ENDOTHELIOPATHY OF TRAUMA
Trauma is the leading cause of death in individuals up to age 46 both in the United States and worldwide, with hemorrhage remaining the number one cause of early trauma deaths in both military and civilian settings (1, 2). Modern resuscitation strategies have significantly reduced early mortality and are based on the experience from combat casualties in recent conflicts. In 2007, Borgman et al. (3) first reported that the ratio of transfused plasma to red blood cells influenced survival. Subsequent civilian studies have confirmed their results and are the foundation for hemostatic-based resuscitation. This strategy incorporates the early use of blood component therapy approximating a 1:1:1 ratio of plasma, platelets, and red blood cells into the initial management of injured patients in hemorrhagic shock (4–8).
Severely injured patients can present coagulopathic. This distinct form of traumatic-induced coagulopathy (TIC) occurs early and is associated with increased transfusions, complications, and mortality (9). The pathophysiology of TIC is attributed to activation of coagulation, hyperfibrinolysis, and consumption coagulopathy. Some argue that the pathophysiological mechanisms are similar to the characteristics of disseminated intravascular coagulation with the fibrinolytic phenotype (10, 11). The decrease in mortality from plasma-based resuscitative strategies appears to extend beyond its ability to correct trauma-induced coagulopathy and provide hemorrhage control and is hypothesized to involve additional protective effects to the post-shock dysfunctional endothelium. Survivors of hemorrhagic shock (HS) demonstrate an “endotheliopathy of trauma (EoT)” which is a systemic response resulting in disturbances of coagulation, inflammation, and endothelial barrier integrity (12). Clinically, this is manifested as a pro-inflammatory state with vascular leak and tissue edema, which contributes to organ dysfunction and late morbidity and deaths. Therefore, restoring the endothelium and barrier integrity is integral to further reducing hemorrhage-related morbidity and mortality.
The endothelial glycocalyx is a complex network of soluble components that projects from the cell surface of the endothelium into the vessel lumen (13–16). This network is a thick (about 0.2–3.0 μm in vivo), negatively charged carbohydrate-rich layer lining the vascular endothelium (17, 18). It is formed by cell membrane-bound sulfated proteoglycans, consisting of a core protein (e.g., transmembrane syndecan, membrane-bound glypican, or matrix-associated perlecan) with glycosaminoglycan side chains (e.g., heparan sulfate, hyaluronic acid, and chondroitin sulfate), and cell membrane glycoproteins attached with sialoproteins (13–16). The syndecan family is comprised of four members (syndecan-1 through -4) and is the major source of cell surface heparan sulfate (13, 14, 19, 20). Cell surface syndecans function primarily as receptors for heparan sulfate-binding molecules or as modulators when their extracellular domain is released from the cell surface by ectodomain shedding (13, 14, 20–23). Syndecan-1 is found on endothelial cells and believed to account for endothelial integrity (18, 24–28). Under physiologic conditions, positively charged soluble components (such as plasma proteins, enzymes, growth factors, cytokines, amino acids, and cations) and water are trapped in the glycocalyx forming an extended endothelial surface layer (16, 17, 22, 23). The glycocalyx serves as an effective barrier to prevent leukocyte-endothelial cell and platelet-endothelial cell adhesion by providing steric hindrance between receptor and ligand, thereby blocking adhesion to the underlying endothelium (18, 19–21, 24, 25). Although much of the literature assumes that shed syndecan-1 is a marker of injury to the endothelial glycocalyx, it also found on epithelial cells including in the intestine and lung (29). Additionally, both platelets and red blood cells contain a glycocalyx, though much less is understood regarding their functions (30–33).
