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Background: The beneficial effects of estrogens on survival from hemorrhage have been suggested in some preclinical models. This study investigated the effects of ethynylestradiol-3-sulfate (EE-3-S) on coagulation, metabolism and survival in pigs following traumatic hemorrhage., Methods: Twenty-six pigs were randomized into: normal saline group (NS, n = 10), EE-3-S group (EE-3, n = 11) groups, and no resuscitation group (NR, n = 5). Femur fracture was performed in each pig's left leg, followed by hemorrhage of 55% of estimated blood volume and a 10-minute shock period. Afterward, pigs were resuscitated with a small volume of either NS alone (4 mL/kg) or EE-3-S with NS (1 mL/kg at concentration of 1 mg/mL, plus NS solution of 3 mL/kg). Pigs in NR group were not resuscitated with any fluid. All pigs were then monitored for 6 hours or until death, with hemodynamics and survival times recorded. Blood samples were taken during the study for measurements of oxygen metabolism (oxygen delivery, extraction, and consumption) and coagulation function (using Rotem with Extem reagents)., Results: All baseline measurements were similar among the three groups. In the NS group, femur fracture and hemorrhage immediately reduced mean arterial pressure (MAP, 74 ± 3 mm Hg to 44 ± 4 mm Hg) and increased heart rate (97 ± 5 bpm to 218 ± 14 bpm, both p < 0.05). Similar changes in MAP and heart rate were observed in the EE-3 and NR groups. There were no differences observed in changes of Rotem ® measurements or oxygen metabolism among the groups during the study. At 6 hours, four pigs in NS, four pigs in EE-3-S, and two pigs in the NR group survived to the end of the study. The mean survival times were similar among the NS (212 ± 43 minutes), EE-3 (212 ± 39 minutes), and NR (223 ± 63 minutes) groups ( p = 0.9845)., Conclusion: Following severe traumatic hemorrhage, hypotensive resuscitation with EE-3-S did not impact coagulation, metabolism, or survival in pigs., (Copyright © 2023 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Background: Valproic acid (VPA) has been extensively used for treatment of anxiety and seizure. Recent studies have shown that VPA has cellular protective effects in preclinical models following severe hemorrhage. This study investigated the effects of VPA on coagulation and survival in pigs after traumatic hemorrhage and hypotensive resuscitation., Methods: Following baseline measurements, femur fracture was performed in 20 anesthetized and instrumented pigs (41 ± 2 kg), followed by hemorrhage of 55% of the estimated blood volume and a 10-minute shock period. Pigs were then resuscitated for 30 minutes with normal saline (NS) alone (NS group, n = 10, 4 mL/kg) or VPA solution (VPA group, n = 10, 90 mg/kg, 2 mL/kg of 45 mg VPA/mL, plus 2 mL NS/kg). All pigs were then monitored for 2 hours or until death. Hemodynamics were recorded, and blood samples were taken for blood and coagulation analysis (Rotem) at baseline, after hemorrhage, resuscitation, and 2 hours or death., Results: Femur fracture and hemorrhage caused similar reductions in mean arterial pressure and cardiac output, and increase in heart rate in both groups. Resuscitation with NS or VPA did not return these measurements to baseline. No differences were observed in hematocrit, pH, lactate, base excess, or total protein between the groups. Compared with NS, resuscitation with VPA decreased platelet counts and prolonged activated partial thromboplastin time, with no differences in fibrinogen levels, prothrombin time, or any of the Rotem measurements between the two groups. Neither survival rates (NS, 7 of 10 pigs; VPA, 7 of 10 pigs) nor survival times after resuscitation (NS, 97 ± 40 minutes; VPA, 98 ± 43 minutes) differed between the groups., Conclusion: Following traumatic hemorrhage and hypotensive resuscitation in pigs, VPA provides no benefit toward improving coagulation function or survival times., (Copyright © 2022 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Background: Poloxamer 188 (P188) is a copolymer surfactant with plasma membrane stabilizing action. This study investigated the effects of P188 on blood volume and coagulation in pigs after traumatic hemorrhage and hypotensive resuscitation., Methods: Femur fracture was performed in 17 anesthetized pigs, followed by hemorrhage of 55% of estimated blood volume and a 10 min shock period. Afterwards, pigs were randomized to be resuscitated with either normal saline (n = 9, 4 mL/kg, NS group) or P188 (n = 8, 1.33 mL/kg at 150 mg/mL, plus 2.67 mL NS/kg, P188 group). Pigs were monitored for 2 h or until death. Hemodynamics were recorded and blood samples were taken at baseline (BL), after hemorrhage, shock, resuscitation, and at 2 h for blood and coagulation analysis using Rotem®., Results: All but one pig in each group survived to 2 h. Femur fracture and hemorrhage reduced mean arterial pressure to half of the BL and elevated heart rate to double of the BL (both P < 0.05). Resuscitation with NS or P188 did not return these measurements to BL. Compared to NS, resuscitation with P188 resulted in a smaller reduction of blood volume (76 ± 3% in P188 and 60 ± 2% in NS); higher base excess (3.3 ± 0.9 vs. 0.5 ± 0.9 mM); and lower hematocrit (24 ± 1 vs. 28 ± 1%) and Ca++ (24 ± 1 vs. 28 ± 1 mM). Resuscitation with P188 prolonged aPTT (43 ± 12 vs. 22 ± 3 s, all P < 0.05)., Conclusions: Following traumatic hemorrhage and hypotensive resuscitation, P188 improved circulation volume and base deficit, but induced slower clotting initiation in pigs. Thus, P188 may have limited benefit as an initial small volume resuscitation adjunct following hemorrhage., Competing Interests: All authors, WZM, HX, IT, and MAD (deceased), have no conflicts of interest to report. This manuscript is not an endorsement of any commercial products by the authors or the U.S. Army., (Copyright © 2021 by the Shock Society.)

