Monocytes and macrophages have been found to play a vital role in inflammatory diseases-induced immunothrombosis ( Figure 1 ). Upon stimulation by PAMPs, DAMPs or proinflammatory mediators (17, 18), monocytes are the main source of circulating TF (19, 20). TF is deemed critical for survival, as deletion in mice leads to universal embryonic death (21), and defects in TF gene expression are associated with differing clinical outcomes in patients with sepsis (22). The binding of lipopolysaccharide to transmembrane receptors in monocytes induces TF mRNA expression via NF-κB activation (23). The interaction of pathogen components either with TLRs or directly with intracellular pathways in monocytes result in inflammasome activation and subsequent TF release via pyroptosis (18, 23, 24). Moreover, pore formation on the cell membrane also induce calcium influx, which triggers phosphatidylserine exposure on the membrane, followed by TF activation (25). Sphingomyelin, another membrane lipid, is also involved in the activation of TF to its procoagulant form (26). Additionally, monocyte activation by PAMPs and DAMPs is followed by increased P-selectin glycoprotein ligand 1 (PSGL-1) expression and the release of TF- and PSGL-1-bearing microparticle (MPs). These MPs can fuse in vitro with platelets, leading to increased TF activity (19, 27). Pancreatic disruption leads to direct exposure of TF to the blood (28).

TF expression and release triggers the extrinsic coagulation pathway by binding factor VII/factor VIIa (FVII/FVIIa) to form TF-FVIIa complex, converting factor X (FX) to factor Xa (FXa). Then, FXa is incorporated into FXa-factor Va-Ca2+-phospholipids (FXa-FVa-Ca2+-PLs) complex known as the prothrombinase (29). Thrombin is formed, leading to fibrin clots ( Figure 1 ). The process seems to be the most potent pathway leading to coagulation cascade activation and DIC (30–34). Studies of experimental or human AP have demonstrated a central role of the TF/FVIIa system in the initiation of thrombin generation (8, 35, 36). In the early stage of severe AP, TF is highly upregulated (8, 37–39), and it is a favorable predictive marker of severe AP (40, 41). Abrogation of the TF/FVIIa pathway by specific interventions aimed at TF or factor VIIa activity resulted in a complete abrogation of thrombin generation in experimental settings (42). As the initiator of the coagulation cascades, TF might play a large part in the development of severe AP, there needs to be a more basic experimental to explore their relationship.

The endothelium lines the lumen of the entire circulatory system, separating blood and subendothelial, and maintaining vascular health by exerting anticoagulant action via tissue factor pathway inhibitor (TFPI), Protein C (PC) system, Antithrombin (ATIII) and fibrinolysis (43, 44).

TFPI is the inhibitor of TF-mediated coagulation, primarily synthesized by endothelial cells (ECs, Figure 1 ), which binds to ECs via proteoglycans/glycosaminoglycans, inactivates TF-FVIIa-FXa complex and prothrombinase in the early phase of the coagulation process (45, 46). The deficiency of TFPI increases susceptibility to the development of DIC and thrombosis (37). However, in AP patients, the plasma TFPI levels were significantly increased, which might be compensatory to the rise of TF, and can be released from fibrin deposits after thrombosis. Elevation of TFPI delayed TF-initiated thrombin generation, the imbalance of TF/TFPI were markedly related to pancreatic necrosis and organ failure (OF) (38, 47).

The PC system, as main natural anticoagulants, harbors PC and Thrombomodulin (TM), which along with endothelial cell protein C receptor (EPCR) catalyzes the thrombin-mediated PC activation ( Figure 1 ). Activated PC (APC) exerts potent anticoagulation by inactivating FVa and FVIIIa (48). TM is expressed on the endothelial surface, which switches the thrombotic activity of thrombin to antithrombotic through activating PC (49, 50). It is known that soluble TM (sTM), fragments of the extracellular region of membrane-bound TM cleaved by leukocyte-derived proteases or metal loproteases, are released into the circulation in inflammatory diseases (50). Multiple studies have reported the usefulness of measuring sTM to evaluate the severity of DIC. EPCR, a transmembrane glycoprotein present on the surface of ECs, increases the efficiency of APC generation by presenting PC zymogen to thrombin/TM complex (51). However, the PC system is damaged in AP patients characterized by low levels of PC and APC (52, 53), and significantly increased levels of plasma sTM and EPCR (54).