Injury to the glycocalyx and shedding of syndecan-1
Shedding of endothelial glycocalyx components has been shown to occur in response to a variety of stressors (34–37). As recently reviewed by Becker et al. (27), loss of the glycocalyx is mediated by “sheddases,” such as matrix metalloproteases, heparanases, hyaluronidases, and proteases and is responsible for endothelial inflammatory changes and vascular hyperpermeability. Shedding of the syndecan-1 ectodomain has been described in a variety of shock states that are summarized in Table 1(37–49). Ectodomains can function as danger-associated pattern molecules (DAMPs) following trauma (47–49). They are associated with enhanced shock, inflammation, and endothelial damage (31, 50–52) and independently predict mortality in injured patients (35–37). Human studies examining syndecan-1 shedding and outcomes after hemorrhagic shock are reviewed in Table 2(34–36, 53–55).
Mechanism of syndecan-1 inhibition by hemorrhagic shock
The precise mechanism by which hemorrhagic shock affects syndecan-1 remains unclear. Although shed syndecan-1 is considered a systemic biomarker for endothelial injury much less is known about syndecan-1 expression and its effect on the endothelium. We have shown in a rodent model that pulmonary syndecan-1 mRNA is significantly reduced following hemorrhagic shock (13), suggesting that microRNAs (miRNAs) may be important. Preliminary studies from our laboratory have demonstrated that miR-19b targets pulmonary endothelial syndecan-1 and is detrimental after hemorrhagic shock, suggesting it contributes to the endotheliopathy of trauma (56, 57). Studies are in progress to determine if resuscitation can inhibit this miRNA.
PLASMA-BASED RESUSCITATION TO MODULATE THE EOT
Glycocalyx and syndecan-1 restitution
Crystalloids and colloids
We and others have shown that crystalloids, either saline or lactated Ringers (LR), fail to restore glycocalyx functional integrity following hemorrhagic shock (13, 58). In a rat model of fixed volume hemorrhage, Torres et al. (59) demonstrated that Hextend, similar to LR, decreased glycocalyx thickness and increased plasma syndecan-1 levels compared to levels in sham and fresh frozen plasma (FFP) treated animals. This same group then demonstrated that endothelial glycocalyx thickness was only partially restored by albumin, but was completely restored by FFP (60). However, shed syndecan-1 levels were comparably reduced by albumin and FFP. Glycocalyx thickness (negatively), and microvascular permeability (positively), were correlated with plasma syndecan-1 and heparin sulfate levels. Overall, resuscitation with crystalloid solutions (saline or LR) caused glycocalyx damage and worsened permeability. Resuscitation with fresh whole blood or plasma evoked protection, and albumin had an intermediate effect (58–61). Nelson et al. (62) using a rat model of volume-controlled hemorrhage and resuscitation with either FFP, albumin, or LR found that both FFP and albumin restored plasma volume, whereas LR did not. Systemic heparan sulfate levels were lower in the FFP and albumin groups but syndecan-1 levels did not differ.
To begin to investigate the effect of plasma-based resuscitation on the glycocalyx, our laboratory initially employed a pressure-controlled model of hemorrhagic shock followed by resuscitation with FFP or LR (13). Pulmonary syndecan-1 mRNA expression was higher in animals resuscitated with plasma in comparison with shock alone or LR and positively correlated with the intensity of cell surface syndecan-1 immunostaining and lung histopathology. To specifically examine the effect of plasma on the endothelium, we infused small bowel mesentery with a lanthanum-based solution then visualized the endothelial glycocalyx in post capillary venules by electron microscopy (13, 14, 34). We demonstrated degradation of the glycocalyx after hemorrhagic shock, which was partially restored at 3 h by plasma but not LR (13, 14, 34). Additionally, a clinically relevant effect of plasma was suggested by the observation that plasma resuscitation required significantly less volume to maintain the mean arterial pressure (MAP) (13, 14, 34).