Introduction: This experimental in vitro study aimed to identify and characterize hypothermia-associated coagulopathy and to compare changes in mild to severe hypothermia with the quantitative measurement of rotational thromboelastometry (ROTEM) and multiple-electrode aggregometry (MULTIPLATE). Methods: Whole blood samples from 18 healthy volunteers were analyzed at the target temperatures of 37, 32, 24, 18, and 13.7°C with ROTEM (ExTEM, InTEM and FibTEM) and MULTIPLATE using the arachidonic acid 0.5 mM (ASPI), thrombin receptor-activating peptide-6 32 µM (TRAP) and adenosine diphosphate 6.4 µM (ADP) tests at the corresponding incubating temperatures for coagulation assessment. Results: Compared to baseline (37°C) values ROTEM measurements of clotting time (CT) was prolonged by 98% (at 18°C), clot formation time (CFT) was prolonged by 205% and the alpha angle dropped to 76% at 13.7°C ( p < 0.001). At 24.0°C CT was prolonged by 56% and CFT by 53%. Maximum clot firmness was only slightly reduced by ≤2% at 13.7°C. Platelet function measured by MULTIPLATE was reduced with decreasing temperature ( p < 0.001): AUC at 13.7°C -96% (ADP), -92% (ASPI) and -91% (TRAP). Conclusion: Hypothermia impairs coagulation by prolonging coagulation clotting time and by decreasing the velocity of clot formation in ROTEM measurements. MULTIPLATE testing confirms a linear decrease in platelet function with decreasing temperatures, but ROTEM fails to adequately detect hypothermia induced impairment of platelets., Competing Interests: BS was employed by the company Alexion Pharma Austria GmbH, Wien, Austria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wallner, Schenk, Paal, Falk, Strapazzon, Martini, Brugger and Fries.)

Background: Severe burn injury results in profound catabolic deterioration. Although burn-related catabolism has been well stated, it is unclear when the catabolic response begins. This study characterized acute changes of muscle protein breakdown at the admission and the day after in severely burned adults., Methods: Twelve patients (43 ± 19 years old) with 40% ± 21% total body surface area burns were prospectively enrolled into an observational study approved by institutional review board. Urinary samples were collected on admission day and the day after (day 1). Patient demographic and clinical data of vital signs, blood gas and chemistry, and coagulation status were collected. Catabolic changes of muscle breakdown were quantified by urinary excretion of 3-methylhisitidine, determined by gas chromatography and mass spectrometry analysis., Results: Compared with admission day, burned patients had elevated mean ± SD arterial pressure (from 90 ± 5 mm Hg to 108 ± 7 mm Hg) and heart rate (from 102 ± 7 beats per minute to 119 ± 4 beats per minute both p < 0.05) after 24 hours. Their 24-hour urinary output was 1,586 ± 813 mL at admission day to 1,911 ± 1,048 mL on day 1. The 24-hour urea excretion was elevated from 172 ± 101 mg/kg per day at admission day to 302 ± 183 mg/kg per day on day 1 (both p < 0.05), with no change in creatinine excretion. Urinary 3-methylhisitidine excretion increased from 0.75 ± 0.74 mg/kg per day at admission to 1.14 ± 0.86 mg/kg per day on day 1 (p < 0.05). The estimated skeletal muscle protein breakdown was increased from 1.1 ± 1.0 g/kg per day at admission day to 1.6 ± 1.2 g/kg per day on day 1 (p < 0.05). There were no changes in prothrombin time, activated partial thromboplastin time, or platelets., Conclusion: In severely burned patients, catabolic muscle protein breakdown is elevated within 24 hours after admission and before changes in coagulation. These findings suggest that early interventions may be needed to effectively attenuate the catabolic responses in burn patients., Level of Evidence: Prospective and observational study, level II., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: This study compared the resuscitation effects of platelets and fibrinogen concentrate (FC) on coagulation and hemodynamics in pigs with traumatic hemorrhage and reduced platelet counts., Methods: Thirty pigs (40 ± 3 kg) were anesthetized and catheterized with an apheresis catheter to remove platelets using the Haemonetics 9000 (Haemonetics, Braintree, MA). Afterward, a femur fracture was induced, followed by hemorrhage of 35% the estimated blood volume. Pigs were then randomized to be resuscitated with 5% human albumin (12.5 mL/kg), FC (250 mg/kg, 12.5 mL/kg), or platelets collected from apheresis (11.0 ± 0.5 mL/kg). Animals were monitored for 2 hours or until death. Blood samples were collected before (baseline [BL]) and after apheresis, after hemorrhage, and after resuscitation to assess changes in hemodynamics and coagulation using Rotem., Results: No change in mean arterial pressure (MAP) or heart rate (HR) was observed by platelet apheresis. Hemorrhage reduced MAP to 57% ± 5% and elevated HR to 212% ± 20% of BL (both p < 0.05). Resuscitation with albumin, FC, or platelets did not revert MAP or HR to BL. Platelet counts were reduced by apheresis from BL 383 ± 20 × 10/μL to 141 ± 14 × 10/μL and were reduced further after resuscitation with albumin (88 ± 18 × 10/μL) or FC (97 ± 13 × 10/μL, all p < 0.05), but improved with platelet resuscitation (307 ± 24 × 10/μL). Fibrinogen concentration was reduced by apheresis from BL 225 ± 9 mg/dL to 194 ± 8 mg/dL, fell after albumin infusion (134 ± 11 mg/dL), increased to 269 ± 10 mg/dL after FC resuscitation (all p < 0.05), and was not affected by platelet resuscitation. Rotem α-angle decreased from 79 ± 2 degrees to 69 ± 1 degrees by apheresis and hemorrhage (p < 0.05), and recovered similarly by resuscitation with FC (87 ± 1 degrees) or platelets (78 ± 2 degrees), but not by albumin (63 ± 3 degrees). Similar responses were observed in Rotem maximum clot firmness., Conclusion: In this traumatic hemorrhage swine model, low-volume resuscitation with FC or platelets was similarly effective in restoring coagulation.

