Inflammation-induced thrombosis, referred to as immunothrombosis, offers host defense advantages by limiting the dissemination of pathogens within the bloodstream. However, platelets and the coagulation cascade also modulate inflammatory responses through their interactions with immune cells, the endothelium, and the complement system. These interactions can escalate inflammation, leading to a state of intensified inflammatory activity driven by thrombotic processes, known as thromboinflammation (11).

The term immunothrombosis was first introduced in 2013 by Engelmann and Massberg to describe how the innate immune system can initiate thrombotic events (1). It involves the interplay between immune cells, coagulation factors, and immune effector proteins, resulting in the formation of microvascular thrombi. These thrombi act as a physical barrier to trap pathogens and prevent their spread while simultaneously activating immune responses (7).

In contrast, the term thromboinflammation predates immunothrombosis and was initially used to describe the role of platelets in inflammatory processes. Today, the concepts of immunothrombosis and thromboinflammation are recognized as interconnected, with their relationships often understood as a cause-and-effect dynamic: Immunothrombosis refers to the immune system’s role in thrombus formation, while thromboinflammation reflects the reciprocal impact of thrombotic events on immune responses (11) ( Figure 1 ).

Cellular signaling plays a central role in sepsis, a condition characterized by profound interactions between inflammation and coagulation. The integrity of endothelial cells is equally crucial, as the loss of their normal antithrombotic and anti-inflammatory functions disrupt homeostasis. This endothelial dysfunction contributes to dysregulated coagulation, complement activation, platelet activation, and immune cell recruitment within the microvasculature (12).

Effective control of an inflammatory response relies on precise communication between involved cells, with cytokines, chemokines, and adhesion molecules serving as key mediators of inflammation and immune regulation (13). Cytokines, small glycoproteins released by one cell and recognized by corresponding receptors on target cells, play a central role in inflammation. This diverse family includes interferons, interleukins, growth factors, and chemokines, all of which initiate complex signaling cascades in responsive cells. These cascades drive critical cellular processes such as proliferation, differentiation, and the release of enzymes or additional mediators, which collectively regulate the cellular environment and sustain the effects of cytokines (14).

In sepsis, coordinated communication between leukocytes, platelets, and endothelial cells is essential for the formation of immunothrombosis, where cell adhesion enables intercellular binding and signal transfer. Platelet-leukocyte interactions are initiated by signals from various receptors, including toll-like receptors (TLRs) and protease activated receptors (PARs) (15). Additionally, vascular endothelial cells play an important role in regulating leukocyte trafficking by expressing adhesion molecules. The selectin family of adhesion molecules, including P- and E-selectin on endothelial cells, mediates leukocyte tethering and rolling, while immunoglobulin superfamily adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), orchestrate firm leukocyte adhesion (16).

Upon activation, platelets interact with monocytes and neutrophils through key adhesion molecules, particularly P-selectin. When expressed on the platelet surface, P-selectin binds to P-selectin glycoprotein ligand-1 (PSGL-1) on monocytes, facilitating platelet-monocyte adhesion (17). This initial binding is followed by integrin-mediated interactions involving platelet endothelial cell adhesion molecule-1 (PECAM-1), which contribute to scaffolding, signal transduction, and cellular responses (18). Together, these multistep adhesion pathways likely drive the biological and pathological mechanisms underlying thrombosis and inflammation in sepsis (19).

Endothelial cells also release a variety of paracrine mediators, including lipid mediators, nitric oxide, growth factors, and cytokines (20). These signals activate surrounding cells, including monocytes, which upregulate tissue factor (TF) expression under inflammatory conditions. In response, neutrophils release NETs, further promoting coagulation and contributing to the formation of microthrombi, known as immunothrombosis (21). During sepsis, activated leukocytes secrete chemokines and damage-associated molecular patterns (DAMPs) to nearby cells and the adherent endothelium, intensifying inflammatory signaling and exacerbating oxidative stress (22). In COVID-19, endothelial dysfunction, along with the induction of cytokines and growth factors, likely plays a critical role in platelet activation, coagulopathy and thromboembolic complications (23).