We verified our findings in a mouse model of volume-controlled hemorrhagic shock (28). Hemorrhage was associated with systemic shedding of syndecan-1, which correlated with decreased pulmonary syndecan-1 and increased pulmonary vascular hyperpermeability and inflammation. FFP resuscitation, compared with LR resuscitation, abrogated these injurious effects by reducing endothelial cell hyperpermeability and inflammation and restoring pulmonary syndecan-1 expression. Modulation of pulmonary syndecan-1 expression may therefore mechanistically be attributed to the beneficial effects FFP. Additionally, protection by plasma via syndecan-1 was not limited to the lungs. Gut injury and inflammation were significantly increased by hemorrhage compared with shams (63). Resuscitation with LR decreased both injury and inflammation, but these effects were further lessened by FFP.
To verify the role of syndecan-1 in plasma's protection we used siRNA to silence syndecan-1 in vitro and found that while FFP enhanced pulmonary endothelial syndecan-1 expression in a time- and dose-dependent manner, the loss of syndecan-1 expression in pulmonary endothelial cells actually increased permeability and stress fiber formation after exposure to FFP (24). Loss of syndecan-1 in vivo using syndecan-1 null mice led to equivalency between LR and FFP in restoring pulmonary injury, inflammation, and permeability after shock. Similarly, syndecan-1 null mice displayed worsened gut injury and inflammation after HS compared with wild-type mice; and LR and FFP equivalently inhibited injury and inflammation in null mice, again demonstrating the link between protection by plasma and syndecan-1.
These observations were confirmed by the work of Torres et al. (58, 59). Using a 40% blood volume hemorrhage model, the authors resuscitated rats using LR. The authors confirmed that the endothelial glycocalyx was significantly damaged by hemorrhagic shock and restored only with FFP, as assessed by circulating syndecan-1 levels and glycocalyx thickness. Second, a clinically beneficial effect of plasma-based resuscitation was indicated by the fact that FFP corrected metabolic acidosis significantly better than LR and hextend, as assessed by pH, base excess, and lactate. This was associated with an improved microcirculation and a lesser degree of hemodilution by FFP compared to LR and hextend. This latter point was also observed in a recent publication of Nelson et al. (62) who demonstrated that resuscitation with FFP resulted in circulating volume expansion equaling the volume of blood loss, while circulating volume expansion by LR was less effective.
Risks and challenges with the use of plasma
The use of plasma is not without risks and challenges. Blood components are still subject to contamination from a variety of pathogens such as viruses, bacteria, and parasites, including emerging pathogens such as the Zika virus. To reduce the risk of transfusion-transmitted infectious disease (TTID), pathogen reduction technologies have been developed. There are two technologies licensed in the United States for plasma (amotosalen-UVA and solvent detergent) and one for platelet concentrates (amotosalen-UVA). A third is still under development; riboflavin (vitamin B2-UV) (64). The INTERCEPT Blood System (Cerus Corporation, Concord, Calif) uses amotosalen (a psoralen) and UVA light to target nucleic acid forming adducts to inhibit replication and inactivation of blood borne pathogens and leukocytes with minimal generation of reactive oxygen species (65). It is intended for use with whole blood derived or apheresis plasma and platelets to reduce the risk of TTID. Among riboflavin-based systems that target pathogen nucleic acids, the Mirasol Pathogen Reduction Technology (Mirasol PRT; CaridianBCT, Lakewood, Colo) uses riboflavin and UV light to introduce irreparable lesions into nucleic acids by reactive oxygen species thereby inhibiting pathogen and white blood cell replication and reducing the load of infectious pathogens (66). There is also commercially available solvent/detergent-treated pooled plasma, OctaplasLG (Octapharma AG, Lachen, Switzerland) that has recently been shown to reduce glycocalyx and endothelial injury compared with FFP in a pilot clinical trial of patients undergoing emergency surgery for thoracic aorta dissection (67).