Introduction: Hypothermia has notable effects on platelets, platelet function, fibrinogen, and coagulation factors. Common laboratory techniques cannot identify those effects, because blood samples are usually warmed to 37°C before analysis and do not fully reflect the in vivo situation. Multiple aspects of the pathophysiological changes in humoral and cellular coagulation remain obscure. This in vitro experimental study aimed to compare the measurements of thromboelastometry (TEM), multiple-electrode aggregometry (MEA) and Real Time Live Confocal Imaging for the purpose of identifying and characterizing hypothermia-associated coagulopathy., Methods: Blood samples were drawn from 18 healthy volunteers and incubated for 30 min before being analyzed at the target temperatures (37, 32, 24, 18, and 13.7°C). At each temperature thromboelastometry and multiple-electrode aggregometry were measured. Real Time Live Confocal Imaging was performed at 4, 24, and 37°C. The images obtained by Real Time Live Confocal Imaging were compared with the functional results of thromboelastometry and multiple-electrode aggregometry., Results: Thromboelastometry standard parameters were impaired at temperatures below baseline 37°C (ANOVA overall effect, p < 0.001): clotting time was prolonged by 27% at 13.7°C and by 60% at 18°C ( p < 0.044); clot formation time was prolonged by 157% ( p < 0.001). A reduction in platelet function with decreasing temperatures was observed ( p < 0.001); the area under the curve at 13.7°C was reduced by 96% (ADP test), 92% (ASPI test), and 91% (TRAP test) of the baseline values. Temperature-associated changes in coagulation were visualized with Real Time Live Confocal Imaging. Molecular changes such as the temperature-associated decrease in the fibrin network are paralleled by cellular effects like the lesser activity of the platelets as a result of decreased temperature. The maximum clot firmness (MCF) in TEM only changed slightly within the temperature range tested., Conclusion: The inhibitory effects of temperature on clot formation were visualized with Real Time Live Confocal Microscopy and compared with standard point-of-care testing. Inhibition of clotting factors and impaired platelet function are probably a result of hypothermia-induced impairment of thrombin. Measurement of MCF in TEM does not fully concur with Real Time Live Confocal Microscopy or MEA in hypothermia., (Copyright © 2020 Wallner, Schenk, Hermann, Paal, Falk, Strapazzon, Martini, Brugger and Fries.)

This study investigated changes in plasma fibrinogen metabolism and changes in coagulation in severely burned adults. Ten patients (27 ± 3 years; 91 ± 6 kg) with 51 ± 3% TBSA were consented and enrolled into an institutional review board-approved prospective study. On the study day, stable isotope infusion of 1-13C-phenylalanine and d5-phenylalanine was performed to quantify fibrinogen production and consumption. During the infusion, vital signs were recorded and blood samples were drawn every hour. Coagulation was measured by thromboelastograph (TEG). Ten normal healthy volunteers (37 ± 7 years; 74 ± 4 kg) were included as the control group. Burned adults had elevated heart rates (120 ± 2 vs 73 ± 5 [control] beats/minute), respiration rates (23 ± 2 vs 15 ± 1 breaths/minute), plasma glucose (127 ± 10 vs 89 ± 2 mg/dl), and fibrinogen levels (613 ± 35 vs 239 ± 17 mg/dl); and decreased albumin (1.3 ± 0.2 vs 3.7 ± 0.1 g/dl) and total protein (4.4 ± 0.2 vs 6.8 ± 0.1 g/dl, all P < .05). Fibrinogen breakdown was elevated in the burn group (2.3 ± 0.4 vs. 1.0 ± 0.3 µmol/kg/minute); and fibrinogen synthesis was further enhanced in the burn group (4.4 ± 0.7 vs 0.7 ± 0.2 µmol/kg/minute, both P < .05). Clotting speed (TEG-alpha) and clot strength (TEG-MA) were increased in the burn group (62 ± 4 vs 50 ± 4°, and 76 ± 2 vs 56 ± 2 mm, respectively, both P < .05). Fibrinolysis of TEG-LY60 was accelerated in the burn group (16 ± 6 vs 3 ± 1) and so was the increase in D-dimer level in the burn group (4.5 ± 0.4 vs 1.9 ± 0.3 mg/l, both P < .05). The hypercoagulable state postburn is in part a result of increased fibrinogen synthesis, over and above increased fibrinogen breakdown., (Published by Oxford University Press on behalf of the American Burn Association 2019.)

Objectives: This study was designed to assess the stability and functional activity of fibrinogen concentrates subjected to the changes in temperature and duration observed in field conditions., Methods: Fibrinogen concentrate was stored at -20°C (12 vials), 22°C (12 vials), and 50°C with 80% humidity (12 vials), for up to 6 mo. At each temperature, three vials of fibrinogen concentrate were taken out at 0, 1, 3, and 6 mo and reconstituted. On analysis days, blood samples were taken from a single healthy donor to collect plasma samples. The donor plasma was mixed with commercial fibrinogen-deficient plasma to make fibrinogen-adjusted plasma (FAP). An aliquot of the reconstituted fibrinogen concentrate was used for quantification of stored fibrinogen content (using STA-R) and function (Rotem - Fibtem) in FAP., Results: At 22°C for 0, 1, 3, and 6 mo, there were no significant changes observed in fibrinogen content (1,223 ± 42 mg/vial, 1,286 ± 86 mg/vial, 1,234 ± 76 mg/vial, and 1,178 ± 64 mg/vial), prothrombin time (13.5 ± 0.1 s, 13.7 ± 0.6 s, 13.3 ± 0.4 s, and 13.7 ± 0.2 s), or activated partial thromboplastin time (31.1 ± 0.2 s, 32.0 ± 0.2 s, 31.5 ± 0.2 s, and 32.0 ± 0.8 s), respectively. There were also no significant changes observed in any of the Fibtem measurements. Similarly, no differences were observed in these variables over time at -20°C and 50°C with 80% humidity., Conclusions: Fibrinogen concentrate maintained its content and function when stored at -20°C to 50°C with up to 80% humidity for 6 mo.