ATIII, as a serine protease inhibitor, which inactivates TF-FVIIa-FXa Complex, FIXa, FXIa, thrombin, and is the most abundant and most important physiological anticoagulant (55). ATIII makes complexes not only with thrombin but also bind to heparan sulfate of the glycocalyx at the ECs surface (56) ( Figure 1 ). ATIII deficiency can result in severe venous thromboembolism, plasma levels of ATIII activity is positively correlated with the severity of DIC (57). In AP patients, the level of ATIII decreases as severity increases, which is rather pronounced in cases of biliary AP (52). This phenomenon could be ascribed to a combination of impaired synthesis because of the negative acute phase response, degradation by elastase, and consumption because of thrombin generation (37, 58).

Tissue-type plasminogen activator (t-PA) and urokinase-type PA (u-PA) released by ECs are the main activators in the fibrinolysis, which transform plasminogen into plasmin, and then catalyze clot dissolution and fibrinolysis. PA inhibitor, type 1 (PAI-1) and thrombin activated fibrinolytic inhibitor (TAFI) are the regulators of the fibrinolysis, of which PAI-1 is the principal inhibitor (59) ( Figure 1 ). It has been shown that the production of PAI-1 is affected by proinflammatory, anti-inflammatory cytokines and the elevated levels sustain longer (60). In healthy volunteers, endotoxin induces a rapid activation in the coagulation system with a concurrent rise in tPA. This temporal activation in fibrinolysis is subsequently counteracted by a greater and sustained rise in PAI-1 (61), The marked increase in PAI-1 level causes fibrinolysis shutdown, subsequently failing to counteract the systemic deposition of fibrin clots during system inflammatory reaction syndrome, leading to thrombosis and DIC (59). Patients with OF have significantly higher plasma levels of PAI-1, and non-survivors demonstrate more potent suppression of fibrinolysis than survivors (36). In severe AP, the level of TAFI also rises at the onset of the disease (58), inhibits fibrinolysis by separating carboxyterminal lysine residues and preventing binding to plasminogen (62).

Cytokines (63) and thrombin (64–67) activate platelets by DAMP receptors (68) ( Figure 1 ), myeloid differentiation factor 88 (MyD88) and cGMP-dependent protein kinase intracellular pathways (67), as well as protease associated receptors (64, 65). Upon platelet activation, dense and α-granules fuse with the cell membrane (65), dense granules are rich in adenosine diphosphate, which further stimulates and amplifies platelet activation via receptors P2Y1 and P2Y12, whilst α-granules contain P-selectin that mediates activation of leukocytes via binding to PSGL-1, chemokines, and pro-coagulant factors. Glycoproteins IIb/IIIa (GPIIb/IIIa) and Iba (GPIba), expressed on the surface of activated platelets (69), bridged by fibrinogen or von Willebrand Factor (vWF), which constitute another receptor category that promotes platelet degranulation and aggregation (69, 70). VWF, from α-granules, facilitate platelet adhesion to the endothelium (71) ( Figure 1 ). FcgRIIa triggers an intracellular pathway for platelet activation by phosphorylating the tyrosine kinases Src, Syk, and phospholipase c gamma 2 (PLCg2) (70). Mechanical interactions are potentiated by change from discoid to stellate shape (72). Activated platelets have also been found to release polyphosphate (PolyP), an inorganic polymer that exerts procoagulant activity. In vitro, PolyP initiates the contact pathway by FXII activation (73). Further, activated platelets aggregate with leucocytes to form platelet-leucocyte aggregates (PLA) (74), PLA in turn cause release of platelets-activating neutrophil extracellular traps (NETs), which form a vicious cycle. Platelets are essential cellular components of the coagulation system in AP animal models (8). In AP, thrombocytopenia is associated with increased disease severity and an ominous prognosis (75).

