Tissue factor expression. It is widely acceptable that the most important trigger of coagulation in sepsis and trauma-associated DIC is excessive tissue factor (TF) expression by circulating monocytes and its exposure from the vascular sub-endothelium following injury ⁷⁵ . This is supported by the observation that patients with DIC have significantly higher levels of circulating TF compared with controls ⁷⁶ . Exaggerated TF expression in septic and trauma DIC was historically attributed to systemic inflammation triggered by the invading microorganisms or their toxins (such as lipopolysaccharides) ^{39– 41} . However, two recent studies have shown that extracellular histones can directly induce TF expression in a dose- and time-dependent manner on the surface of endothelial cells and macrophages via Toll-like receptor-4 (TLR-4) and TLR-2 and activation of the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1) pathways ^{12, 42} . With regard to NETs, recent studies have reported that NETs bear TF and contribute to thrombosis in myocardial infarction ⁷⁷ and anti-neutrophil cytoplasmic antibody-associated vasculitis ^{78, 79} . One study in the cancer setting reported significant interactions and correlations between NET components (primarily circulating DNA and nucleosomes) and TF-bearing microparticles culminating in overt DIC ⁴³ . Interestingly, both TF-bearing NETs and TF-bearing microparticles were found to be triggered by complement C5a ⁷⁹ , highlighting a significant interaction between the coagulant and innate immune systems that include complement in contributing to pathology.

Pro-inflammatory cytokine involvement. In sepsis and trauma, the DIC process is directly linked into, and triggered by, the systemic inflammatory host response, of which pro-inflammatory cytokines play critical roles beyond induction of TF expression ⁴⁴ . Excessive cytokine activities disrupt the fine balance and cross talk between coagulant, anti-coagulant, and inflammatory pathways to augment the pro-coagulant phenotype ^{44, 45} . Histones can directly induce the release of several pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-alpha (TNF-α) ^{10, 46– 48} . NETs act as scaffolds to potentiate IL production and activation ^{80– 82} . Conversely, cytokines can induce NETosis ⁸³ in a bi-directional relationship akin to that between histones and NETs ( Figure 1).

Platelet activation. Platelets are important for both the initial burst and further sustenance of thrombin generation by acting as scaffolds on which further coagulation activation takes place ⁸⁴ and through platelet-derived polyphosphate activation of factor XI ⁸⁵ . Platelets also promote a pro-coagulant phenotype via P-selectin expression which enables adherence to the vascular endothelium and leukocytes while also augmenting TF expression ⁴⁹ and phosphatidylserine exposure on monocytes ^{50, 51} which collectively enhances thrombin generation ⁵¹ . Histones can directly induce platelet activation via calcium influx with subsequent platelet aggregation and consumption in vitro and in vivo ^{13, 52, 86} . Histones can also promote thrombin generation in a platelet-dependent manner via P-selectin expression, phosphatidylserine exposure, and FV/Va availability on platelet surfaces ⁸⁷ . Histone-induced TF expression and subsequent thrombin generation can further activate platelets. In terms of clinical relevance, a case control study in intensive care patients recently illustrated a strong association between high histone levels and subsequent platelet consumption and thrombocytopenia to translate the findings of histone-induced thrombocytopenia in animal models ⁸⁸ . In parallel, NETs can interact with platelets to induce platelet aggregation, polyphosphate release, and subsequent thrombin generation to cause intravascular coagulation in septic mice ²⁶ . Conversely, platelets can directly induce NETosis ^{33, 89, 90} ( Figure 1) and contribute to pathology, including that of transfusion-associated acute lung injury ^{33, 90} .