The healthy endothelium plays a crucial role in maintaining vascular homeostasis by expressing molecules that are both antithrombotic and anti-inflammatory (12, 24). During sepsis, however, the endothelium becomes activated either directly by pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi or indirectly through neutrophil extracellular traps (NETs) and proinflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 (IL-1) (4). Due to its constant exposure to circulating blood, the endothelium is equipped with mechanisms to counteract thrombosis and inflammation (25). Key examples include heparanoid proteoglycans, prostacyclins, ectonucleotidases like CD39 and CD73, the protein C receptor, and tissue factor pathway inhibitor, which all work to maintain an antithrombotic surface (26). Furthermore, the endothelium functions as a selective barrier, regulating the passage of molecules and cells between the bloodstream and surrounding tissues. When activated, this typically quiescent structure loses its antithrombotic properties and adopts a pro-inflammatory phenotype (10).

Upon activation, vascular endothelial cells interact with leukocytes and platelets, playing a crucial role in immunothrombosis, a systemic response to infections that limits pathogen spread (24). These immune and thrombotic responses are complex, creating procoagulant, proadhesive, and proinflammatory conditions that result in glycocalyx damage, upregulation of adhesion molecules, release of von Willebrand factor (vWF), and vascular tone impairment, all of which facilitate the formation of immunothrombosis (27). When activated, endothelial cells release the contents of their Weibel-Palade bodies, including vWF (28), which interacts with platelet GPIbA, promoting platelet-neutrophil interactions through neutrophil β2 integrins, with evidence suggesting that vWF enhances these interactions. Additionally, CCL5 and CXCL4 (platelet factor 4) have been shown to immobilize on the endothelium, attracting neutrophils and monocytes to sites of thromboinflammation (29).

Vascular endothelial cells also express innate immune receptors, such as toll-like receptors (TLRs) and protease-activated receptors (PARs). Activation of TLRs by agonists like lipopolysaccharides and peptidoglycans upregulates the expression of inflammatory and procoagulant mediators in endothelial cells (30). Additionally, TLR activation modulates microvascular permeability and increases the expression of adhesion molecules (19).

A substantial body of evidence indicates that sepsis-induced endothelial activation leads to increased tissue factor (TF) expression, secretion, and heightened TF activity. Complement components, particularly C5a, have also been shown to stimulate endothelial cells to produce active TF. In addition to TF, other mechanisms contributing to immunothrombosis include the impaired fibrinolytic and anticoagulant functions of the endothelium (4). The process of TF “decryption”, which refers to the crucial post-translational activation step that regulates TF activity, is considered a pivotal factor in driving TF-mediated immunothrombosis (31). Bacterial endotoxins further enhance TF expression while increasing plasminogen activator inhibitor (PAI-1) levels, thereby inhibiting fibrinolysis and creating a procoagulant endothelial surface. Across all cases, endothelial dysfunction is marked by a downregulation of key components of the natural anticoagulant system (12).

Given the important role of the endothelium in regulating hemostasis, fibrinolysis, and vessel wall permeability, endothelial dysfunction in the pulmonary vasculature serves as a trigger for immunothrombosis, leading to the coagulopathy seen in COVID-19 patients. Increasing evidence suggests that COVID-19 is primarily an endothelial disease (32), characterized by elevated levels of PAI-1 and vWF, heightened platelet activation, and a hypercoagulable state, resulting in venous, arterial, and microvascular thrombosis. Although the precise triggers of this endotheliopathy are not yet fully understood, potential factors include direct viral invasion, immune cell infiltration (by neutrophils and macrophages), platelet activation, and hypoxemia, which upregulates TF expression and fosters fibrin-based clot formation, supporting a thromboinflammatory feedback loop. Additional mechanisms may involve complement-mediated damage and surges in pro-inflammatory cytokines, such as IL-1 and IL-6, which lead to direct endothelial injury (2).

