Trauma-induced coagulopathy (TIC) is present in up to 25% of severely injured patients upon hospital admission (1). The exact pathophysiology of TIC still remains unclear. However, disturbance of endothelial homeostasis is the key feature of several mechanisms potentially linked to the pathogenesis of TIC (2, 3). Early correction of intravascular blood loss and aggressive hemostatic treatment is the first-line strategy to fight circulatory failure and to prevent and treat TIC (4). The current standards for hemostatic therapies during trauma resuscitation involve transfusion with high ratios of plasma and platelet concentrates, purified coagulation factor concentrates (CFC), and antifibrinolytic agents such as tranexamic acid (5–7). Many European trauma centers favor the concept of goal-directed coagulation therapy. Integral to this approach is the individualized use of fibrinogen concentrates and prothrombin complex concentrates, which resulted in a decline of fresh frozen plasma (FFP)-based resuscitation (8). Apart from its hemostatic potential, preclinical studies have linked the beneficial effects of plasma to its ability to mitigate inflammation and protect the endothelium, thereby actively restoring the “endotheliopathy of trauma” (9–11). Following traumatic injury, the integrity of the vascular endothelium and its luminal layer, the endothelial glycocalyx, are particularly disrupted—a condition associated with vascular hyperpermeability (12), poor flow dynamics (13), and increased mortality (14). Excessive symphato-adrenal activation is proposed to be an essential driver of endothelial cell and glycocalyx damage (15). Concomitant inflammation and coagulopathy are suspected to be mediated by the breakdown of glycocalyx, given that resolution of inflammation is hampered by dysfunctional endothelium (16, 17). The disintegration of glycocalyx components such as syndecan-1 and heparan sulfate is termed shedding, a process that refers to the cleavage of the ectodomain from the membrane spanning syndecan-1 by endothelial matrix metalloproteinases (18). In experimental studies of hemorrhagic shock, restoration of the endothelial glycocalyx and recovery of microvascular flow have been attributed to resuscitation based on blood derived products (e.g., FFP) and colloids (9–11). In contrast, resuscitation with crystalloids showed to aggravate shock-induced endothelial glycocalyx damage, which has been assessed by increased levels of shed glycocalyx components, decreased endothelial glycocalyx thickness, and increased vascular permeability (9, 10). Work by Pati et al. (19) further demonstrated that compared with FFP, prothrombin-complex concentrate (PCC) exhibits similar effects on the vascular endothelium in both in vitro and in vivo shock models of vascular hyperpermeability. Additionally, fibrinogen concentrate (FC) is a key element of hemostatic therapy (8, 20). Apart from its dominant role in hemostasis, fibrin(ogen) mediates the inflammatory cascade by recruiting leukocytes to the site of injury and promoting transendothelial migration (21).
The objective of this study was to evaluate the role of FC and PCC on endothelial injury, glycocalyx degradation, and sympatho-adrenal activation in shock-induced endothelial dysfunction studied in a rat model. We hypothesized that both FC and PCC, in particular in combination with colloids, exert a regenerative effect on the damaged endothelial glycocalyx.
MATERIAL AND METHODS
Male Sprague-Dawley rats (n = 109) weighing 440 to 480 g (Charles River, Germany) were used in all experiments. Rats were kept under a regular 12h-light/12h-dark cycle in standard housing condition with access to food and water ad libitum. Health status was monitored daily by veterinarians. Seven days before undergoing the experiment the animals arrived at the facility for the purpose of adaptation. Animal use was approved by the committee of the city of Vienna, Austria, (protocol number: MA58-605466/2015/12) to be carried out at Ludwig Boltzmann Institute for Experimental and Clinical Traumatology (Austria) in accordance with the guidelines of the NIH on the use of Laboratory Animals.
Anesthesia and instrumentation
Figure 1 illustrates the experimental timeline. Anesthesia and instrumentation were performed as described previously (22). Anesthesia was induced with oxygen-isoflurane composite (3%) for primary instrumentation, which was then switched to an i.v. ketamine (60 mg/kg/h, Pfizer, Vienna, Austria) and i.m. xylazine protocol (2.5 mg/kg, Bayer Healthcare, Wuppertal, Germany). Animals were breathing spontaneously throughout the procedure. A line was placed in the femoral artery which was used for hemodynamic monitoring, blood gas analysis, and blood removal.