P-selectin stored in granular structures of ECs and platelets can be quickly mobilized towards the cell surface upon stimulation (76). P-selectin and its ligand, PSGL-1 linking is the first step for platelet adhesion (56) ( Figure 1 ). PSGL-1 expressed on platelets, monocytes, and neutrophils mediate leukocyte and platelet rolling on the vascular wall as well as platelet-neutrophil and platelet–platelet aggregations to link inflammatory infiltration and thrombus formation (77). The expression of P-selectin on the platelet membrane not only mediates the adherence of platelets to leukocytes and endothelial cells but also enhances the expression of TF on monocytes (78). Notably, monocyte-derived, TF containing MPs fail to incorporate in thrombi when infused into P-selectin null mice, indicating that the accumulation of leukocyte-derived TF in growing thrombi is mediated by PSGL-1 on the MPs (79).

The levels of P-selectin are related to the development and course of AP, it’s value on admission may play a pivotal role as indicators of overall prognosis (80). The elevated level of P-selectin markedly strengthens the leukocyte–endothelium interaction and the thrombosis (81). Suppressing P-selectin inhibits leukocyte and platelet rolling in postcapillary venules of the inflamed pancreas (82), protecting against thrombosis (83) and improving pancreatic microcirculation and histopathology of acinar necrosis without causing any bleeding complications (84). Escopy et al. reviewed both preclinical and clinical trials that have evaluated therapeutic potential of biologic and small-molecule inhibitors as well as antibodies of P-selectin in a variety of diseases linked to immunothrombosis and coagulopathy (85). Wherein, crizanlizumab, a monoclonal antibody of P-selectin, has been evaluated for the treatment of vaso-occlusive crises with sickle cell disease (Food and Drug Administration approval in 2019) and COVID-19 vasculopathy (NCT04435184 and NCT04505774). Inclaclumab, a newly developed monoclonal antibody of P-selectin, was noted to reduce myocardial damage of non-ST-elevation in patients with myocardial infarction in a phase 2 clinical trial (NCT01327183). Currently, a multicenter phase 3 trial is in progress to determine whether inclaclumab could reduce the frequency of vaso-occlusive crises (Thrive-131; NCT04935879) and to evaluate its long-term safety (Thrive-133 open-label extension; NCT05348915). Except for these anti-P-selectin antibodies, PSI-697, PSI-421, as small-molecule inhibitors of P-selectin, have also been widely studied (85, 86). P-selectin and P-selectin glycoprotein ligand-1 play a fundamental role in aggravating pancreatic inflammation and their antibodies alleviate inflammatory responses in experimental severe AP (82). Therefore, it is believed that further research on the therapeutic potential of these inhibitors of P-selectin and related pathways in severe AP maybe promising.

DAMPs also activate neutrophils, which are typically the first responders to AP. Activated neutrophils exert their antimicrobial activity mainly through three processes: phagocytosis, degranulation, and the release of NETs (87). Neutrophil activation and release of NETs are considered as the initial and indispensable event in thrombus formation (88) ( Figure 1 ). NETs as a meshwork of DNA fibers, comprise histones, antimicrobial proteins, and high-mobility group box 1 (89), which promote endothelial dysfunction (90), increase platelet activation, adhesion, aggregation (89–91), in turn contribute to thrombin-mediated fibrin generation (92, 93). NETs also propagate thrombosis by capturing TF and TF-positive extracellular vesicles from the circulation, further driving coagulation (94). Wherein, thrombin formation consists of NETs-induced platelet-dependent mechanisms and platelet-poor plasma via activation of the intrinsic coagulation pathway (95). Interaction of NETs with membrane-derived MPs released by activated neutrophils further enhanced NET-mediated intrinsic coagulation pathway activation (96). However, the role of NET components, or intact NETs on thrombosis is debatable, and merit further investigation. Except for prothrombotic role, NETs were also shown to interfere with the endogenous anticoagulant mechanisms. More specifically, extracellular nucleosomes within NETs facilitated TFPI degradation by neutrophil elastase on the surface of activated neutrophils (97), neutrophil elastase bound to DNA complexes was also shown to cleave plasminogen into fragments, cell-free DNA was capable of binding to plasmin and fibrin at the same time, resulting in decreased plasmin production and impaired fibrinolysis (98). H3 and H4, could also interact with TM and PC, leading to the inhibition of APC generation (99).