SARS-CoV-2 directly infects vascular endothelial cells, causing cellular damage and apoptosis, thereby reducing the antithrombotic function of the normal endothelium (33, 34). In severe cases of COVID-19, endothelial cells in the lung’s blood vessels are further activated by elevated levels of pro-inflammatory cytokines (IL-1, IL-6 and TNF) and ferritin, exacerbating a vicious cycle of immunothrombosis. This hyperinflammatory state induces a hypercoagulable condition, contributing to thrombosis within the pulmonary microvasculature (2).

The efficient and coordinated activation of intravascular coagulation in response to blood-borne pathogens and circulating products from damaged host cells facilitates the biological process of immunothrombosis. During this process, innate immune cells leverage their procoagulant mechanisms, which include the local release of active tissue factor (TF), the degradation of endogenous anticoagulants, and the formation of a procoagulant matrix composed of extracellular nucleosomes (1). TF is expressed by various cell types, including fibroblasts, pericytes, epithelial cells, monocytes and neutrophils, and also circulates in the blood as microparticles (35, 36). Monocyte-expressed TF, along with TF delivered by monocyte-derived microparticles and possibly neutrophils, is considered the key trigger of coagulation in immunothrombosis (37).

Notably, the activation of intravascular TF is directly linked to leukocyte recognition of pathogens or damaged cells. For instance, the detection of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by monocyte receptors (including TLRs and CD14), enhances TF gene transcription and protein expression. Additionally, TF is activated by damage-associated molecular patterns (DAMPs) released from injured host cells. Therefore, intravascular TF activity is upregulated in response to both pathogens and damaged cells through de novo synthesis of the TF protein (1).

Upon exposure to the bloodstream following vascular injury, TF forms a complex with activated factor VII (FVIIa), initiating the extrinsic pathway of the coagulation cascade. The release of TF is associated with elevated levels of cytokines and chemokines. The TF-FVIIa complex activates factor X (FXa), which converts prothrombin into thrombin, further amplifying FX activation through the coagulation feedback loop. Thrombin, in turn, initiates the conversion of fibrinogen into fibrin, leading to the formation of a stable clot (7).

The fibrinolytic system, responsible for breaking down blood clots, plays a vital role in regulating inflammation and maintaining the balance between proinflammatory and anti-inflammatory processes. Activated by tissue plasminogen activator (tPA) and urokinase (uPA), this system facilitates the effective resolution of fibrin clots, restoring blood flow following vessel injury. Fibrin serves as the primary substrate for plasmin, promoting the interaction between tPA and plasminogen on its surface, thus enabling its own degradation. Various cell types, including endothelial cells, monocytes, macrophages, and neutrophils, contribute to fibrinolysis by expressing cell surface receptors with fibrinolytic activity, acting as co-factors for plasmin generation, and protecting against circulating fibrinolysis inhibitors. During fibrinolysis, several fibrin degradation products, such as D-dimer, are released and exhibit immunomodulatory and chemotactic functions (11).

Fibrinolysis is primarily controlled by the interplay between tPA and plasminogen activator inhibitor 1 (PAI-1). Upon endothelial injury or exposure to thrombin, tPA is released from the Weibel-Palade bodies of endothelial cells, triggering a surge in fibrinolytic activity. This initial phase of enhanced fibrinolysis, however, is quickly counteracted by increased production of PAI-1 by endothelial cells. The elevated levels of PAI-1 shift the balance away from fibrinolysis, promoting microthrombus formation and contributing to organ dysfunction (38). In sepsis, dysregulated endothelial production of PAI-1 may impair fibrinolysis, promoting thromboinflammation and procoagulant effects (19).