Hemorrhagic shock model
We performed a pressure-controlled and blood gas-guided hemorrhagic shock as described previously (22). Following a midline laparotomy, rats were bled to a mean arterial pressure (MAP) of 30 to 35 mm Hg. The initial 10 mL of shed blood were drawn at a flow rate of 1 mL/min via a pump. MAP was kept at 30 to 35 mm Hg by manual blood removal using a syringe. In order to ensure a similar degree of shock in all animals, we used a predefined cutoff for base deficit (≥ 5.5 mmol/L and arterial lactate (≥ 2.2 mmol/L) prior to resuscitation. Provided that both endpoints were validated by intermittent blood gas analysis, shock was terminated accordingly (end-of-shock; EOS). In order to circumvent the differing individual response of each animal, the model is controlled by biological endpoints. The cut-off values in the present study are based on our previous findings, which demonstrated reproducible alterations of glycocalyx integrity (22). As indicated in Figure 2, a number of nine animals did not complete the study. Yet this had no effect on the statistical results by means of small sample size. Due to the nature of the established HS model, further removal of blood for additional analyses resulted in an immediate decompensation. Thus, eight animals were terminated at the end-of-shock in order to obtain sufficient amounts of blood for analyzing epinephrine, sVEGFR1, syndecan-1, and heparan sulfate. These data were exclusively used as reference values (indicated in Fig. 3Figs. 1 and 3, dashed line) and are excluded from further statistical comparisons. The total blood volume (EBV) of each animal was estimated using a formula described by Lee et al. (23). The amount of blood withdrawn during shock is expressed as percentage of the EBV.
Our resuscitation model consists of seven different groups:
- 1. Human Albumin 5% 15 mL/kg (CSL Behring, Marburg, Germany)
- 2. Rat Fresh Frozen Plasma 15 mL/kg (obtained from donor animals n = 16)
- 3. Lactated Ringer's 75 mL/kg (Fresenius Kabi, Graz, Austria)
- 4. Lactated Ringer's 75 mL/kg + Fibrinogen Complex Concentrate 70 mg/kg (Haemocompletan, CSL Behring, Marburg, Germany)
- 5. Lactated Ringer's 75 mL/kg + Prothrombin Complex Concentrate 25 IU/kg (Beriplex, CSL Behring, Marburg, Germany)
- 6. Human Albumin 5% 15 mL/kg +Fibrinogen Complex Concentrate 70 mg/kg (Haemocompletan, CSL Behring, Marburg, Germany)
- 7. Human Albumin 5% 15 mL/kg + Prothrombin Complex Concentrate 25 IU/kg (Beriplex, CSL Behring, Marburg, Germany)
At the end-of-shock, the animals in the CFC groups were initially resuscitated with a bolus injection of either FC (35 mg/kg/min) or PCC (12.5 IU/kg/min) to recapitulate an early aggressive hemostatic therapy in the emergency room (6, 24). In the CFC-free groups, a bolus of the substance assigned to a given group was administered in a volume equivalent to the amount of factor concentrates. Resuscitation was then maintained for 60 min with a continuous infusion of either the same fluid or the paired vehicle. After resuscitation, animals were monitored for another 60 min until the end-of-observation. Vehicle volumes for Ringer's lactate and colloids (e.g., albumin and FFP) differ in a ratio 5:1, due to the higher volume effect of colloids (25, 26). The volume effect of lactated Ringer's is approximately 20%, hence the chosen ratio aimed to generate an identical intravasal volume as in the colloid-based groups (25). Blood gas analysis was performed at baseline, at the end-of-shock, and at the end-of-observation.
Circulating markers of endothelial damage (sVEGFR1), glycocalyx shedding (syndecan-1, heparan sulfate), and sympatho-adrenal activation (epinephrine) were measured at baseline and at the end-of-observation.
