The Effects of Heparan Sulfate Infusion on Endothelial and Organ Injury in a Rat Pneumosepsis Model.

Septic shock is characterized by endothelial dysfunction, leading to tissue edema and organ failure. Heparan sulfate (HS) is essential for vascular barrier integrity, possibly via albumin as a carrier. We hypothesized that supplementing fluid resuscitation with HS would improve endothelial barrier function, thereby reducing organ edema and injury in a rat pneumosepsis model. Following intratracheal inoculation with Streptococcus pneumoniae, Sprague Dawley rats were randomized to resuscitation with a fixed volume of either Ringer's Lactate (RL, standard of care), RL supplemented with 7 mg/kg HS, 5% human albumin, or 5% human albumin supplemented with 7 mg/kg HS (n = 11 per group). Controls were sham inoculated animals. Five hours after the start of resuscitation, animals were sacrificed. To assess endothelial permeability, 70 kD FITC-labelled dextran was administered before sacrifice. Blood samples were taken to assess markers of endothelial and organ injury. Organs were harvested to quantify pulmonary FITC-dextran leakage, organ edema, and for histology. Inoculation resulted in sepsis, with increased lactate levels, pulmonary FITC-dextran leakage, pulmonary edema, and pulmonary histologic injury scores compared to healthy controls. RL supplemented with HS did not reduce median pulmonary FITC-dextran leakage compared to RL alone (95.1 CI [62.0-105.3] vs. 87.1 CI [68.9-139.3] µg/mL, p = 0.76). Similarly, albumin supplemented with HS did not reduce pulmonary FITC-dextran leakage compared to albumin (120.0 [93.8-141.2] vs. 116.2 [61.7 vs. 160.8] µg/mL, p = 0.86). No differences were found in organ injury between groups. Heparan sulfate, as an add-on therapy to RL or albumin resuscitation, did not reduce organ or endothelial injury in a rat pneumosepsis model. Higher doses of heparan sulfate may decrease organ and endothelial injury induced by shock.

The effects of resuscitation with different plasma products on endothelial permeability and organ injury in a rat pneumosepsis model.

BACKGROUND: Endothelial injury and permeability are a hallmark of sepsis. Initial resuscitation of septic patients with crystalloids is associated with aggravation of endothelial permeability, which may be related either to low protein content or to volume. We investigated whether initial resuscitation with different types of plasma or albumin decreases endothelial dysfunction and organ injury in a pneumosepsis rat model compared to the same volume of crystalloids. STUDY DESIGN AND METHODS: Sprague-Dawley rats were intratracheally inoculated with Streptococcus pneumoniae. Twenty-four hours after inoculation, animals were randomized to 2 control groups and 5 intervention groups (n = 11 per group) to receive resuscitation with a fixed volume (8 mL/kg for 1 h) of either Ringer's Lactate, 5% human albumin, fresh frozen plasma derived from syngeneic donor rats (rFFP), human-derived plasma (hFFP) or human-derived solvent detergent plasma (SDP). Controls were non-resuscitated (n = 11) and healthy animals. Animals were sacrificed 5 h after start of resuscitation (T = 5). Pulmonary FITC-dextran leakage as a reflection of endothelial permeability was used as the primary outcome. RESULTS: Inoculation with S. Pneumoniae resulted in sepsis, increased median lactate levels (1.6-2.8 mM, p < 0.01), pulmonary FITC-dextran leakage (52-134 µg mL-1, p < 0.05) and lung injury scores (0.7-6.9, p < 0.001) compared to healthy controls. Compared to animals receiving no resuscitation, animals resuscitated with rFFP had reduced pulmonary FITC leakage (134 vs 58 µg/mL, p = 0.011). However, there were no differences in any other markers of organ or endothelial injury. Resuscitation using different human plasma products or 5% albumin showed no differences in any outcome. CONCLUSIONS: Resuscitation with plasma did not reduce endothelial and organ injury when compared to an equal resuscitation volume of crystalloids. Rat-derived FFP may decrease pulmonary leakage induced by shock.

Anti-high-mobility group box-1 treatment strategies improve trauma-induced coagulopathy in a mouse model of trauma and shock.