Fibrinolytic proteins are also crucial in regulating the immune response, particularly through their role in clearing proinflammatory fibrin. Plasminogen activators, such as tPA and urokinase, modulate the innate immune response through both fibrinolytic and non-fibrinolytic mechanisms. Plasminogen itself has diverse functions in controlling proinflammatory processes, including the recruitment of monocytes and lymphocytes during inflammation, as well as enhancing macrophage phagocytosis. PAI-1, an acute phase protein, is upregulated in response to bacterial infections, aiding in pathogen clearance and thereby helping to limit inflammation. Endothelial cells produce PAI-1, with its release further increased by inflammatory cytokines (11).

In addition to the fibrinolytic system, the body has an endogenous anticoagulation system that includes tissue factor pathway inhibitor (TFPI), activated protein C, and antithrombin. These natural anticoagulants help prevent widespread systemic coagulation. Like fibrinolytic agents, these proteins also contribute to the immune response. Research has shown that TFPI exhibits antimicrobial properties, while protein C, through the activation of protease-activated receptor 1 (PAR-1), promotes the release of pro-inflammatory cytokines from monocytes, which in turn drives neutrophil chemotaxis (7).

Fibrinogen, the key structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, where its presence correlates with disease severity (39). Fibrin, the final product of coagulation, not only provides a scaffold for immune cells but also facilitates the recruitment and activation of inflammatory cells (11). Additionally, fibrin contributes to bacterial containment by trapping pathogens within the clot, further enhancing the immune response by promoting chemotaxis and supporting the adhesion of leukocytes, including macrophages, dendritic cells, and neutrophils (7).

It has been shown that fibrin binds to the SARS-CoV-2 spike protein, leading to the formation of proinflammatory blood clots that contribute to systemic thromboinflammation in COVID-19. Additionally, the spike protein disrupts fibrin polymerization, degradation, and its inflammatory properties, suggesting that it delays fibrinolysis. These findings align with observations of dense, fibrinolysis-resistant blood clots in COVID-19 patients, highlighting fibrinogen’s role as a SARS-CoV-2 spike-binding protein that accelerates the formation of abnormal, highly inflammatory clots (39).

The complement system comprises over 50 proteins that work together through protease activity to enhance immune defenses. These proteins facilitate the recruitment of inflammatory cells, promote opsonization, and aid in the clearance of pathogens, playing a crucial role in both protecting the body from infections and removing damaged cells (40). This intricate network of plasma proteins operates through a cascade of interactions, activated via three primary pathways: the classical, alternative, and lectin pathways. Upon activation, the complement system initiates a series of reactions that culminate in the formation of membrane attack complexes (MACs) on the surface of pathogens or infected cells. These complexes can directly destroy invading microbes, promote phagocytosis by immune cells, and stimulate inflammatory responses, thereby amplifying the immune defense (11).

The complement system can be triggered by various stimuli, ultimately converging at the generation of C5a – a potent chemotactic and pro-inflammatory protein – and the assembly of the MAC, comprising C5b, C6, C7, C8, and C9 (10). There are intricate interactions between the complement system and platelets, which influence both immune responses and coagulation. Upon binding to platelet receptors, complement proteins can initiate a cascade of events that recruit and activate immune cells at sites of injury or infection (11). Platelets express several complement receptors, including cC1qR, gC1qR, C3aR, and C5aR, while complement components like C1q, C3, C4, and C9 can bind to activated platelet surfaces (41). This interaction activates platelets, promoting the surface expression of P-selectin, which facilitates neutrophil adhesion to the endothelium, enhancing immune cell recruitment (42).

The complement system plays a key role in neutrophil recruitment, as proteins like C3a and C5a can upregulate TF activity, further activating neutrophils (7). Additionally, complement activation has been shown to induce rapid TF decryption, increasing its procoagulant function. Complement factor C3 is crucial for platelet activation, and in turn, thrombin can activate both C3 and C5, leading to elevated TF expression on endothelial cells through interactions with C5a and the MAC (C5b-9) (31). Among the complement factors, C3 and C5 are particularly significant in driving thromboinflammation. C3a and C5a promote the production of inflammatory mediators by binding to their respective receptors (38). Moreover, C5a transforms the leukocyte membrane into a procoagulant surface, enhancing neutrophil release of NETs and accelerating coagulation (43, 44).