In AP patients, the plasma levels of NET components increase significantly compared to the controls (100). Platelets regulate the formation of NETs and NET-MPs aggregations (100–102) and in turn, NETs recruit platelets and neutrophils, reinforcing each other and injuring the endothelium within pancreatic microvasculature (100, 103). Exosomes adhere to NETs in vitro where they have a dose-dependent pro-coagulant effect. NETs also activate the intrinsic coagulation pathway via autoactivation of Factor XII (102). NETs are complex structures composed of DNA and cytotoxic granule proteins, including myeloperoxidase and neutrophil elastase. NETs targeting, either preventing their formation or degrading the NETs that have already formed, has been proved to prevent tissue damage and reduce risk of thrombus formation in the context of infections (104). Preventing NETs formation by inhibition protein-arginine deaminase 4, neutrophil elastase, or gasdermin D, has been proved to be effective in several preclinical inflammatory disease models. While whether inhibition of NETs formation has a detrimental effect on host defense mechanisms has not been determined (105). Another strategy could be to interfere with NETs that have already formed: a recombinant human deoxyribonuclease I (rhDNase) is already used for the treatment of cystic fibrosis with safety confirmed, making it a very viable option for other diseases. A phase Ib study for patients with systemic lupus erythematosus (SLE) showed that DNase was well tolerated without severe adverse effects. Significant recent developments in the field of rhDNase targeting the NET have led to testing of new NET-targeted drugs in clinical trials of patients with COVID-19 (NCT04409925, NCT04541979 and NCT05139901) (105). Heparins, as a class of anticoagulant drugs, has been proposed to destabilize NETs by dissociating histones from the chromatin backbone of the extracellular traps as well as to prevent phorbol myristate acetate-induced NET formation (106). Colchicine, destabilization of actin cytoskeleton in NETs has been tested in Gout. N-acetyl cysteine, a ROS scavenger, improved the condition of patients with SLE and acute liver failure. Anti-TNF monoclonal antibodies have been used for the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease, and anti-IL-17 antibodies have also shown some efficacy for the treatment of RA (107). The involvement of NETs has been well established in the pathobiology in experimental models and patients of severe AP (100, 108, 109). All the aforementioned therapeutic agents targeting NETs are expected to have significant potential to mitigate severe AP in transitional research (105).

The complement system shares a common origin with the coagulation system and influences each other. It is activated through proteolytic cascades (110), leading to the formation of membrane attack complexes, ultimately polymerizing and inducing lysis of the cellular target (111). Recent studies have shown specific crosstalk between complement and coagulation in AP patients (112). First, in addition to activation by serine proteases, granzyme B and trypsin also cleave the central complement components, generating C3a and C5a (113). Second, C3a and C5a release TF from monocytes and ECs and promote platelet activation, leading to thrombogenesis (110, 111). Moreover, C3 is essential for the recruitment of neutrophils into the pancreas and NET formation (114). After C3a and C5a complement activation, the direct products also stimulate the platelets and promote coagulation by stimulating phosphatidylserine exposure (111) ( Figure 1 ), enhancing the activation of platelets, granulocytes, and ECs, increasing the microcirculation thrombosis and pancreatic injury.