Blood samples were collected in EDTA (EDTA-tube; 3.2%; Sarsted, Wedel, Germany) vials at baseline, at the end-of-shock, and at the end-of-observation, subsequently processed and stored at −80°C until analysis. Blood gas analysis was performed with an ABL 800 Flex System (Radiometer Medical A/S, Copenhagen, Denmark) using heparinized arterial blood, respectively.
Syndecan-1 was determined according to the manufacturer's instructions with a commercially available sandwich enzyme linked immunoassay (ELISA) (Cat. no. seb966ra, Cloud-Clone Corp, Houston, Tex) using an antibody specific to rat syndecan-1 proteoglycan. The lower limit of detection (LLD) was 1.56 ng/mL.
Plasma epinephrine was measured with a commercially available competitive ELISA (Cat. no. abx257158, Abbexa Ltd, UK) using an antibody specific to epinephrine in combination with a biotin-conjugated antibody for detection according to the manufacturer's instructions. The LLD was 31.25 pg/mL.
Vascular endothelial growth factor receptor 1
VEGFR1 was measured according to the manufacturer's instructions with a sandwich ELISA (Cat. no. abx255660, Abbexa Ltd, UK) using a combination of an antibody specific for VEGFR1 and a biotin-conjugated antibody for detection. The LLD was 0.156 ng/mL.
Heparan sulfate was determined with a commercial ELISA kit (UCL-E0623Ge, WuhanEIAab, China) according to the manufacturer's instructions. Given that pilot experiments revealed a considerable background effect in an undiluted EDTA plasma, protein was denatured at 60°C for 30 min, put immediately on ice and spun down at 2,500 × G for 15 min. 100 μL of the supernatant were then added per well and the procedure was carried out according to the kit's manual.
An operator blind to the different experimental groups performed each assay for all animals at once. All samples were analyzed with EDTA plasma thawed at room temperature. Syndecan-1 was measured with undiluted plasma, whereas plasma for the determination of epinephrine and sVEGFR1 was diluted 1:5. Heparan sulfate measurements were performed as described above.
A priori sample size calculation was performed using G-Power V220.127.116.11 based on results of a prior study analyzing changes of syndecan-1 after hemorrhage and reperfusion (10). Given an effect size of 0.55, α= 0.05, and a power of 0.95, a total sample size of 10 is required. All data were evaluated for normality using the D’Agostino Pearson test.
We did (Multivariate) Analyses of Variance ((M)ANOVA) for all between-group analyses (Shed blood and shock duration; MAP and heart rate; base deficit and arterial lactate; epinephrine; sVEGFR1; heparan sulfate; syndecan-1). For within group analyses (epinephrine; VEGFR1; heparan sulfate; syndecan-1) we did Repeated Measures ANOVAS. Furthermore, we analyzed differences between groups with a post hoc Tamhanes T2 test and checked for contrasts in between group analyses. We also conducted the non-parametric equivalents of these tests (Friedman test or Kruskal–Wallis test with corresponding post hoc analyses respectively).
A P value < 0.05 was considered statistically significant. Data are presented as mean±SD. Statistical analysis was performed with SPSS 24.0 and graphs were plotted with Prism version 5.01 (La Jolla, Calif).
Bleeding volume and shock duration
In general, the magnitude of the induced shock was similar to all rats; neither bleeding volume % nor shock duration demonstrated statistical difference among the groups. Table 1 depicts values for bleeding volume and shock duration parameters.
Hemodynamics and metabolic changes
Colloid-based resuscitation significantly increased MAP compared with all RL-based groups. In line with the hemodynamics after reperfusion, the group displayed higher MAP at end-of-observation compared with the crystalloid-based groups (all P < 0.05 in a MANOVA and the non-parametric equivalents). The type of fluid resuscitation had no consistent effect on heart rate at end-of-observation. Table 2 lists hemodynamic and metabolic data obtained at baseline, at the end-of-shock, and at the end-of-observation.