Introduction: Platelet apheresis is a routine clinical practice, but the physiological impact on the donors has been incompletely characterized. This study measured the effects of platelet apheresis on hemodynamics, oxygen metabolism, and coagulation in pigs to assess its impact before employing the animals in experimental studies., Methods: Forty pigs (39.8 ± 0.6 kg) were anesthetized and catheterized with an apheresis catheter in the femoral vein. During the platelet apheresis process, blood was withdrawn from the pig to separate platelets, and the remaining red blood cells and plasma returned back to the pigs, using the Haemonetics MCS+9000 system. A total of 12 cycles of blood withdrawn and return were performed during the entire apheresis procedure to reduce platelet counts to a target of 50% of baseline. During the process, hemodynamics was recorded in each cycle. Blood samples were collected before and after apheresis to assess changes in oxygen metabolism and coagulation by prothrombin time, activated partial thromboplastin time (STA-R Evolution Stago), and using Rotem thrombelastometry, and platelet aggregation using a Chrono-Log 700 aggregometer., Results: During each cycle of the apheresis, mean arterial pressure (MAP) was decreased and heart rate was increased by blood withdrawal, but both recovered after blood return. On the completion of the apheresis, platelet count decreased from baseline 345 ± 15 10 9 /L to 141 ± 14 10 9 /L and fibrinogen levels were reduced from 124 ± 5 to 99 ± 4 mg/dL (both p < 0.05). Although oxygen delivery remained unchanged, oxygen consumption was decreased from 4.0 ± 0.2 to 3.2 ± 0.0 mL O 2 /kg/min (p < 0.05). Rotem alpha (clotting speed) decreased from 79 ± 0 to 69 ± 1° and maximum clot firmness (MCF or clot strength) decreased from 71 ± 1 to 57 ± 1 mm (both p < 0.05). No changes were observed in prothrombin time or activated partial thromboplastin time. Platelet aggregation induced by arachidonic acid or collagen was decreased to 28 ± 6% or 71 ± 3% of baseline values (p < 0.05), respectively., Conclusion: Platelet apheresis caused significant fluctuations in hemodynamics, reduced oxygen consumption, in addition to the compromised platelet aggregation and clotting function expected. The observations warrant consideration in humans undergoing apheresis over extended periods., (Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.)

Traumatic injury is one of the leading causes of death, with uncontrolled hemorrhage from coagulation dysfunction as one of the main potentially preventable causes of the mortality. Hypothermia, acidosis, and resuscitative hemodilution have been considered as the significant contributors to coagulation manifestations following trauma, known as the lethal triad. Over the past decade, clinical observations showed that coagulopathy may be present as early as hospital admission in some severely injured trauma patients. The hemostatic dysfunction is associated with higher blood transfusion requirements, longer hospital stay, and higher mortality. The recognition of this early coagulopathy has initiated tremendous interest and effort in the trauma community to expand our understanding of the underlying pathophysiology and improve clinical treatments. This review discusses the current knowledge of coagulation complications following trauma.

Introduction: Ibuprofen is commonly used by warfighters in the deployed environment. This study investigated its dose effects on in vitro coagulation in human and pig blood., Methods: Blood samples were collected from 6 normal volunteers and 6 healthy pigs and processed to make platelet-adjusted samples (100 × 10(3)/μL, common transfusion trigger in trauma). Ibuprofen was added to the samples at concentrations of 0 μg/mL (control), the concentration from the highest recommended oral dose (163 μg/mL, 1×), and 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Platelet aggregation by Chrono-Log aggregometer and coagulation by rotational thrombelastogram (Rotem) were assessed at 15 minutes after the addition of ibuprofen., Results: A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested in human or pig blood. Collagen-stimulated platelet aggregation was inhibited starting at 1× in human blood and 4× in pig blood. Rotem measurements were similarly compromised in pig and human blood starting at 16×, except clot formation time was prolonged at 1× in human blood (all p < 0.05)., Conclusion: Ibuprofen inhibited platelet aggregation at recommended doses, and compromised coagulation at higher doses. Human blood was more sensitive to ibuprofen inhibition. Further effort is needed to investigate ibuprofen dose responses on coagulation in vivo., (Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.)

Background: Fibrinogen plays a central role in coagulation and falls to critical levels early after trauma. Administration of fibrinogen concentrate (FC) to improve hemostasis after severe bleeding seems beneficial, but it is unclear whether its use introduces excessive fibrinogen with a potential risk of thrombosis. This study investigated changes of endogenous fibrinogen metabolism from FC administration following traumatic hemorrhage in pigs., Methods: Anesthetized, instrumented pigs were randomized into lactated Ringer's (LR) solution only and LR plus FC groups (n = 7 each). Femur fracture of each pig's left leg was followed by hemorrhage of 60% total blood volume and resuscitation with LR (3× bled volume, LR group) or LR plus FC at 250 mg/kg (LR-FC group). Afterward, a constant infusion of stable isotopes 1-C-phenylalanine (phe, 6 hours) and d5-phe (3 hours) was performed with hourly blood sampling and subsequent gas chromatography-mass spectrometry analysis to quantify fibrinogen synthesis and breakdown rates, respectively. Blood gas and coagulation indices (thromboelastography) were measured on intermittent blood samples, and hemodynamics was continuously monitored. Animals were euthanized after the 6-hour isotope period., Results: Mean arterial pressure decreased by 50% after hemorrhage but improved after LR resuscitation in both groups. Hemorrhage and LR resuscitation reduced total protein, hematocrit, fibrinogen, and platelets to 50% of baseline values. Moreover, hemorrhage and resuscitation decreased fibrinogen concentration (207 ± 6 vs. 132 ± 7 mg/dL) and clot strength (72 ± 2 vs. 63 ± 2 mm) in both groups (p < 0.05). FC administration restored plasma fibrinogen concentrations and clot strength within 15 minutes, while no changes occurred in the LR group. Fibrinogen synthesis rates in the LR-FC group (1.3 ± 0.2 mg/kg/h) decreased versus the LR group (3.1 ± 0.5; p < 0.05), whereas fibrinogen breakdown rates were similar., Conclusion: Our data suggest an effective feedback mechanism that regulates host fibrinogen availability and thereby suggests that acute thrombosis from FC administration is an unlikely risk.

The aim of this study was to determine whether visceral protein levels increase under positive nitrogen balance during times of decrease in acute-phase reactant levels in patients with burn injury. This was a post hoc analysis of a prospective, interventional study approved by the local institutional review board. A total of 10 subjects between the ages of 18 and 72 with ≥ 20% total body surface area burn were enrolled over a 14-month period. Data were collected for five subjects (average age of 28 ± 8 years and total body surface area burn of 69 ± 15%) who met the inclusion criteria. Changes in visceral protein levels were examined along with nitrogen balance and acute-phase reactants when the subjects were on enteral nutrition, and the proteins were not examined during times of acute kidney injury. Descriptive statistics were performed, and linear regression was used to analyze the association of visceral proteins and nitrogen balance during times that acute-phase reactant levels were decreasing. The subjects received an average of 3044 ± 1613 kcal/day (39 ± 20 kcal/kg), meeting 72% of caloric goals and achieving positive nitrogen balance during 68% of the 40 weekly measurements, with 174 ± 85 g of protein intake per day (2.2 ± 1.1 g/kg). There was a weak relationship between nitrogen balance and changes in visceral protein levels during times that the acute-phase reactant levels were decreasing (P > .05). Visceral proteins were found to be poor markers of nutritional status. This study is unique because the subjects were able to achieve positive nitrogen balance despite severe burns.

