Previous research has indicated that coagulation disturbances exhibit dynamic changes throughout the progression of burn injuries7. Early alterations in the coagulation system may be less discernible in patients with smaller burn areas. However, in patients with extensive burns, these changes can manifest within 24 hours of injury. The precise pathophysiology underlying early coagulation changes in severe burn patients remains uncertain, with endothelial injury and tissue hypoperfusion currently the main thought drivers8. Extensive vascular endothelial damage and capillary leakage lead to a self-protective activation of coagulation pathways within the body. Damage-associated molecular patterns (DAMPs), molecular fragments generated in stressed or damaged tissues following burn injuries, are detected by pattern recognition receptors on the surfaces of a wide array of cells throughout the body9. The binding of DAMPs not only induces TF expression and activates external coagulation pathways but also triggers complement cross-linking with the coagulation system10. Within the context of systemic multisystem crosstalk, coagulation exhibits significant heterogeneity.
Due to the limited specificity, we proceeded to identify DECRGs with potential effects on survival outcomes at the transcriptional level. These 28 DECRGs encompass regulators and proteins associated with diverse coagulation pathways, encompassing the complement system, hemostatic processes, and fibrinolysis. GO analysis of the six DECRGs identified as survival-related through univariate Cox regression unveiled a predominant involvement of neutrophils and their associated functional components. In the setting of widespread activation of coagulation and immunoinflammatory systems following burn injuries in vivo, DAMPs induced early in the course of burn injuries trigger the expression of TF on the surface of innate immune cells and activate external coagulation pathways11. Neutrophils, as pivotal components of the innate immune system, serve as essential mediators and play a significant role in fibrin formation during the early post-burn period12. In a laser-induced vascular endothelial injury model, neutrophils are the initial cells to accumulate within the vessel wall, interact with and aggregate around damaged endothelial cells, and contribute to thrombus formation13. Importantly, DAMPs also stimulate neutrophil degranulation, leading to the release of serine proteases (such as elastase and histone G) and the formation of neutrophil extracellular traps14. Previous research has primarily associated the impact of neutrophil serine proteases on the coagulation process with their role in promoting the inactivation of TFPI and fibrin production12. Early activation of neutrophils contributes to hemostasis and local inflammatory responses, but excessive dysregulation can result in coagulation depletion and a systemic immune-inflammatory response that is more likely to lead to adverse outcomes.
The expression of genes associated with fatal outcomes, namely P2RX1 and CYP4F2, seems to contribute to the process of neutrophil hyperactivation. P2X1 receptors, which are ATP-gated ion channels, are expressed on both platelets and neutrophils15,16. ATP ligands increase in the extracellular space of the endothelium following tissue injury. This channel exhibits high permeability to Ca2+, with approximately 10% of the action potential mediated by these ions17. In platelets, P2X1 receptors mediates the activation of ERK2 phosphorylation via increased intracellular Ca2+, thereby enhancing the aggregation response to thrombin18. Additionally, the accumulation and pro-thrombotic capacity of neutrophils rely on the P2X1 receptor. Mice lacking the P2X1 receptor exhibited a substantial reduction in neutrophil chemotaxis and fibronectin production at the site of endothelial injury when compared to wild mice15. CYP4F2 serves as a vitamin K1 oxidase and an ω-hydroxylase of menaquinone 4 (MK-4)19,20. Moreover, genetic polymorphisms in the CYP4F2 gene impact plasma concentrations of vitamin K1 and MK-4. Vitamin K, functioning as a cofactor for gamma-glutamyl carboxylase, plays a crucial role in the hepatic metabolism of proteins C and S, along with the synthesis of coagulation factors II, VII, IX, and X21. Furthermore, genetic polymorphisms in CYP4F2 are linked to the necessary dosage of warfarin22.
Following MR analysis, it was observed that plasma TF levels decreased after exposure to burns, while there was an increase in the expression of negatively regulated factors such as TFPI, protein S, and DAF. TFPI plays a pivotal role as a major inhibitor of the FVIIa-TF complex within the exogenous coagulation pathway, and protein S functions as an inhibitory cofactor that enhances the inhibitory capacity of TFPI1. DAF serves as a negative regulator within the complement cascade23. In summary, following burn injuries, genetic variation perspectives indicate a somewhat heightened activation of negative regulatory mechanisms. This aligns with findings from previous studies focusing on early trauma24; however, further examination is required within the context of burn patients. Given the dynamic nature of burn injuries, the insights we have presented may necessitate additional validation through rigorous animal or clinical trials.