Vascular endothelial injury. The vascular endothelium, one of the biggest organs in the body, has a natural anti-coagulant surface mediated by the generation of APC ⁹¹ , tissue factor pathway inhibitor (TFPI) ⁹² , and expression of heparan sulfate and glycosaminoglycans that convey anti-thrombin (AT) activity ⁹³ . Endothelial cells also participate in fibrinolysis through release of tissue plasminogen activator (tPA) upon activation ^{94, 95} . Therefore, damage or dysfunction of the vascular endothelium is another vital aspect of DIC pathogenesis. To this end, histone-induced toxicity on the vascular endothelium is well documented ⁹ and further extended in clinical studies with strong correlations observed between levels of circulating histones and soluble TM, a marker of endothelial cell injury, in critically ill patients ¹⁰ . In addition, histones have been reported to induce the release of ultra-large von Willebrand factor (vWF) multimers ⁹⁶ , which are involved in platelet adhesion, platelet consumption, and microvascular thrombosis. Reports in patients with sepsis have indeed documented elevated levels of ultra-large multimers of vWF together with deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), which is responsible for degradation of these multimers ^{53, 54} , but the direct effect of histones on ADAMTS-13 is not currently known. As to NETs, a recent study showed that vascular endothelial cells have limited phagocytic capacity for NETs, which could result in poor NET clearance and subsequent damage to endothelial tight junctions to increase vascular permeability ⁹⁷ . Another study illustrated that matrix metalloproteinases within NETs contribute to endothelial dysfunction ⁶⁷ . Indeed, activated endothelial cells can also induce NETosis ⁹⁸ . As such, these histone- and NET-induced changes are also likely to play relevant roles in DIC pathogenesis.

A physical consequence of cellular injury induced by histones and NETs is the exposure of negatively charged phospholipid surfaces from membrane disruption ⁹⁹ . Availability of such phospholipid surfaces is highly pro-coagulant and can accelerate the prothrombinase reaction by 250,000-fold ¹⁰⁰ . In addition, histones can directly bind prothrombin and cause its auto-activation into thrombin. The implications would be that histones can directly generate thrombin without requiring coagulation activation ¹⁰¹ . Although this reaction takes about eight hours and therefore may not be physiologically relevant, the available evidence points to the diversity in how histones directly contribute to thrombin generation and disseminate coagulation activation.

Reduced endogenous anti-coagulant activity. The three endogenous anti-coagulant pathways—AT, PC, and TFPI—play a critical role in regulating the extent of clot generation. All of these pathways are significantly compromised in DIC. Declining trends in AT and PC levels can be used to identify patients in a non-overt stage of DIC before full decompensation ⁶ . Low AT and PC levels have been validated as strong predictors of mortality in patients with sepsis and DIC ^{55, 102} .

Although low levels of endogenous anti-coagulants can be due to pathological consumption by the excessive coagulopathy in DIC, other mechanisms also contribute to reduced activity and levels ¹⁰³ . Related to histones and NETs are two studies showing that histones can disrupt the PC pathway by downregulating TM ¹² or dampening TM-dependent PC activation ⁵⁶ . This would impair APC anti-coagulant, anti-inflammatory, and cyto-protective functions ⁵⁷ , including its ability to proteolytically cleave histones ⁹ . NET-associated elastases can potently degrade AT and TFPI ^{58– 60} . In addition, two other potential mechanisms for endogenous anti-coagulant loss are reduced synthesis by the liver and loss to the extravascular space from enhanced vascular permeability ⁶⁰ . Histones can potentially contribute to both of these mechanisms by inducing liver injury and inflammation ^{16, 61} and significantly increasing vascular permeability through endothelial damage ^{9, 10} .

Intrinsic pathway activation. In physiological terms, the intrinsic pathway of coagulation plays an important role in increasing thrombin generation and accelerating hemostatic clot formation. This is well exemplified by the significant bleeding issues in patients without factor VIII or IX. cfDNA- and NET-bound DNA can exert pro-coagulant effects through activating the intrinsic pathway of coagulation via FXI and FXII ^{62, 63} . Likewise, histones can activate the intrinsic pathway through an FXII-dependent mechanism, and histone-DNA complexes significantly contribute to elevated FXII in patients with overt DIC ¹⁰⁴ . Indirectly, histone-induced release of platelet polyphosphate can stimulate factor XI auto-activation as well as accelerate its thrombin-mediated activation ¹⁰⁵ .