BACKGROUND: Trauma-induced coagulopathy is associated with platelet dysfunction and contributes to early mortality after traumatic injury. Plasma concentrations of the damage molecule high-mobility group box-1 (HMGB-1) increase after trauma, which may contribute to platelet dysfunction. We hypothesised that inhibition of HMGB-1 with a monoclonal antibody (mAb) or with recombinant thrombomodulin (rTM) improves trauma-induced coagulopathy in a murine model of trauma and shock. METHODS: Male 129S2/SvPasOrlRJ mice were anaesthetised, mechanically ventilated, and randomised into five groups: (i) ventilation control (VENT), (ii) trauma/shock (TS), (iii) TS+anti-HMGB-1 mAb (TS+AB), (iv) TS+rTM (TS+TM), and (v) TS+anti-HMGB-1 mAb+rTM (TS+COMBI). Primary outcome was rotational thromboelastometry EXTEM. Secondary outcomes included tail bleeding time, platelet count, plasma HMGB-1 concentration, and platelet activation. RESULTS: Trauma and shock resulted in a hypocoagulable thromboelastometry profile, increased plasma HMGB-1, and increased platelet activation markers. TS+AB was associated with improved clot firmness after 5 min compared with TS (34 [33-37] vs 32 [29-34] mm; P=0.043). TS+COMBI was associated with decreased clot formation time (98 [92-125] vs 122 [111-148] s; P=0.018) and increased alpha angle (77 [72-78] vs 69 [64-71] degrees; P=0.003) compared with TS. TS+COMBI also reduced tail bleeding time compared with TS (P=0.007). The TS+TM and TS+COMBI groups had higher platelet counts compared with TS (P=0.044 and P=0.041, respectively). CONCLUSIONS: Inhibition of HMGB-1 early after trauma in a mouse model improves clot formation and strength, preserves platelet count, and decreases bleeding time.

Coagulopathy Underlying Rotational Thromboelastometry Derangements in Trauma Patients: A Prospective Observational Multicenter Study.

BACKGROUND: Viscoelastic hemostatic assays such as rotational thromboelastometry (ROTEM) are used to guide treatment of trauma induced coagulopathy. The authors hypothesized that ROTEM derangements reflect specific coagulation factor deficiencies after trauma. METHODS: This was a secondary analysis of a prospective cohort study in six European trauma centers in patients presenting with full trauma team activation. Patients with dilutional coagulopathy and patients on anticoagulants were excluded. Blood was drawn on arrival for measurement of ROTEM, coagulation factor levels, and markers of fibrinolysis. ROTEM cutoff values to define hypocoagulability were as follows: EXTEM clotting time greater than 80 s, EXTEM clot amplitude at 5 min less than 40 mm, EXTEM lysis index at 30 min less than 85%, FIBTEM clot amplitude at 5 min less than 10 mm, and FIBTEM lysis index at 30 min less than 85%. Based on these values, patients were divided into seven deranged ROTEM profiles and compared to the reference group (ROTEM values within reference range). The primary endpoint was coagulation factors levels and fibrinolysis. RESULTS: Of 1,828 patients, 732 (40%) had ROTEM derangements, most often consisting of a combined decrease in EXTEM and FIBTEM clot amplitude at 5 min, that was present in 217 (11.9%) patients. While an isolated EXTEM clotting time greater than 80 s had no impact on mortality, all other ROTEM derangements were associated with increased mortality. Also, coagulation factor levels in this group were similar to those of patients with a normal ROTEM. Of coagulation factors, a decrease was most apparent for fibrinogen (with a nadir of 0.78 g/l) and for factor V levels (with a nadir of 22.8%). In addition, increased fibrinolysis can be present when the lysis index at 30 min is normal but EXTEM and FIBTEM clot amplitude at 5 min is decreased. CONCLUSIONS: Coagulation factor levels and mortality in the group with an isolated clotting time prolongation are similar to those of patients with a normal ROTEM. Other ROTEM derangements are associated with mortality and reflect a depletion of fibrinogen and factor V. Increased fibrinolysis can be present when the lysis index after 30 min is normal.

The effect of shock duration on trauma-induced coagulopathy in a murine model.