Introduction: Ibuprofen is commonly used by Soldiers in the deployed environment. This study investigated its dose-effects on in vitro coagulation., Methods: Blood samples were collected from 4 normal healthy pigs and were processed to make platelet-adjusted (100×10(3)/μL) blood samples. Ibuprofen was added to the samples at doses of 0 μg/mL (control), recommended oral dose (163 μg/mL, 1×), 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Arachidonic acid or collagen-stimulated platelet aggregation was assessed at 15 minutes after the addition of ibuprofen. Coagulation was assessed with measurements of prothrombin time (PT) and activated partial thromboplastin time (aPTT), and thrombelastography by Rotem., Results: A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested. Collagen-stimulated platelet aggregation was inhibited to 71%±5% and 10%±5% of the control values at ibuprofen doses of 4× and 20×, respectively (both p<0.05). No changes were observed in PT at any dose, but aPTT was prolonged at dose of 16× and 20×. Rotem measurements of coagulation time, clot formation time, maximum clot firmness, and A10 were compromised at dose 16× and 20× (all p<0.05)., Conclusion: Ibuprofen inhibited platelet aggregation at recommended doses, but did not compromise aPTT or coagulation profile until at 16 times the recommended doses and higher. Further effort is needed to clarify whether there are different dose-responses between human and pig blood samples in trauma situations., (Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.)

Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

Introduction: Changes of plasma albumin and fibrinogen after various insults have been described as acute phase responses. This study investigated the acute changes of hepatic synthesis of albumin and fibrinogen after hemorrhage and resuscitation with lactated Ringer's (LR) solution or normal saline (NS) in pigs., Methods: Twenty anesthetized pigs were randomized into control (n = 6), LR solution (n = 7), and NS (n = 7) groups. Hemorrhage of 60% estimated blood volume was induced in the LR and NS groups by removing blood from the left femoral artery with a computer-controlled pump, followed by resuscitation with either LR solution at three times the bled volume, or NS to reach the same mean arterial pressure as in the LR group. Stable isotope 1-C-phenylalanine was infused for 6 h with hourly blood sampling and subsequent gas chromatography and mass spectrometry analysis to quantify hepatic protein synthesis., Results: Hemorrhage decreased mean arterial pressure and increased heart rate. Resuscitation with LR solution or NS corrected these changes. Compared with baseline, hemorrhage and resuscitation decreased albumin levels to 49% ± 2% and 44% ± 3% and fibrinogen levels to 50% ± 2% and 53% ± 2% in LR solution and NS (all P < 0.05), respectively. Albumin synthesis was impaired from 8.8 ± 1.4 mg/kg per hour (control) to 5.3 ± 0.8 mg/kg per hour in LR solution and 3.9 ± 0.6 mg/kg per hour in NS (both P < 0.05). No changes were observed in fibrinogen synthesis after hemorrhage and resuscitation with LR solution (4.4 ± 0.7 mg/kg per hour) or NS (3.3 ± 0.4 mg/kg per hour), compared with the control (3.5 ± 0.3mg/kg per hour)., Conclusions: Hemorrhage and resuscitation compromised albumin synthesis, but not fibrinogen synthesis. There were no differences in hepatic synthesis of albumin or fibrinogen between LR solution and NS resuscitation.

Background: Ongoing improvements in trauma care now recommend earlier use of blood products as part of damage control resuscitation, but generally these products are not available at far forward battlefield locations. For the military, questions continue to arise regarding efficacy of normal saline (NS) vs. lactated Ringer's (LR). Thus, this study compared the effects of LR and NS after severe hemorrhage in pigs., Methods: 20 anesthetized pigs were randomized into control (n = 6), LR (n = 7), and NS (n = 7) groups. Hemorrhage of 60% estimated total blood volume was induced in LR and NS groups by removing blood from the left femoral artery using a computer-controlled pump. Afterwards, the pigs were resuscitated with either LR at 3 times the bled volume or the volume of NS to reach the same mean arterial pressure (MAP) as in LR group. Hemodynamics were measured hourly and blood samples were taken at baseline (BL), 15 min, 3 h and 6 h after resuscitation to measure changes in coagulation using thrombelastograph®., Results: MAP was decreased by hemorrhage but returned to BL within 1 h after resuscitation with LR (119 ± 7 ml/kg) or NS (183 ± 9 ml/kg, p < 0.05). Base excess (BE) was decreased by hemorrhage; resuscitation with LR recovered BE but not with NS. Total peripheral resistance was decreased with NS and LR, with a larger drop shown in NS. Serum potassium was increased with NS, but not affected with LR. Coagulation changes were similar between LR and NS., Conclusions: NS may be inferior to LR in resuscitation due to its vasodilator effects and the risks of metabolic acidosis and hyperkalemia.

Background: This study compared coagulation function after resuscitation with Hextend and lactated Ringer's (LR) solution in pigs with tissue injury and hemorrhagic shock., Methods: Pigs were randomized into control (n = 7 each), LR, and Hextend groups. Femur fracture was induced using the captive bolt stunner at midshaft of the pigs' left legs, followed by hemorrhage of 60% total blood volume and resuscitation with either Hextend (equal to bled volume) or LR to reach the same mean arterial pressure. Pigs in the control group were not bled or resuscitated. Hemodynamics was monitored hourly for 6 hours. Blood samples were taken at baseline (BL), after hemorrhage, 15 minutes, 3 hours, and 6 hours after resuscitation for blood and coagulation measurements., Results: Mean arterial pressure decreased to 50% of BL by the 60% hemorrhage but returned to near BL within 1 hour after LR or Hextend resuscitation. Heart rate was increased (from 91 ± 4 beats per minute to 214 ± 20 beats per minute) by hemorrhage and decreased after resuscitation but remained elevated above BL in both groups. Resuscitation with Hextend (42 mL/kg) or LR (118 ± 3 mL/kg) reduced hematocrit, total protein, fibrinogen, and platelet counts, with greater decreases shown in the Hextend group. Clot strength was lower but returned to BL by 3 hours in the LR group, whereas it remained reduced for the 6-hour period after Hextend. The overall clotting capacity after LR was decreased after hemorrhage and resuscitation but returned to BL by 3 hours, whereas it remained low after Hextend for the 6-hour experiment period., Conclusion: After traumatic hemorrhage, coagulation function was restored within 6 hours with LR resuscitation but not with Hextend. The lack of recovery after Hextend is likely caused by greater hemodilution and possible effects of starches on coagulation substrates and further documents the need to restrict the use of high-molecular-weight starch in resuscitation fluids for bleeding casualties.