Timing of plasma administration
Naumann et al. (68) demonstrated that the endotheliopathy of trauma occurred within 5 to 8 min of injury. This finding suggests that the early use of plasma after injury may be beneficial. Indeed, Diebel et al. (69) demonstrated in their in-vitro biomimetic model of endothelial vascular barrier dysfunction following shock that the early use of plasma restored the endothelial glyocalyx and reduced syndecan-1 shedding, while the late use was comparable to shock alone. This supports the concept that the early transfusion of plasma is important to outcomes after hemorrhage. Two prospective randomized trials have in fact examined the effect of plasma transfusion on mortality in the prehospital setting. A study by Sperry et al. (70) demonstrated significantly lower 30-day mortality in injured patients at risk for hemorrhagic shock who received prehospital plasma compared with standard of care therapy with crystalloids. However, a similar study, but with rapid ground rescue, showed no survival benefit (71).
Alternative plasma preparations
In large urban civilian trauma centers, thawed plasma with 5-day shelf life is now frequently available immediately upon patient arrival in emergency rooms. However, in military and remote or austere civilian settings, the rapid availability of plasma is limited (72). Fresh frozen plasma requires approximately 30 min to thaw and then must be stored at 4°C and used within 5 days. These logistical challenges have led to intense developments of dried plasma products, which can be lyophilized or spray dried. To date, there are no dried plasma products available that are FDA approved, though a number are in development. A French product, FLYP, is currently being used by the United States Special Operations Command (SOCOM) under an expanded use authorization. This product is lyophilized and pathogen reduced by amotosalen-UVA treatment (73). A retrospective study from France recently demonstrated that use of this product was associated with earlier and a more rapid transfusion ratio (plasma: RBC) improvement compared with FFP (74). We and others have been interested in the endothelial protective effects of dried plasma products. Both in vitro and in vivo studies have thus far confirmed similar protective effects to FFP (75–78). Pati et al. (79) evaluated the prothrombin complex factor concentrate, Kcentra (CSL Behring), in a mouse model of hemorrhagic shock-induced pulmonary vascular leak. Interestingly, Kcentra and FFP; but not albumin, inhibited vascular permeability in their model. Kcentra contains nearly 100 proteins, including albumin, prothrombin, factors VII, IX, and X, proteins C and S, and antithrombin III. As in the case of FFP, the precise proteins in Kcentra responsible for the observed effects remain uncertain.
FIBRINOGEN RESUSCITATION TO MODULATE THE EOT
Fibrinogen stabilization of syndecan-1
Fibrinogen is a 340-kDa homodimeric glycoprotein comprised of two alpha, two beta, and two gamma polypeptide chains linked by disulfide bridges (80, 81). The 15–42 sequence of the beta chain of fibrinogen has been shown to mediate platelet and endothelial cell spreading, fibroblast proliferation, and capillary tube formation (82–84). Interaction between this sequence and the endothelial cell receptor, vascular endothelial (VE)-cadherin, has been shown to promote capillary tube formation and angiogenesis (85). We recently investigated the contribution of fibrinogen to the endothelial protection afforded by plasma. When pulmonary endothelial cells were incubated in plasma or fibrinogen-deficient plasma, fibrinogen depleted plasma-treated cells demonstrated comparable levels of permeability to LR-treated cells (25). This finding was associated with both an increase in stress fiber formation and a decrease in pulmonary syndecan-1 immunostaining. In addition, we confirmed a comparable decrease in permeability in vitro with FFP and fibrinogen. This led us to further explore the mechanism by which fibrinogen may interact with syndecan-1 on the cell surface. Using colocalization and co-immunoprecipitation experiments, we demonstrated that fibrinogen associates with syndecan-1. We hypothesize that this occurs between the beta chain of fibrinogen that has known heparan sulfate binding sites and the heparan sulfate moieties attached to syndecan-1 (83, 85). Potential mechanisms by which fibrinogen could stabilize cell surface syndecan-1 and provide endothelial protection may include modulation of sheddases or regulation of gene expression. Binding of fibrinogen to syndecan-1 may shield the cleavage sites of syndecan-1 by its known sheddases, thus stabilizing syndecan-1 on the cell surface. The second mechanism is that fibrinogen binding may activate an intracellular signaling pathway which positively feeds back to increase syndecan-1 gene expression. Our preliminary studies suggest that the PAK1 pathway may be important (86). These results demonstrate an endothelial barrier protective effect for fibrinogen, which may support the early use of fibrinogen as a therapeutic intervention for hemorrhagic shock. Indeed, other studies also demonstrated that fibrinogen-derived peptide B-beta (15–42) preserves endothelial barrier function in shock (87, 88). Figure 1 illustrates a proposed pathway for protection of the endothelium by fibrinogen following hemorrhagic shock.