BACKGROUND: Trauma-induced coagulopathy (TIC) is a life-threatening condition associated with high morbidity and mortality. TIC can present with different coagulation defects. In this study, the aim was to determine the effect of shock duration on TIC characteristics. We hypothesized that longer duration of shock leads to a more hypocoagulable rotational thromboelastometry (ROTEM) profile compared to a shorter duration of shock. METHODS: Male B57BL/6J(c) mice (n = 5-10 per group) were sedated and mechanically ventilated. Trauma was induced by bilateral lower limb fractures and crush injuries to the liver and small intestine. Shock was induced by blood withdrawals until a mean arterial pressure of 25-30 mmHg was achieved. Groups reflected trauma and shock for 30 min (TS30) and trauma and shock for 90 min (TS90). Control groups included ventilation only (V90) and trauma only (T90). RESULTS: Mice in the TS90 group had significantly increased base deficit compared to the V90 group. Mortality was 10% in the TS30 group and 30% in the TS90 group. ROTEM profile was more hypocoagulable, as shown by significantly lower maximum clot firmness (MCF) in the TS30 group (43.5 [37.5-46.8] mm) compared to the TS90 group (52.0 [47.0-53.0] mm, p = 0.04). ROTEM clotting time and parameters of clot build-up did not significantly differ between groups. CONCLUSIONS: TIC characteristics change with shock duration. Contrary to the hypothesis, a shorter duration of shock was associated with decreased maximum clotting amplitudes compared to a longer duration of shock. The effect of shock duration on TIC should be further assessed in trauma patients.

Plasma and rhADAMTS13 reduce trauma-induced organ failure by restoring the ADAMTS13-VWF axis.

Trauma-induced organ failure is characterized by endothelial dysfunction. The aim of this study was to investigate the role of von Willebrand factor (VWF) and its cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) in the occurrence of endothelial permeability and organ failure in trauma. In an observational study in a level-1 trauma center, 169 adult trauma patients with clinical signs of shock and/or severe injuries were included. Trauma was associated with low ADAMTS13 and high VWF antigen levels, thus generating an imbalance of ADAMTS13 to VWF. Patients who developed organ failure (23%) had greater ADAMTS13-to-VWF imbalances, persistently lower platelet counts, and elevated levels of high-molecular-weight VWF multimers compared with those without organ failure, suggesting microthrombi formation. To investigate the effect of replenishing low ADAMTS13 levels on endothelial permeability and organ failure using either recombinant human ADAMTS13 (rhADAMTS13) or plasma transfusion, a rat model of trauma-induced shock and transfusion was used. Rats in traumatic hemorrhagic shock were randomized to receive crystalloids, crystalloids supplemented with rhADAMTS13, or plasma transfusion. A 70-kDa fluorescein isothiocyanate-labeled dextran was injected to determine endothelial leakage. Additionally, organs were histologically assessed. Both plasma transfusion and rhADAMTS13 were associated with a reduction in pulmonary endothelial permeability and organ injury when compared with resuscitation with crystalloids, but only rhADAMTS13 resulted in significant improvement of a trauma-induced decline in ADAMTS13 levels. We conclude that rhADAMTS13 and plasma transfusion can reduce organ failure following trauma. These findings implicate the ADAMTS13-VWF axis in the pathogenesis of organ failure.

Platelet-to-red blood cell ratio and mortality in bleeding trauma patients: A systematic review and meta-analysis.

BACKGROUND: In traumatic bleeding, transfusion practice has shifted toward higher doses of platelets and plasma transfusion. The aim of this systematic review was to investigate whether a higher platelet-to-red blood cell (RBC) transfusion ratio improves mortality without worsening organ failure when compared with a lower ratio of platelet-to-RBC. METHODS: Pubmed, Medline, and Embase were screened for randomized controlled trials (RCTs) in bleeding trauma patients (age ≥16 years) receiving platelet transfusion between 1946 until October 2020. High platelet:RBC ratio was defined as being the highest ratio within an included study. Primary outcome was 24 hour mortality. Secondary outcomes were 30-day mortality, thromboembolic events, organ failure, and correction of coagulopathy. RESULTS: In total five RCTs (n = 1757 patients) were included. A high platelet:RBC compared with a low platelet:RBC ratio significantly improved 24 hour mortality (odds ratio [OR] 0.69 [0.53-0.89]) and 30- day mortality (OR 0.78 [0.63-0.98]). There was no difference between platelet:RBC ratio groups in thromboembolic events and organ failure. Correction of coagulopathy was reported in five studies, in which platelet dose had no impact on trauma-induced coagulopathy. CONCLUSIONS: In traumatic bleeding, a high platelet:RBC improves mortality as compared to low platelet:RBC ratio. The high platelet:RBC ratio does not influence thromboembolic or organ failure event rates.