Background: Hemorrhagic shock contributes to coagulopathy after trauma. We investigated daily changes of coagulation components and coagulation function for 5 days in hemorrhaged and resuscitated pigs., Methods: Fourteen pigs were randomized into the sham control (C) and the hemorrhage and lactated Ringer's resuscitation (H-LR) groups. On day 1, hemorrhage was induced in the H-LR group by bleeding 35% of the total blood volume, followed by LR resuscitation at three times the bled volume. Pigs in the C group were not hemorrhaged or resuscitated. Hemodynamics and coagulation were measured daily after H-LR on day 1 to day 5., Results: No changes in hemodynamics and coagulation function occurred in C. Hemorrhage decreased mean arterial pressure and increased heart rate. LR resuscitation corrected these changes within 2 hours. Compared with the baseline values (BL) on day 1, fibrinogen levels were decreased to 76% ± 6% by H-LR on day 1, increased to 217% ± 16% on day 2, and remained increased thereafter; platelet counts were decreased to 63% ± 5% by H-LR on day 1 and remained lower on days 2 and 3 but returned to BL by days 4 and 5 (all p < 0.05). Thrombin generation was decreased by H-LR on days 1 and 2 but then increased to above BL on days 4 and 5. Coagulation factor levels were decreased by H-LR on day 1 but returned to BL on day 3 except for factor XIII. Clot strength was decreased by H-LR on day 1 and returned to BL by day 2. Clot rapidity did not change on day 1 but was decreased on days 2 and 3 and returned to BL on days 4 and 5., Conclusion: Hemorrhage and resuscitation reduced coagulation components and compromised coagulation function, which showed different recovery profiles over the 5-day study period.

This study's objective was to investigate the daily dynamics of fibrinogen metabolism and coagulation function for 5 days after hemorrhagic shock in pigs. Sixteen pigs were randomized into the control (C) and the hemorrhage (H) groups. On day 1, hemorrhage was induced in H by bleeding 35% of the estimated total blood volume, followed by resuscitation with lactated Ringer's solution at three times the bled volume. Then, a primed constant infusion of stable isotopes was performed in both groups daily for 5 days to measure changes in fibrinogen metabolism, together with changes in hemodynamics and coagulation function. Hemorrhage caused a decrease in mean arterial pressure and an increase in heart rate. Fluid resuscitation corrected these changes. Compared with baseline day 1, fibrinogen levels in H were decreased to 76% ± 6% by hemorrhage and resuscitation on day 1, increased to 217% ± 16% on day 2, and remained elevated afterward; clot strength in H was decreased by hemorrhage on day 1 and returned to baseline values on day 2 and afterward. Compared with day 1 control value (1.3 ± 0.1 mg/kg per hour), fibrinogen synthesis in H was increased to 3.6 ± 0.1, 5.1 ± 0.5, 2.6 ± 0.4, 2.7 ± 0.5, and 2.3 ± 0.3 mg/kg per hour on days 1 through 5 (all P < 0.05); fibrinogen breakdown in H was elevated on days 1 and 2 but returned to control values afterward. Hemorrhage caused acute decreases in fibrinogen concentration and clot strength, followed by an increase in fibrinogen concentration and recovery of clot strength. The increase in fibrinogen appeared primarily due to a sustained increase in fibrinogen synthesis.

Background: Hemorrhagic shock and trauma are associated with acidosis and altered coagulation. A fall in pH has been reported to attenuate the activity of recombinant activated Factor VII (rFVIIa) in vitro. However, it is not known if acidosis induced by hemorrhagic shock or infusion of HCl attenuates FVIIa activity in vivo. The purpose of this study was to determine if acidosis, induced by two methods, affects recombinant FVIIa (rFVIIa) activity in swine, and if correction of the pH restores rFVIIa activity to normal., Methods: Acidosis was induce in anesthetized swine in two separate models: 1) HCl infusion (n=10) and 2) hemorrhage/hypoventilation (n=8). Three groups per model were used: Control (pH7.4), Acidosis (arterial pH7.1) and Acidosis-Corrected (bicarbonate infusion to return pH from 7.1 to 7.4). Pigs were then injected with rFVIIa (90 μg/kg) or vehicle (saline) at target pH and arterial blood samples were taken for measurement of coagulation function, including Thromboelastography -TEG, Thrombin Generation, Activated Clotting Time, Prothrombin Time, activated Partial Thromboplastin Time, Fibrinogen Concentration and Platelet count before and 5min after injection of rFVIIa., Results: Acidosis led to a hypocoagulation as measured by almost all coagulation parameters in both models. Furthermore, the change in coagulation function produced after infusion of rFVIIa was not different between control, acidosis and acidosis-corrected groups for all coagulation parameters measured., Conclusion: Acidosis associated with hemorrhagic shock or HCl infusion led to a hypocoagulation that was not corrected with bicarbonate infusion. Furthermore, acidosis did not affect rFVIIa function, and correction of the acidosis with bicarbonate had no effect on rFVIIa function in these models. This suggests that in vivo acidosis did not diminish rFVIIa function.

Background: As part of our overall interest in the mechanisms and treatment related to the development of the lethal triad of hypothermia, acidosis, and coagulopathy seen in trauma patients, the purpose of this study was to determine whether acidosis, inducible either by HCl infusion or hemorrhage/hypoventilation, leads to coagulopathy, and if correction of the acidosis will alleviate this coagulopathy., Methods: In two separate experiments, acidosis was induced in anesthetized swine by (1) HCl infusion (n = 10) or (2) hemorrhage/hypoventilation (n = 8). Arterial blood samples were taken before HCl infusion or hemorrhage (arterial pH 7.4), after HCl infusion or hemorrhage (pH 7.1), and after bicarbonate infusion to return pH to 7.4. Arterial pH and blood gases were measured every 15 minutes., Results: Acidosis (arterial pH 7.1) led to a hypocoagulation as measured by several coagulation parameters. In both experiments, acidosis was associated with a significant decrease in the maximum strength of the clot and the rate at which the clot formed. There was a significant decrease in endogenous thrombin potential and maximum thrombin concentration after acidosis in both groups (thrombin generation assay). However, the activated clotting time, prothrombin time, and activated partial thromboplastin time were significantly elevated only in the HCl-infused group. Fibrinogen concentration and platelet count were significantly reduced in both groups after acidosis. The hypocoagulation that was induced by either hemorrhage/hypoventilation or HCl infusion was not immediately corrected after returning pH to 7.4 with bicarbonate injection., Conclusions: These data suggest that acidosis induced by HCl infusion or by hemorrhage/hypoventilation leads to hypocoagulation. Simple correction of the arterial pH with bicarbonate is not sufficient to correct this coagulopathy.