Base deficit and arterial lactate
As response to hemorrhage and ischemia, a strong increase in arterial lactate and base deficit were evident in all groups. Significant improvement of lactate levels was observed in all groups at end-of-observation. Base deficit at end-of-observation, was significantly lower in animals resuscitated with FFP (0.5 ± 1.2 mmol/L) compared with all other groups (all P < 0.05 in a MANOVA and the non-parametric equivalents) (Table 2).
Sympatho-adrenal activation and endothelial damage
Epinephrine strongly (796%) increased from baseline (Table 3) (pooled data) to end-of-shock in all groups. At end-of-observation, epinephrine concentration was significantly lower in animals resuscitated with HA+FC compared with rats resuscitated with RL (Fig. 3A). No other statistically relevant differences were recorded (in an ANOVA and the non-parametric equivalents).
Hemorrhagic shock was associated with a 50% increase of sVEGFR1 as seen in the evidence by the shock reference group. Changes from baseline to end-of-observation were significant in all groups (using a Repeated Measures ANOVA and the non-parametric equivalents). No consistent effect of any type of resuscitation fluids was observed. In general, sVEGFR1 levels for the HA ± CFC groups were lower in comparison with animals resuscitated with RL+ CFCs (HA 14.1 ± 1.7 ng/mL vs RL+FC 19.7 ± 3.3 ng/mL P < 0.01 in an ANOVA and the non-parametric equivalents). Interestingly, animals resuscitated with FFP revealed similar values for sVEGFR1 compared with RL+ CFCs (Fig. 3B).
Circulating heparan sulfate was elevated by 91% at end-of-shock compared with baseline (pooled BL: 32.4 ng/mL vs. EOS: 62 ng/mL). Resuscitation with FFP resulted in full restoration of heparan sulfate to baseline. All the remaining groups demonstrated a further significant increase of circulating heparan sulfate concentration until end-of-observation (using a Repeated Measures ANOVA and the non-parametric equivalents). FFP-treated rats showed significantly lower level of heparan sulfate (35,9 ng/mL) than rats treated with HA (66.4 ng/mL) and all the RL groups in an ANOVA and the non-parametric equivalents (RL: 89.4 ng/mL, RL+FC: 90.9 and RL+PCC: 78 ng/mL; all P < 0.001; Fig. 4A).
Hemorrhagic shock led to an increase of circulating syndecan-1 as indicated by the shock reference group (pooled BL: 3.6 ng/mL vs. EOS: 8.6 ng/mL). At end-of-observation, syndecan-1 remained significantly elevated (lowest by 50% (HA+PCC), highest by 283% (HA+FC)) compared with BL, except for BL+PCC+RL (using a Repeated Measures ANOVA and the non-parametric equivalents).
Compared to other treatment groups, we did not find any significant differences (tested by an ANOVA and the non-parametric equivalents, see Figure 3). Of note, the highest elevation of circulating syndecan-1 was recorded in the HA+FC group (12.07 ng/mL at end-of-observation) but it was burdened with a large data variation (Fig. 4B).
While many studies have demonstrated beneficial effects of FFP on the endotheliopathic microvasculature, there is still a paucity of data regarding the influence of CFCs on endothelial glycocalyx damage. This is of particular interest as CFCs such as FC and PCC are increasingly used as an initial hemostatic therapy in order to treat trauma-induced coagulopathy (24, 27).
Most recently, we have shown that severe hemorrhagic shock itself causes endothelial injury even prior to the resuscitation (22). Moreover, the intensity of endotheliopathy was found to be associated with shock severity and high sympatho-adrenal activation. In the present study, shock-mediated endotheliopathy was evident by a sustained increase of circulating sVEGFR1 and glycocalyx degradation markers such as syndecan-1 and heparan sulfate. Compared with crystalloids, colloid-based resuscitation protocols revealed a higher MAP, improved base deficit and lactate as well as lower concentrations of epinephrine and heparan sulfate. The composition of the resuscitation fluids had no effect on sVEGFR1 and syndecan-1 and co-administration of CFCs did not improve any of these variables.