Bosutinib reduces endothelial permeability and organ failure in a rat polytrauma transfusion model.

BACKGROUND: Trauma-induced shock is associated with endothelial dysfunction. We examined whether the tyrosine kinase inhibitor bosutinib as an adjunct therapy to a balanced blood component resuscitation strategy reduces trauma-induced endothelial permeability, thereby improving shock reversal and limiting transfusion requirements and organ failure in a rat polytrauma transfusion model. METHODS: Male Sprague-Dawley rats (n=13 per group) were traumatised and exsanguinated until a MAP of 40 mm Hg was reached, then randomised to two groups: red blood cells, plasma and platelets in a 1:1:1 ratio with either bosutinib or vehicle. Controls were randomised to sham (median laparotomy, no trauma) with bosutinib or vehicle. Organs were harvested for histology and wet/dry (W/D) weight ratio. RESULTS: Traumatic injury resulted in shock, with higher lactate levels compared with controls. In trauma-induced shock, the resuscitation volume needed to obtain a MAP of 60 mm Hg was lower in bosutinib-treated animals (2.8 [2.7-3.2] ml kg-1) compared with vehicle (6.1 [5.1-7.2] ml kg-1, P<0.001). Lactate levels in the bosutinib group were 2.9 [1.7-4.8] mM compared with 6.2 [3.1-14.1] mM in the vehicle group (P=0.06). Bosutinib compared with vehicle reduced lung vascular leakage (W/D ratio of 5.1 [4.6-5.3] vs 5.7 [5.4-6.0] (P=0.046) and lung injury scores (P=0.027). CONCLUSIONS: Bosutinib as an adjunct therapy to a balanced transfusion strategy reduced resuscitation volume, improved shock reversal, and reduced vascular leak and organ injury in a rat polytrauma model.

Targeting Endothelial Dysfunction in Acute Critical Illness to Reduce Organ Failure.

During hyperinflammatory conditions that can occur in acute critical illness, such as shock or hypoperfusion, inflammatory mediators activate the endothelium, fueling a proinflammatory host-response as well as procoagulant processes. These changes result in shedding of the glycocalyx, endothelial hyperpermeability, edema formation, and lead to disturbed microcirculatory perfusion and organ failure. Different fluid strategies that are used in shock may have differential effects on endothelial integrity. Collectively, low protein content fluids seem to have negative effects on the endothelial glycocalyx, aggravating endothelial hyperpermeability, whereas fluids containing albumin or plasma proteins may be superior to normal saline in protecting the glycocalyx and endothelial barrier function. Targeting the endothelium may be a therapeutic strategy to limit organ failure, which hitherto has not received much attention. Treatment targets aimed at restoring the endothelium should focus on maintaining glycocalyx function and/or targeting coagulation pathways or specific endothelial receptors. Potential treatments could be supplementing glycocalyx constituents or inhibiting glycocalyx breakdown. In this review, we summarize mechanisms of endothelial dysfunction during acute critical illness, such as the systemic inflammatory response, shedding of the glycocalyx, endothelial activation, and activation of coagulation. In addition, this review focuses on the effects of different fluid strategies on endothelial permeability. Also, potential mechanisms for treatment options to reduce endothelial hyperpermeability with ensuing organ failure are evaluated. Future research is needed to elucidate these pathways and to translate these data to the first human safety and feasibility trials.

The use of cryopreserved platelets in a trauma-induced hemorrhage model.