Hemorrhagic coagulopathy (without neurological injuries) constitutes 40% of injury-related death in civilian hospitals and on the battlefield, and the underlying contributing mechanisms remain unclear. The purpose of this study is to investigate the effects of fibrinogen availability on coagulation function after hemorrhage in pigs. Sixteen crossbred commercial Yorkshire swine were randomized into the control group (group C) (n = 8) and hemorrhage group (group H) (n = 8). Hemorrhage was induced in group H by bleeding 35% of the estimated total blood volume, followed by resuscitation with lactated Ringer solution at three times the bled volume. Pigs in group C were not hemorrhaged or resuscitated. Blood samples were withdrawn at baseline, 15 min, 3 h, 6 h, and 24 h after hemorrhage and lactated Ringer (LR) resuscitation (H-LR). Coagulation was assessed by using thrombelastography. All baseline measurements were similar between groups C and H. Hemorrhage caused a decrease in mean arterial pressure and an increase in heart rate in group H, but LR resuscitation corrected these changes within 1 h. Compared to baseline values, fibrinogen concentrations in group H decreased at 15 min, 3 h and 6 h after H-LR, but increased to double that of the baseline value at 24 h; platelet counts decreased throughout the study; clot strength was decreased at 15 min, 3 h and 6 h, but returned to baseline value at 24 h after H-LR. Hemorrhage caused decreases in fibrinogen and platelets, and compromised clot strength. The rebound of fibrinogen at 24 h restored clot strength despite platelet deficit. These data suggest the potential compensatory role of fibrinogen in restoring coagulation function in vivo after hemorrhagic shock.

Albumin plays an important role in maintaining physiological homeostasis. Although decreased albumin concentration has been well described as an acute-phase response following injury, it is unclear whether the decrease is due to compromised synthesis of albumin, dilution, or imbalance between synthesis and breakdown rates, particularly after injury. We investigated changes in albumin synthesis in severely burned patients using stable isotope infusion techniques. Five patients (29 ± 3 years; 80 ± 7 kg) with burn of 48% ± 4% total body surface area (TBSA) were enrolled and studied in the ICU at the Burn Unit of the US Army Institute of Surgical Research. Five age- and sex-matched healthy volunteers (33 ± 5 years; 81 ± 6 kg) were included as controls. On the study day (13 ± 3 days after burn), a primed constant infusion (4 h) of stable isotope d5-phenlylalanine and d3-ketoisocaproic acid was given. Hourly arterial blood samples were drawn during the infusion to determine albumin synthesis rates, using gas chromatography-mass spectrometry analysis. Burned patients had higher heart and respiration rates. Plasma total protein in burn patients (4.5 ± 0.3 g · dL-1) was lower compared with controls (6.8 ± 0.2 g · dL-1). Plasma albumin concentration in burn patients (1.1 ± 0.1 g · dL-1) was also lower compared with controls (3.8 ± 0.1 g · dL-1; both P < 0.05). Albumin synthesis rate in burn patients (4.6 ± 0.2 mg · kg-1 · h-1) was enhanced compared with controls (2.2 ± 0.2 mg · kg-1 · h-1; P < 0.05). Despite the decrease in albumin concentration, albumin synthesis was enhanced in severely burned patients during the flow phase.

Objectives: To investigate the hemostatic status of critically ill, nonbleeding trauma patients. We hypothesized that a hypercoagulable state exists in patients early after severe injury and that the pattern of clotting and fibrinolysis are similar between burned and nonburn trauma patients., Materials: Patients admitted to the surgical or burn intensive care unit within 24 hours after injury were enrolled. Blood samples were drawn on days 0 through 7. Laboratory tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), levels of activated factor XI, D-dimer, protein C percent activity, antithrombin III percent activity, and thromboelastography (TEG)., Results: Study subjects were enrolled from April 1, 2004, to May 31, 2005, and included nonburn trauma patients (n = 33), burned patients (n = 25), and healthy (control) subjects (n = 20). Despite aggressive thromboprophylaxis, three subjects (2 burned and 1 nonburn trauma patients [6%]) had pulmonary embolism during hospitalization. Compared with controls, all patients had prolonged PT and aPTT (p < 0.05). The rate of clot formation (alpha angle) and maximal clot strength were higher for patients compared with those of controls (p < 0.05), indicating a hypercoagulable state. Injured patients also had lower protein C and antithrombin III percent activities and higher fibrinogen levels (p < 0.05 for all). Activated factor XI was elevated in 38% of patients (control subjects had undetectable levels)., Discussion: Thromboelastography analysis of whole blood showed that patients were in a hypercoagulable state; this was not detected by plasma PT or aPTT. The high incidence of pulmonary embolism indicated that our current prophylaxis regimen could be improved.

