Remarkably, there is an imbalance of low concentrations of blood coagulation factors (e.g., factor V and VIII) and high concentrations of anticoagulant molecules (e.g., TFPI, antithrombin) in the lymph. The production and release of tissue plasminogen activator tPA and PAI-1by lymphatic endothelial cells exhibit high fibrinolytic activity. The fibrin generation is largely counteracted by the unavailability of cell surface anionic phospholipids such as those physiologically present on blood platelets, combined with only low levels of coagulation factors, and the strong inhibitory activity of heparin, antithrombin, and tissue factor pathway inhibitor (46). The above mechanisms may be the reasons why lymphatic coagulation is slower than blood coagulation (52, 53).

Theoretically, any known cause of lymphatic vessel occlusion due to internal obliteration, external compression, or increased lymphatic pressure may predispose an individual to localized lymphatic thrombosis (46). The venous pressure is higher than the lymphatic pressure, and this may prevent further reflux of blood into the lymph vessels to protect lymphatic function in cases of lymphovenous valve dysfunction (54, 55). However, lymphatic valves dysfunction may cause human lymphedema or reflux of blood into the terminal parts of lymph ducts, followed by coagulation. A recent experimental study revealed a new pathway for platelet activation induced by the endothelial layer of the lymph vessels. Platelet aggregation stabilizes thrombin to prevent retrograde blood flow, resulting in thrombus formation at the lymphovenous valve (56).

Lymphatic vasculature displays remarkable heterogeneity in structure and function. The primarylymphaticsare right lymphatic duct (RLD) and thoracic duct (TD). TD is the largest lymphatic vessel that drains chylous and lymphatic fluid from most of the human body including the limbs and abdomen (57). The liver produces about 25–50% of the lymph flowing through the TD (58). The liver is also the primary site of synthesis of all clotting factors and their inhibitors, as well as several proteins involved in fibrinolysis and anticoagulation (59, 60). This may explain why the TD contains far more proteins and clotting factors than the lymphatic ducts in the limbs, including the axillary or inguinal lymphatics.

The analyses of lymphatic vessels inembryos, an dual origin theory proposed that lymphatic endothelial cells originated from two sources (61, 62). The study of mouse embryos and Xenopus tadpoles also provide evidence that lymphatic endothelial cells origin from both the veins and scattered mesodermal precursor cell (63, 64). These studies demonstrate the existence of a separate population of lymphatic endothelial cells with distinct molecular and function identity that forms local lymphatic vessels (65). Understanding the different lymphatic endothelial cells sources is crucial, as distinct lymphatic endothelial cells will likely contribute differentially to lymphatic thrombosis.

In contrast to blood, which is not in direct contact with the cellular layers of parenchymal organs, the lymph is derived directly from the organ interstitialfluid, whichbathes each organ's cellular layers (66, 67). That is, the lymph can actually provide the different organ's specific metabolic signature. The analysis of the proteome, lipidome, and metabolome of lymph collection from different parenchymal organs demonstrated differences in the distribution of lymphatic fluid from different anatomical areas (1). The composition of coagulation factors in lymph also depends on the interstitial fluid in the surrounding tissues. In addition, hemodynamic forces, pathological conditions, and the extracellular environment may affect the lymphatic system, leading to the adaptive changes (68). This may result in distinct levels and patterns of coagulation of lymphatic vessels in specific organs.

Although lymphatic thrombosis occurs in a variety of diseases, it has rarely been reported. For cancer lymphatic thrombosis, the leading causes include external tumor compression, neoplastic obliteration by metastatic cells, or lymphatic injury after lymph node dissection. Patients with fungal, bacterial and viral infections in tissues near lymphatic vessels may have lymphatic thrombosis and progressive lymphatic destruction. Lymphatic thrombosis was also found in sporadic cases of complications of central venous thrombosis or injury during cardiothoracic surgery, chronic venous insufficiency and thoracic outlet syndrome, which accompanied with lymphatic venous valve injury or obstruction of thoracic catheter drainage. If the lymphatics thrombosis causes accumulation of interstitial fluid, the lymphatico-venous anastomosis is expected to reduce the inner pressure of the lymphatic vessel and reconstruct lymph flow. For patients with severe, unexplained edema, clinicians may consider the possibility of the lymphatic thrombosis. Indocyanine green lymphography and radionuclide lymphoscintigraphy may locate lymphatic obstruction, while laboratory tests are still limited. Many cases of lymphatics thrombosis were found in lymphatic biopsy. Further studies are needed to understand the mechanisms of lymphatic thrombosis and develop novel diagnostic and therapeutic strategies.

JLiu conceived and designed the review. JLiu, WZ, and JT wrote the first draft. JLi, JLia, and XQ participated in writing of the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the National Nature Science Foundation of China (81873473 and 91939110), Academic Promotion Program of Shandong First Medical University (2019QL014) and Shandong Taishan Scholarship (JLiu).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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