Complement and coagulation activation are closely linked to COVID-19 severity (31), and abnormal complement activation has been previously associated with thromboinflammatory responses and microangiopathy (45). In severe cases, enhanced activation of the alternative complement pathway correlates with markers of endothelial injury (e.g. angiopoietin-2) and hypercoagulability (e.g., thrombomodulin and von Willebrand factor), highlighting complement activation as a defining feature of COVID-19 (46). Histopathological analysis of skin and lung tissues from patients with severe disease reveals extensive microvascular deposition of terminal complement components, including C5b-9 (membrane attack complex), C4d, and MASP-2, indicative of sustained, systemic complement activation (47). Since complement can both mediate tissue injury and be activated in response to it, the markers of complement activity observed in severe COVID-19 may be both a consequence and a cause of ongoing complement activation (48).

Platelets are anucleated blood cells derived from megakaryocytes in the bone marrow. Once released into the bloodstream, they circulate for 7–10 days before being primarily cleared by macrophages in the liver (49, 50). Upon vessel damage, platelets are activated, adhere, change their shape, secrete their granule contents and start to form aggregates (51). Platelets contain three distinct types of storage granules – alpha granules, delta granules, and lysosomes – which house a variety of receptors, soluble proteins, and bioactive molecules. These components play critical roles not only in hemostasis but also in regulating inflammation and immune responses (52).

Platelets are increasingly recognized for their roles beyond hemostasis and thrombosis, particularly in modulating immune responses. For instance, platelet-leukocyte interactions lead to bidirectional immune crosstalk and transactivation, contributing to vascular inflammation (10). Some researchers even classify platelets as immune cells, given their surface expression of pathogen sensors and their ability to enhance immune functions, such as cytokine production and the release of clotting factors in response to stimulation (53).

In addition to their role in initiating and propagating coagulation, innate immune cells, and platelets – key components of clots – play a crucial role in facilitating immunothrombosis through several mechanisms. Platelets promote the recruitment of innate immune cells and enhance the expression of TF, particularly on monocytes. Moreover, activated platelets have evolved antimicrobial strategies that directly support the innate immune response during immunothrombosis and beyond (1). Equipped with a variety of pattern-recognition receptors, including the complete LPS receptor complex, platelets can actively bind circulating bacteria and present these pathogens to neutrophils and other immune cells (54). In response to bacterial products, platelets engage directly with neutrophils, triggering the formation of NETs (55, 56). Although platelets facilitate and accelerate NETosis, NET formation can also occur independently of them (57).

Toll-like receptors (TLRs) are expressed by various immune cells, including platelets, which display TLRs both on their membrane and intracellularly (8). Through these receptors, platelets are able to detect pathogens such as bacteria and viruses. Activation of platelet TLRs triggers not only platelet aggregation but also a pro-inflammatory response. Therefore, TLRs on platelets play a dual role in promoting aggregation and contributing to thromboinflammation (29). TLRs recognize Damage-Associated Molecular Patterns (DAMPs) and Pathogen-Associated Molecular Patterns (PAMPs), leading to cellular activation that generates oxygen and nitrogen radicals and produce cytokines (58). When platelets encounter DAMPs or PAMPs, they become activated and mediate immune responses (8). TLR activation induces immunothrombosis via multiple pathways after PAMP recognition. The surface of activated platelets facilitates thrombin generation, followed by fibrin and clot formation (59).