Fibrinogen for hemostasis
Fibrinogen plays a key role in hemostasis by acting as an endogenous substrate for fibrin formation, promoting clot formation and platelet aggregation by binding platelet glycoprotein IIb/IIIa receptors. Hypofibrinogenemia is known to be associated with worse outcomes after trauma (89, 90); and the degree of hypofibrinogenemia is correlated with increased injury severity (91). There are an increasing number of reports describing the limitations of FFP for treating ongoing severe hypofibrinogenemia in critically injured patients (92, 93). In the RETIC trial, a prospective randomized pilot trial in Austria, patients received either fibrinogen concentrate and/or prothrombin concentrate or FFP once low fibrinogen was confirmed in bleeding patients. The trial was stopped early for futility due to possible harm in patients receiving plasma (94).
As fibrinogen is the first coagulation factor to fall below a critical value during massive bleeding, it seems plausible that it should be the first protein to be given to patients with trauma. A systematic review of 91 studies evaluating the outcome of FFP or fibrinogen concentrate administration to patients in a perioperative or massive trauma setting concluded that the use of fibrinogen concentrate was associated with improved outcomes, whereas the evidence for an FFP effect was inconclusive (93). In another retrospective analysis of trauma patients comparing FFP administration to the administration of fibrinogen concentrate, the authors found that red blood cell (RBC) transfusion was reduced in 29% of patients in the fibrinogen group (n = 80) compared with only 3% in the FFP group (n = 601) (95). A meta-analysis of six randomized controlled trials involving 248 patients undergoing elective surgery found that the administration of fibrinogen concentrate led to reduced RBC transfusion without an increase in thrombotic events or mortality (96). Thus, fibrinogen concentrate appears to be an effective and safe alternative to FFP in reducing the need for allogeneic blood transfusion in bleeding patients.
There are three randomized controlled trials in progress or recently completed investigating early fibrinogen concentrate replacement in injured patients in hemorrhagic shock. The early fibrinogen concentrate therapy for major hemorrhage in trauma (E-FIT1) trial in the United Kingdom enrolled injured patients requiring activation of a major hemorrhage protocol (97). Patients were randomized to fibrinogen concentrate or placebo. The primary outcome was the feasibility of fibrinogen concentrate administration early after patient arrival, defined as > 90% of patients receiving product within 45 min. Despite the use of a lyophilized fibrinogen concentrate with no thaw time, only 69% of patients received the study intervention (fibrinogen concentrate or placebo) within the designated period. This was primarily attributed to the study design with time required for administration of the blinded placebo in addition to the known time of reconstitution of fibrinogen concentrate. In this small study of 39 patients, there were no differences in mortality or transfusion requirements. The fibrinogen in the initial resuscitation of severe trauma (FiiRST) trial was a similar pilot feasibility study of patients randomized to fibrinogen concentrate or placebo to determine the proportion of patients receiving the intervention within 1 h (98). In this study, 96% of patients received the intervention within 1 h. Finally, the PROOF-ith (Pilot Randomized Trial of Fibrinogen in Trauma Haemorrhage, NCT02344069) study is a single-center study also randomizing patients to a pre-emptive dose of fibrinogen concentrate or placebo to determine if fibrinogen concentrate as first-line therapy can increase clot strength as evaluated by thromboelastography (99).