It has been proposed that early changes in severe burns involve the activation of coagulation pathways, a decrease in natural anticoagulants (protein C and protein S), and the initiation of fibrinolysis1. Nonetheless, some scholars contend that coagulation experiences heightened activation, leading to microvascular thrombosis subsequent to coagulation dysregulation caused by consumptive coagulation disorders25. This results in a state of hypocoagulability and increased fibrinolysis. In our study, we observed prolonged PT and APTT in the death group, supporting the notion that early hypocoagulation may hold predictive value for survival outcomes.
Importantly, early fibrinolytic activation has been consistently observed in the majority of studies, and it represents a significant factor contributing to mortality in trauma patients, including those with burns26–28. This phenomenon may be attributed to the release of substantial levels of free tissue-type fibrinogen activator from damaged endothelial cells, ultimately resulting in an upregulation of fibrinogenesis29. In our multicenter population-based study, D-dimer, a marker reflecting activation of fibrinolysis, was significantly elevated in the death group and was an independent predictor of survival outcome. Previous investigations have demonstrated that D-dimer levels are elevated upon admission in burn patients25. Moreover, early elevation in D-dimer levels in trauma patients is believed to correlate not only with the severity of injury but also to predict multiple adverse outcomes, including hemorrhage, disseminated intravascular coagulation (DIC), and death30. In severely traumatized patients, Hayakawa et al identified an optimal cutoff value of 38 mg/L for 24-hour D-dimer levels associated with mortality outcomes using receiver operating characteristic (ROC) analysis28. In our study, we observed an association between D-dimer levels exceeding 1.418 mg/L and mortality outcomes, with a corresponding increase in the risk of death as D-dimer levels rose. Given that endothelial injury, tissue hypoperfusion, and systemic inflammation frequently co-occur in severe burns, a mild fibrinolytic status may serve as a predictive marker for adverse outcomes.
Besides D-dimer, serum calcium levels below 2.03 mmol/L were also linked to an elevated risk of mortality. As a pivotal element of P2X1 receptors in the coagulation pathway, excessive depletion of blood calcium independently contributes to mortality risk. In addition, neutrophil activation after injury leads to increased intracellular calcium mobilization31. While previous studies have observed a decrease in serum calcium levels following trauma, they did not identify an association with adverse outcomes32. Likewise, in our study, we initially did not observe any disparity in calcium levels between the groups that survived and those that did not. Furthermore, no statistical significance was evident in the univariate Cox analysis. However, positive findings surfaced following variable screening. This may warrant further consideration of the interaction of serum calcium with other factors, which is also more reflective of the real-world impact of serum calcium levels.
When combined with transcript levels and clinical characteristics, it was observed that patients in the death group exhibited elevated levels of both fibrinolysis and thrombosis. Furthermore, this phenomenon is associated with the early activation of neutrophils. Based on this observation, we formulated a hypothesis that adverse outcomes may be linked to early secondary coagulation disorders arising from hyperactivation of the coagulation system. Excessive coagulation consumption could potentially manifest as the depletion of various factors. However, it is important to note that the present study did not comprehensively and meticulously collect indicators such as coagulation factors and fibrinolysis from a large number of patients. This is an aspect that we intend to address more rigorously in our future investigations. Given the intricate interplay with various immune-inflammatory systems, it appears challenging to intervene directly in the coagulation pathway solely based on alterations in patient coagulation screening indicators. Furthermore, it is worth noting that extensive assessments of thrombosis and hemostasis have not yet been undertaken at the clinical level. Utilizing four valuable predictive indicators allows for the identification of individuals at risk, thereby enabling the implementation of additional interventions and tailored care for this population.
There are also some limitations in this paper. Firstly, we encountered limitations in obtaining GWAS data for all DECRGs candidates, and our MR analysis was consequently restricted to the available metrics. Secondly, our study did not involve the analysis of variations in mRNA and protein expression levels of the core genes through cellular experiments. The current lack of open studies of burned patients still exists, and thus genetic diversity and potential differences between the different population cohorts in our study are still not well avoided. However, we provide a method of analysis that may be flawed with the current data, but also provides direction for future research. Lastly, we could only gather information regarding the patients' prior medical histories, particularly concerning coagulation deficiencies and pre-hospitalization anticoagulant medications, through verbal inquiries.