Platelets have been found to be hyperactivated in patients with COVID-19, potentially exacerbating the thromboinflammatory cascade through interactions with neutrophils. Notably, activated platelets play a critical role in the formation of NETs, which are key components of immunothrombosis. These findings support the hypothesis that endothelial dysfunction and platelet activation are central features of COVID-19 associated coagulopathy, potentially driving the severe damage seen in critical cases (2). This COVID-19-specific platelet hyperactivation, characterized by elevated P-selectin expression and increased circulating platelet–neutrophil aggregates, likely contributes to the heightened immunothrombotic response observed in severe disease (60).

Upon activation, platelets release alpha granule-stored molecules, which contribute to the recruitment of additional monocytes and neutrophils. Monocytes then interact with platelets, forming platelet-monocyte complexes that induce TF expression in monocytes. Meanwhile, hyperactivated neutrophils form platelet-neutrophil aggregates, promoting further NET formation and fueling the cycle of prothrombotic activity (7). This sequence of events, combined with platelet aggregation and fibrin deposition, leads to thromboinflammation and vessel occlusion, even in the absence of direct vascular injury (61).

Leukocytes are critical cellular components in the defense against infections, with neutrophils and monocytes playing key roles in thromboinflammation. While neutrophils and monocytes share many common receptors, their functions differ significantly. Monocytes act as the conductors of inflammation, coordinating immune responses, whereas neutrophils are frontline defenders, directly attacking and immobilizing pathogens.

In response to various stimuli, monocytes become prothrombotic, releasing proinflammatory cytokines and triggering innate immune responses. Activated neutrophils amplify inflammation by releasing proteases and oxygen radicals, ultimately undergoing different forms of cell death, such as necrosis, necroptosis, and pyroptosis. Cellular components released from dying cells, particularly DAMPs like DNA and histones, are highly proinflammatory and procoagulant (62).

Monocytes and neutrophils play critical roles in thrombosis, with many thrombosis-related host molecules being produced or activated by these cells. Activated monocytes, along with the microparticles they release, express intravascular TF, promoting coagulation during thrombus formation. Intravascular TF may also be expressed by other immune cells, such as neutrophils, eosinophils, and platelets, further contributing to clot development. Both monocytes and neutrophils are rapidly recruited to the vessel wall or become incorporated into growing clots (1).

Both neutrophils and monocytes are capable of releasing extracellular traps – web-like structures composed of DNA and intracellular components. While neutrophil extracellular traps (NETs) have gathered the most attention, monocyte extracellular traps (METs) also play a significant role in the body’s defense against infection and are central mediators of thromboinflammation (38). NETs consist of a meshwork of DNA, histones, and antimicrobial proteins that are expelled from neutrophils upon activation (1, 8). In COVID-19, circulating neutrophils release increased levels of NETs in the blood, trachea, and lungs (63). These NETs contain nucleosomal components, including histones, DNA, extracellular viral micro-RNAs, and TF, all of which contribute to immunothrombosis (64).

Various therapies targeting immunothrombosis have been proposed, including treatments that modulate cell-specific immune responses, inhibit platelet-endothelial interactions, or block platelet activation. These range from well-established therapies to more experimental approaches, some of which remain theoretical. Developing treatments that target endothelial innate immune responses and immunothrombosis holds significant potential for reducing morbidity and mortality in sepsis patients (4). However, no drugs specifically targeting immunothrombosis have been approved for clinical use in sepsis, primarily due to inconclusive efficacy results and an increased risk of side effects (4). Ongoing research continues to explore the effects of new molecules aimed at intracellular inflammatory pathways, NET formation, and complement components.

Patients with severe COVID-19 who experience thromboembolic events often present with elevated inflammation markers, including CRP, IL-6, fibrinogen, and ferritin, suggesting a strong link to immunothrombosis (99). Immunothrombosis is widely regarded as a central pathophysiological mechanism in the development of sepsis-associated disseminated intravascular coagulation (DIC). DIC is characterized by endothelial dysfunction and an imbalance in procoagulant, anticoagulant, and fibrinolytic systems.