Both experimental and clinical data emphasize the role of glycocalyx barrier dysfunction during trauma and major hemorrhage (14, 17). Animal models revealed that endothelial glycocalyx damage necessitates a certain degree of shock intensity (22). Clinical findings of trauma-related endotheliopathy suggest that mortality is closely linked to catecholamine release and endothelial dysfunction (14). In 424 trauma patients, Johansson et al. reported an independent relationship between high plasma epinephrine level and syndecan-1 as a marker of endothelial glycocalyx damage. In our model, hemorrhagic shock caused a sustained epinephrine release as displayed by its elevation in the shock reference group. Furthermore, endothelial glycocalyx damage markers such as sVEGFR1, heparan sulfate, and syndecan-1 significantly increased. This indicates that the hemorrhagic shock protocol employed in our rat model recapitulates (at least partly) the events reported in patients and appears as a clinically-relevant platform for endothelial glycocalyx-oriented studies.
In an experimental model of ischemia and reperfusion, Jacob et al. (28) showed that maintenance of an albumin concentration at the physiological level preserved the integrity of the endothelial glycocalyx as shown by lower levels of heparan sulfate and syndecan-1. In a small cohort of trauma patients, Rahbar et al. (12) observed that the concentration of shed glycocalyx components in patients displaying low plasma colloid oncotic pressure was higher compared with those with normal plasma colloid oncotic pressure. The current study revealed a trend toward lower plasma concentration of heparan sulfate in groups treated with HA compared with RL groups. However, enhanced correction of the shock state in the albumin-based groups, as shown by higher MAP compared with the RL groups, might explain the observed preservation for glycocalyx. It can be assumed that the finding of lower epinephrine in the HA+FC group compared with all other resuscitation arms is more likely due to the colloid-driven MAP increase rather than related to the supplemented CFC. sVEGFR1 and syndecan-1 were not affected by the type of fluid resuscitation. Addition of CFCs had no effects on these variables.
Animals treated with FFP presented with significantly lower values for base deficit compared with all other groups with exception for HA. Based on the similar results for hemodynamic recovery and lactate levels in animals resuscitated with either FFP or HA, we assume that the observed differences in base deficit are likely due to the buffering capacity of FFP (29). This study demonstrated that the (in vitro) buffering capacity of FFP was five times better than the acid buffering of human serum albumin (29).
Acknowledging the difference of distribution volumes inherent to resuscitation fluids appears to be crucial in the context of glycocalyx assessment in the circulation. For example, fast extravasation of crystalloids is a possible confounder in the measurement of plasma concentrations of different glycocalyx damage markers. By measuring plasma volume and calculating the total circulating amount of heparan sulfate, Nelson et al. (30) recently observed that differences in the heparan sulfate level between animals resuscitated with crystalloids and colloids reflect the differences in the distribution volume of the administered substances. Given that the total amount of circulating heparan sulfate did not differ between animals resuscitated with Ringer's acetate, HA, and FFP, the authors argued that the superiority of plasma-based resuscitation is largely due to its volume effect. In our study, plasma heparan sulfate concentration (at end-of-observation) returned to baseline in the FFP group only. HA and RL-based resuscitation led to significantly higher concentrations compared with FFP. Co-administration of CFCs achieved a strong trend toward higher heparan sulfate levels compared with FFP but did not reach statistical significance in HA+FC (P = 0.07) and HA+PCC (P = 0.06). The significant increase of heparan sulfate until the end-of-shock and the subsequent return to the lower detection range would suggest a clearance of heparan sulfate and therefore indicate a restoration of endothelial barrier function during reperfusion in the FFP group. Assuming that HA and FFP are equally effective plasma expanders, our data suggest a specific effect of FFP interacting with the heparan sulfate proteoglycan. In this respect, we cannot exclude an interaction between the plasmatic components (e.g., antithrombin) of FFP and the shed heparan sulfate. Apart from its ability to inhibit glycocalyx degrading enzymes, antithrombin holds a non-catalytic binding domain for heparin and heparan. It has been hypothesized that this steric effect could preclude degrading sheddases from accessing heparan sulfate and prevent degradation (31, 32). The suggested protective effect of resuscitation with FFP in our model remains questionable. In line with Nelson et al., we observed no difference in the syndecan-1 level between RL, HA and FFP, yet we are lacking data corrected for plasma volume in order to confirm this observation. Moreover, FFP-based resuscitation did not reveal any improvement with regard to shedding of sVEGFR1 compared with all other groups.