In the United States and United Kingdom, cryoprecipitate is used to replace fibrinogen as part of massive transfusion protocols to control bleeding after trauma. Cryoprecipitate is prepared from plasma and contains fibrinogen, von Willebrand factor, factor VIII, factor XIII and fibronectin. In the United States, at best, massive transfusion protocols include fibrinogen containing products such as cryoprecipitate only late in the protocol and/or in response to low plasma fibrinogen levels. The mean fibrinogen concentration in plasma is approximately 2 g/L and large volumes of plasma (approximately 30 mL/kg) are required to adequately supplement fibrinogen, thus additional sources of fibrinogen such as cryoprecipitate are frequently required.
The CRYOSTAT I study, recently published by Curry et al. (100), demonstrated that the early administration of cryoprecipitate as part of a major hemorrhage protocol is feasible in trauma patients. They observed that 85% of patients randomized to the cryoprecipitate arm received cryoprecipitate within 90 min and the median time to transfusion was 60 min. This is still longer than in the EFIT-1 trial using fibrinogen concentrate. Currently, the CRYOSTAT2 trial is enrolling patients in the United Kingdom. This is a prospective randomized multicenter study in injured patients in shock to receive early empiric cryoprecipitate as part of a massive hemorrhage protocol. Mortality at 30 days is the primary endpoint. Finally, in Australia there is an ongoing exploratory multicenter randomized study comparing fibrinogen concentrate to cryoprecipitate in bleeding patients, the FEISTY (Fibrinogen Early In Severe Trauma study, NCT02745041) Trial (101). Importantly, in this study, thromboelastography will guide dosing in both groups with the primary endpoint being time to fibrinogen supplementation.
We were interested in exploring whether cryoprecipitate would have endothelial protective effects similar to FFP. We therefore performed preliminary experiments to investigate the effect of cryoprecipitate on the endothelium. As there is now a pathogen-reduced cryoprecipitate, we included both conventional and pathogen-reduced cryoprecipitate. As shown in Figures 2 and 3, both conventional and pathogen-reduced cryoprecipitate had comparable endothelial protective effects.
Cryoprecipitate-reduced plasma is the residual supernatant from the process used to make cryoprecipitated antihemophilic factor (AHF) (102). The FDA has indicated in a guideline published in 2017 that cryoprecipitated AHF can be prepared from pathogen-reduced plasma frozen within 8 h of collection to provide pathogen-reduced derivative components (pathogen-reduced cryoprecipitate (PCR) and pathogen-reduced plasma cryoprecipitate reduced (PRPCR)). The effects of PRPCR on endothelial cell function are largely unknown, but the retention of some fibrinogen and data from prior studies suggests that it may be effective to maintain endothelial cell function (25). Data shown in Figure 4 suggests that it indeed maintains endothelial protective effects comparable to FFP. PRPCR retains antithrombotic protein activities (i.e., normal levels of antithrombin III, protein C, and protein S), while minimizing pro-thrombotic proteins (reduced factor VIII, fibrinogen, and von Willebrand factor) indicating that the risk of excessive prothrombotic risk should be minimal (102). This may in fact be an ideal product to treat endothelial dysfunction in nonbleeding conditions such as sepsis and burns. There are recent preclinical data supporting the use of FFP for both sepsis and burns (103, 104).
Advances in hemostatic resuscitation have not only improved outcomes in injured patients with hemorrhagic shock but have been accompanied by a more in-depth knowledge of how hemostatic blood products mitigate the endotheliopathy of trauma and repair the dysfunctional endothelium. As mechanisms of injury are better understood, research is focusing on a more “personalized” approach to repair the endothelium using not just plasma but newer alternatives such as cryoprecipitate and fibrinogen. Additionally, as plasma becomes safer and more available with pathogen reduction and freeze-fried technologies, the use of plasma and its byproducts may be expanded to treat nonhemorrhagic-related disease states similarly associated with a dysfunctional endothelium.
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