In severe sepsis, elevated plasma levels of thrombin and D-dimer, both markers of coagulation activation, correlate with increased disease severity. Accordingly, decreased levels of natural anticoagulants, such as protein C, protein S, and antithrombin, are commonly observed and are strongly associated with poorer clinical outcomes. Research also indicates that ICU patients with sepsis who exhibit enhanced NET formation are more likely to develop thrombocytopenia, prolonged PT and aPTT, elevated d-dimer levels, and decreased fibrinogen, all of which are predictive of DIC development (4).

In the early stages of the COVID-19 pandemic, certain changes in standard coagulation tests were observed, most notably elevated D-dimer levels and hyperfibrinogenemia (100). D-dimer, an indirect marker of fibrinolysis and fibrin turnover, reflects the systemic breakdown of vascular thrombi through the fibrinolytic process. Studies showed that D-dimer levels rose significantly in the early stages of COVID-19, with a 3- to 4-fold increase linked to poor prognosis (101, 102). Fibrinogen, which is converted to fibrin by thrombin, plays a crucial role as the primary structural component of blood clots. Severe COVID-19 has been associated with significantly elevated fibrinogen levels, leading to excessive fibrin deposition and impaired fibrin degradation (103).

Thrombocytopenia and PT prolongation were uncommon in COVID-19 associated coagulopathy (104). A low platelet count, when observed, was however linked to higher mortality rates (105). Another study revealed a significant increase in vWF activity and decreased ADAMTS-13 levels in COVID-19 patients, suggesting that impaired regulation of vWF and the reduced capacity of its cleavage by ADAMTS-13 could be indicative of an increased thrombosis risk (38).

Viscoelastic testing, mostly employed in trauma and high risk of bleeding surgery to guide resuscitation, has also been used in hypercoagulative states (106, 107). This capability to detect and quantify hypercoagulative states represents a significant advantage of ROTEM over conventional tests (108, 109).

Four key ROTEM variables are commonly analyzed. EXTEM, an extrinsically activated assay using phospholipids and tissue factor, assesses the extrinsic coagulation pathway. INTEM, an intrinsically activated assay with phospholipids and ellagic acid, evaluates the intrinsic pathway. FIBTEM is a fibrin-based assay that isolates the fibrinogen function by incorporating tissue factor and the platelet inhibitor cytochalasin D, which excludes the platelet contribution to clot formation. HEPTEM, is similar to INTEM, but includes heparinase to neutralize the effects of heparin.

Within each ROTEM-variable, five parameters are measured. Clotting time (CT) represents the time to the onset of clot formation, indicating coagulation activation. Clot formation time (CFT) reflects the time required for the clot to reach a 20 mm amplitude, providing information about clot propagation. Maximum clot firmness (MCF) measures the peak amplitude, which assesses clot stability. Clinically, a high MCF is indicative of a hypercoagulable state. Lysis index (LI-30 and LI-60) represents the percentage of clot breakdown occurring 30 and 60 minutes after CT, respectively, indicating the extent of fibrinolysis over time (110–112).

In severe COVID-19, an example of an immunothrombotic state, ROTEM analysis revealed prolonged CT, shortened CFT, increased MCF, and elevated LI60, indicating delayed coagulation initiation, faster clot propagation, increased clot stability, and reduced fibrinolysis. In other words, while clots took longer to form, once initiated, they developed more rapidly, became stronger, and were more resistant to breakdown compared to those in healthy individuals.

Prolonged CT, unlike other ROTEM parameters associated with immunothrombosis, is not indicative of hypercoagulation and presents a somewhat paradoxical finding. The prolongation of INTEM-CT is likely attributed to the effects of heparin, whereas EXTEM-CT prolongation is not. Prolonged EXTEM-CT, similar to prolonged prothrombin time (PT), has been observed in some studies of COVID-19 patients and may indicate deficiencies in clotting factors II, VII, and X, which have been observed in COVID-19 patients (113, 114).