Recent work by Pati et al. (19) suggests that a four-factor PCC (Kcentra) modulates vascular function likewise to FFP. In our study, a bolus application of the same PCC followed by either RL or HA showed no additional net effect on glycocalyx markers compared with treatment with RL or HA alone. The current study revealed that in vivo administration of PCC was unable to correct endotheliopathy. However, there are considerable differences between our protocol and the one by Pati et al.; we administered PCC in a dose of 25 IU/kg/BW, half of the dose Pati et al. used in their study. Additionally, Pati et al. maintained the reperfusion with RL and HA. PCC is a highly potent hemostatic drug with substantial adverse effects when given at higher concentrations (20). Moreover, a recent study by Moe et al. (33) raised concerns on the adverse effects of using PCC alone to treat coagulopathy in hemorrhaged pigs, as some animals developed disseminated intravascular coagulation. It has been demonstrated that thrombin cleaves both syndecan-1 and syndecan-4 in vitro, suggesting a link between pro-inflammatory/hemostatic activation and shedding of endothelial glycocalyx (34). Intuitively, one could think that supplementation with clotting factors would therefore enhance the enzymatic cleavage of syndecans. Since levels for syndecan-1 showed no net effect after PCC administration compared with RL and HA alone, our data do not support this hypothesis.
The present study suffers from several limitations. First, all of the primary endpoints reported were measured 1 h after resuscitation was terminated. Therefore, we cannot exclude that the plasma concentrations were different before and after sample collection, although other studies demonstrated similar results for glycocalyx degradation markers between 1 and 2 h after resuscitation (10, 30). Second, our model of pressure controlled hemorrhagic shock cannot fully rebuild the complexity of trauma-related endotheliopathy, due to the absence of uncontrolled blood loss and the lack of severe mechanical tissue injury. The midline laparotomy in our model only in part mimics tissue injury. However, Ayala et al. (35) found that IL-6 levels significantly increase after performing a midline laparotomy, before the onset of bleeding, compared with unmanipulated animals. The failure of PCC and FC to show an effect on endothelial glycocalyx damage markers could therefore be related to the nature of our model. However, it has been shown previously that hypoperfusion—the main feature of our model—is the key driver of the endotheliopathy in major trauma (15, 36). Third, in order to compare plasma concentrations of circulating markers measured in different resuscitation protocols (e.g., crystalloid vs. colloid), an accurate estimation of plasma volumes is necessary. Therefore, crystalloid versus colloid comparisons do not allow for an exact conclusion regarding plasma levels of glycocalyx components. Fourth, our study did not include a blood-based resuscitation arm, a feature that could have led to different results when combined with coagulation factor concentrates. Furthermore, we used albumin purified from human plasma and cannot rule out influences on our endpoints related to the species dependent differences of the albumin molecule. Immunological reactions caused by xenobiotic effects could have also confounded the results. However, we recorded no clinical evidence of immunological response during the phase of resuscitation and observation.
The current study revealed that plasma-based resuscitation in hemorrhagic shock model normalized the heparan sulfate but not syndecan-1 concentration. Fibrinogen concentrate and prothrombin complex concentrate yielded no significant effects on circulating markers of glycocalyx degradation, suggesting that coagulation factor concentrates do not exert a therapeutic effect upon endothelial glycocalyx in the hemorrhagic shock setting.
MP, SB, HR, VF, and HS designed the study. JZ and MJ performed the analytical part. NH, MA, CK, and AB conducted the experiments. UK computed the data. NH, JZ, FB, SB, HR, and HS interpreted the data. NH, SB, and HS drafted the manuscript. All authors critically reviewed the manuscript and agreed to the final version.
The authors thank Marcin Osuchowski for critically reviewing the final manuscript.
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