Impaired fibrinolysis. Impaired or excessive fibrinolysis is an important aspect of sepsis- and trauma-induced DIC, respectively ^{64 65, 106} . All studies investigating the effects of histones, cfDNA, and NETs on the fibrinolytic system have consistently shown an overwhelmingly anti-fibrinolytic effect ^{66, 68, 107} . This effect is mediated by enhanced clot resistance to fibrinolysis by plasmin and downregulation of plasminogen activation by tPA ^{66, 68, 107} . As such, it appears that the effects of histones and NETs on the fibrinolytic system are relevant for sepsis-induced DIC but may not directly account for the hyper-fibrinolytic phenotype in trauma-associated DIC, although high histone levels in such patients may increase tPA release through significant endothelial stimulation and damage ⁶⁹ .

The development of multiple organ injury further augments thrombin generation and dysfunction in DIC. Microvascular thrombosis can be triggered by the factors discussed above in leading to organ ischemia and failure. There is increasing understanding of the role mediated by circulating histones in particular. In addition to causing direct injury to endothelial and other hematopoietic cells ^{9, 10, 52, 99} , histones have been shown to mediate distant organ injury and dysfunction. In mice models of trauma ¹⁰ and sepsis ¹⁵ , histones are major mediators of lung and cardiac injury and dysfunction, respectively. The clinical relevance of these findings has also been demonstrated in cohorts of critically ill patients with trauma ¹⁰ and sepsis ¹⁵ . The evidence for the distant organ-damaging properties of histones in trauma comes from the consequent development of acute lung injury after significant non-thoracic trauma. Translational relevance is supported by the increased development of acute lung injury in patients with severe non-thoracic trauma with high histone levels. Similarly, sepsis patients without pre-existing cardiac disease were significantly more likely to develop new-onset cardiac arrhythmia (nine-fold increase) and left ventricular dysfunction (two-fold increase) if they had high histone levels ¹⁵ . Equally, circulating histones can mediate renal ¹⁰⁸ , liver ^{61, 109} , and brain ¹⁰⁹ injury. Notably, the incubation of plasma or serum from critically ill patients (with sepsis, trauma, or pancreatitis) with cultured endothelial cells or cardiomyocytes induces cell death, which can be prevented in the presence of an antibody to histones ^{10, 15} .

Cellular damage is associated with microparticle formation and release ^{70, 71} . Their pro-coagulant properties include TF expression ^{43, 78, 79} , as discussed above. Circulating microparticles from damaged or activated hematopoietic cells also have exposed phosphatidylserine and these surfaces provide attachment sites for coagulation factors, which contribute to thrombotic complications in inflammatory disorders ⁷² . Significantly high levels of microparticles from activated endothelial cells and neutrophils were recently demonstrated in septic shock-induced DIC patients in whom elevated levels of NET surrogate markers (for example, nucleosomes and circulating DNA-MPO complexes) were evident ⁷³ . These microparticles may have synergistic pro-coagulant effects with NETs ⁷² and prime neutrophils to undergo NETosis by facilitating a pro-inflammatory environment, including the release of pro-inflammatory cytokines ^{74, 110} .

These histone-induced cytotoxic effects not only are a manifestation of micro- and macro-vascular thrombosis due to the pro-coagulant effects described above but also are from direct cytotoxicity mediated by histone binding to cellular membranes with consequent pore formation, calcium influx, and overload ^{10, 11, 52, 99, 111} . Fattahi et al. have demonstrated that after histone infusion into mice, histones localize (in order of concentration) in the lungs, spleen, kidneys, plasma, liver, heart, and brain ¹¹² . As histones unravel from DNA binding as part of nucleosomes, their cytotoxicity becomes apparent because of their ability to bind cell membrane phospholipids. However, in intact nucleosomes, where histone binding sites are covered by DNA, no cytotoxicity could be elicited ⁸⁶ . Collectively, these data suggest that circulating histones in patients with sepsis- or trauma-associated DIC are major mediators of distant organ injury and adverse clinical outcome.