The shortened CFT and increased MCF, indicating a rapid increase in clot strength, are consistent with elevated fibrinogen levels and enhanced platelet activation observed in severe COVID-19 cases (115). These changes are likely driven by the massive inflammatory response that characterizes the disease and may signal the presence of immunothrombosis.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (APL) in individuals who experience thrombotic events or specific pregnancy-related complications (49). The APLs included in the APS classification criteria are anti-cardiolipin (ACL), anti-β2 glycoprotein 1 (β2GP1) antibodies, and lupus anticoagulant (LA) (116). APL are more commonly identified in individuals with other autoimmune disorders, particularly systemic lupus erythematosus (SLE) (117). The persistent presence of APL induces a prothrombotic state, predisposing patients to thrombosis. According to the “two-hit” model of APS-related thrombosis, a primary “first hit” disrupts the endothelium and creates a predisposition to clot formation, while a “second hit”, triggered by factors such as pregnancy, infections, or traditional cardiovascular risk factors, drives clinical thrombotic events (118). The cornerstone of APS management is anticoagulation therapy, with vitamin K antagonists being the preferred agents supported by the strongest clinical evidence. However, recurrent thrombotic events remain a significant challenge even in patients receiving adequate anticoagulation therapy (119). In Catastrophic Antiphospholipid Syndrome (CAPS), a condition marked by widespread microthrombosis driven by an increased inflammatory response, management typically involves a comprehensive triple therapy approach. This includes anticoagulants, plasmapheresis, and high-dose corticosteroids, with the addition of intravenous immunoglobulins in selected cases (120).

In APS, the systemic prothrombotic and proinflammatory state arises from the impact of APL on various components of the innate immune system, including monocytes, neutrophils, platelets, and endothelial cells (121). This persistent state, which is not fully addressed by anticoagulant therapy alone, underscores the need for therapeutic strategies targeting the innate immune system, as such interventions may provide additional clinical benefits (94).

Monocyte cytokine production and TF expression play a central role in the pathophysiology of APS. APL activate monocytes, driving them into a proinflammatory and procoagulant phenotype characterized by the production of cytokines such as TNF-α and IL-6, along with TF. Notably, monocytes from thrombotic APS patients exhibit higher TF expression and activity compared with monocytes from APS patients without thrombosis. Neutrophils also contribute to the thrombotic process by releasing NETs in response to APL, which further promotes clot formation. APL-induced NET release drives thrombin generation as well as platelet activation and aggregation, reinforcing the prothrombotic cascade. This process is further exacerbated by increased interactions between neutrophils and endothelial cells, mediated through P-selectin glycoprotein ligand-1. Additionally, platelet-leukocyte interactions are enhanced, likely driven by elevated levels of soluble P-selectin and soluble CD40-ligand, further amplifying the prothrombotic state (49).

The enhanced release of NETs in APS is supported by observations that neutrophils from APS patients spontaneously release NETs, with APL targeting β2GP1 significantly amplifying this process (122). NET accumulation in APS is further exacerbated by a reduced capacity to degrade these structures, linked to the presence of anti-NET antibodies. These antibodies are associated with recurrent VTE, emphasizing their role in the disease’s thrombotic burden (123, 124). Taken together, these observations suggest that the dual mechanisms of exaggerated NET formation and impaired NET degradation synergistically intensify the prothrombotic effects of NETs in APS (10).

In COVID-19, elevated APL titers have been associated with increased neutrophil and platelet activity, correlating with more severe respiratory disease (125). However, it remains uncertain whether APL in COVID-19 patients are transient, as observed in other viral infections, or persistent, which would suggest a long-term thrombotic risk (10). It is hypothesized that SARS-CoV-2 may act synergistically with APL to promote the immunothrombotic process, as APL can directly induce NETosis. Evidence suggests that dysregulated cytokine release in COVID-19 may be sustained by crosstalk between neutrophils and macrophages mediated by NETs, leading to a profoundly exaggerated immunothrombotic response (2).