BACKGROUND: Cryopreserved platelet products can be stored for years and are mainly used in military settings. Following thawing, cryopreserved platelets are activated, resulting in faster clot formation but reduced aggregation in vitro, rendering their efficacy in bleeding unknown. Also, concerns remain on the safety of these products. The aim was to investigate the efficacy and safety of cryopreserved platelets in a rat model of traumatic hemorrhage. STUDY DESIGN AND METHODS: After 1 hour of shock, rats (n = 13/group) were randomized to receive a balanced transfusion pack (1:1:1 red blood cell:plasma:platelet) made from syngeneic rat blood, containing either liquid stored platelets or cryopreserved platelets. Primary outcome was the transfusion volume required to obtain a mean arterial pressure (MAP) of 60 mmHg. Secondary outcomes were coagulation as assessed by thromboelastometry (ROTEM®) and organ failure as assessed by biochemistry and histopathology. RESULTS: The transfusion volume to obtain a MAP of 60 mmHg was lower in animals receiving cryopreserved platelets (5.4 [4.1-7.1] mL/kg) compared to those receiving liquid stored platelets (7.5 [6.4-8.5] mL/kg, p < 0.05). ROTEM® clotting times were shorter (45 [41-48] vs. 49 [45-53]sec, p < 0.05), while maximum clot firmness was slightly lower (68 [67-68] vs. 69 [69-71]mm, p < 0.01). Organ failure was similar in both groups. CONCLUSIONS: Use of cryopreserved platelets required less transfusion volume to reach a targeted MAP compared to liquid stored platelets, while organ injury was similar. These results provide a rationale for clinical trials with cryopreserved platelets in (traumatic) bleeding.

Use of a high platelet-to-RBC ratio of 2:1 is more effective in correcting trauma-induced coagulopathy than a ratio of 1:1 in a rat multiple trauma transfusion model.

BACKGROUND: Platelet dysfunction importantly contributes to trauma-induced coagulopathy (TIC). Our aim was to examine the impact of transfusing platelets (PLTs) in a 2:1 PLT-to-red blood cell (RBC) ratio versus the standard 1:1 ratio on transfusion requirements, correction of TIC, and organ damage in a rat multiple trauma transfusion model. METHODS: Mechanically ventilated male Sprague Dawley rats were traumatized by crush injury to the small intestine and liver and a fracture of the femur, followed by exsanguination until a mean arterial pressure (MAP) of 40 mmHg. Animals were randomly assigned to receive resuscitation in a high PLT dose (PLT to plasma to RBC in a ratio of 2:1:1) or a standard PLT dose (ratio of 1:1:1) until a MAP of 60 mmHg was reached (n = 8 per group). Blood samples were taken for biochemical and thromboelastometry (ROTEM) assessment. Organs were harvested for histopathology.Outcome measures were transfusion requirements needed to reach a pretargeted MAP, as well as ROTEM correction and organ failure. RESULTS: Trauma resulted in coagulopathy as assessed by deranged ROTEM results. Mortality rate was 19%, with all deaths occurring in the standard dose group. The severity of hypovolemic shock as assessed by lactate and base excess was not different in both groups. The volume of transfusion needed to reach the MAP target was lower in the high PLT dose group compared to the standard dose, albeit not statistically significant (p = 0.054). Transfusion with a high PLT dose resulted in significant stronger clot firmness compared to the standard dose at all time points following trauma, while platelet counts were similar. Organ failure as assessed by biochemical analysis and histopathology was not different between groups, nor were there any thromboembolic events recorded. CONCLUSIONS: Resuscitation with a high (2:1) PLT-to-RBC ratio was more effective compared to standard (1:1) PLT-to-RBC ratio in treating TIC, with a trend towards reduced transfusion volumes. Also, high PLT dose did not aggravate organ damage. Transfusion strategies using higher PLT dose regiments might be a feasible treatment option in hemorrhaging trauma patients for the correction of TIC.

Endogenous microparticles drive the proinflammatory host immune response in severely injured trauma patients.

INTRODUCTION: Severe trauma affects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. METHODS: A prospective, observational substudy of the ACIT II (Activation of Coagulation and Inflammation in Trauma II) study was performed at our academic level I trauma center. Adult multiple-trauma patients with an injury severity score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole-blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. RESULTS: Ten trauma patients and 10 control subjects were included. Trauma patients were significantly injured with a median injury severity score of 19 (range, 17-45). Patients were neither in shock nor bleeding. On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with control subjects, as reflected by decreased production of interleukin 6 (IL-6), IL-10, and tumor necrosis factor α (P < 0.001). In trauma patients, MP-positive plasma was associated with a significantly higher synthesis of IL-6 and tumor necrosis factor α compared with plasma depleted from MPs (P = 0.047 and 0.002, respectively). Compared with control subjects, the number of circulating MPs was significantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in control subjects. CONCLUSIONS: On admission, trauma patients have a reduced immune response toward endotoxin challenge, which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an inflammatory host response after injury.