Background: Hypothermia and hemorrhagic shock contribute to coagulopathy after trauma. In this study, we investigated the independent and combined effects of hypothermia and hemorrhage with resuscitation on coagulation in swine and evaluated clinically relevant tests of coagulation., Methods: Pigs (n = 24) were randomized into four groups of six animals each: sham control, hypothermia, hemorrhage with resuscitation, and hypothermia, hemorrhage with resuscitation combined. Hypothermia to 32 degrees C was induced with a cold blanket. Hemorrhage was induced by bleeding 35% of total blood volume followed by resuscitation with lactated Ringer's solution. Coagulation was assessed by thrombin generation, prothrombin time (PT), activated partial thromboplastin time (aPTT), activated clotting time (ACT), and thrombelastography (TEG) from blood samples taken at baseline and 4 hour after hypothermia and/or hemorrhage with resuscitation. Data were compared with analysis of variance., Results: Baseline values were similar among groups. There were no changes in any measurements in the control group. Compared with baseline values, hemorrhage with resuscitation increased lactate to 140% +/- 15% (p < 0.05). Hypothermia decreased platelets to 73% +/- 3% (p < 0.05) with no effect on fibrinogen. Hemorrhage with resuscitation reduced platelets to 72% +/- 4% and fibrinogen to 71% +/- 3% (both p < 0.05), with similar decreases in platelets and fibrinogen observed in the combined group. Thrombin generation was decreased to 75% +/- 4% in hypothermia, 67% +/- 6% in hemorrhage with resuscitation, and 75% +/- 10% in the combined group (all p < 0.05). There were no significant changes in PT or aPTT by hemorrhage or hypothermia. ACT was prolonged to 122% +/- 1% in hypothermia, 111% +/- 4% in hemorrhage with resuscitation, and 127% +/- 3% in the combined group (all p < 0.05). Hypothermia prolonged the initial clotting time (R) and clot formation time (K), and decreased clotting rapidity (alpha) (all p < 0.05). Hemorrhage with resuscitation only decreased clot strength (maximum amplitude [MA], p < 0.05). TEG parameters in the combined group reflected the abnormal R, K, MA, and alpha observed in the other groups., Conclusion: Hypothermia inhibited clotting times and clotting rate, whereas hemorrhage impaired clot strength. Combining hypothermia with hemorrhage impaired all these clotting parameters. PT, aPTT were not sensitive whereas ACT was not specific in detecting these coagulation defects. Only TEG differentiated mechanism related to clotting abnormalities, and thus may allow focused treatment of clotting alterations associated with hypothermia and hemorrhagic shock.

Background: To treat the coagulopathy of trauma, some have suggested early and aggressive use of cryoprecipitate as a source of fibrinogen. Our objective was to determine whether increased ratios of fibrinogen to red blood cells (RBCs) decreased mortality in combat casualties requiring massive transfusion., Methods: We performed a retrospective chart review of 252 patients at a U.S. Army combat support hospital who received a massive transfusion (>or=10 units of RBCs in 24 hours). The typical amount of fibrinogen within each blood product was used to calculate the fibrinogen-to-RBC (F:R) ratio transfused for each patient. Two groups of patients who received either a low (<0.2 g fibrinogen/RBC Unit) or high (>or=0.2 g fibrinogen/RBC Unit) F:R ratio were identified. Mortality rates and the cause of death were compared between these groups, and logistic regression was used to determine if the F:R ratio was independently associated with survival., Results: Two-hundred and fifty-two patients who received a massive transfusion with a mean (SD) ISS of 21 (+/-10) and an overall mortality of 75 of 252 (30%) were included. The mean (SD) F:R ratios transfused for the low and high groups were 0.1 grams/Unit (+/-0.06), and 0.48 grams/Unit (+/-0.2), respectively (p < 0.001). Mortality was 27 of 52 (52%) and 48 of 200 (24%) in the low and high F:R ratio groups respectively (p < 0.001). Additional variables associated with survival were admission temperature, systolic blood pressure, hemoglobin, International Normalized Ratio (INR), base deficit, platelet concentration and Combined Injury Severity Score (ISS). Upon logistic regression, the F:R ratio was independently associated with mortality (odds ratio 0.37, 95% confidence interval 0.171-0.812, p = 0.013). The incidence of death from hemorrhage was higher in the low F:R group, 23/27 (85%), compared to the high F:R group, 21/48 (44%) (p < 0.001)., Conclusions: In patients with combat-related trauma requiring massive transfusion, the transfusion of an increased fibrinogen: RBC ratio was independently associated with improved survival to hospital discharge, primarily by decreasing death from hemorrhage. Prospective studies are needed to evaluate the best source of fibrinogen and the optimal empiric ratio of fibrinogen to RBCs in patients requiring massive transfusion.

Objective: Uncontrolled bleeding from coagulopathy signals imminent death in severely injured patients. Acidosis is an important predictor of coagulopathy, but the underlying contributing mechanisms are unclear. This study was designed to investigate the effects of acidosis on fibrinogen metabolism and coagulation function in a swine model., Methods: Twelve pigs were randomly divided into the control (n = 6) and acid (n = 6) groups. Acidosis of pH 7.1 was induced by infusion of 0.2 M HCl in lactated Ringer solution in the acid group. Afterward, an infusion of stable isotope 1-13C-phenylalanine (6 hours) and d5-phenylalanine (4 hours) was performed. Blood samples were withdrawn hourly to quantify fibrinogen synthesis and degradation rates using gas chromatograph and mass spectrometry analysis. To correlate changes in fibrinogen metabolism, coagulation changes were assessed by prolonged prothrombin time, partial activated thromboplastin time, activated clotting time, and thrombelastograph (TEG)., Results: Acidosis caused decreases in mean arterial pressure, arterial bicarbonate concentration, base excess, fibrinogen concentration, and platelet counts. Acidosis increased fibrinogen degradation rate from the control value of 4.3 +/- 1.0 mg/kg/h to 11.8 +/- 1.4 mg/kg/h (P < 0.05), with no effect on fibrinogen synthesis. Prolonged prothrombin time, partial activated thromboplastin time, activated clotting time were consistently prolonged by acidosis (all P < 0.05). Clotting rapidity (angle alpha in TEG) was decreased from a baseline value of 73.3 +/- 1.1 degree to 63.0 +/- 2.4 degree (P < 0.05). Clot strength (maximum amplitude in TEG) was decreased from a baseline value of 72.2 +/- 1.4 mm to 56.2 +/- 3.1 mm (P < 0.05)., Conclusions: Acidosis compromised the clotting process and accelerated fibrinogen consumption with no effect on fibrinogen production, resulting in a deficit in fibrinogen availability.

Objective: To investigate the effect of tris-hydroxymethylaminomethane (THAM) pH neutralization on reversing coagulation abnormalities caused by acidosis., Design: Random and controlled study., Setting: Animal research facility and laboratory., Subjects: Yorkshire swine (n = 18)., Interventions: Acidosis was induced in 12 pigs by infusing 0.2 M hydrochloric acid (HCl). When the target pH of 7.1 was achieved, the pigs were infused with either 0.3 M THAM to achieve pH of 7.4 (intervention group) or an equal volume of lactated Ringer's solution (acid control group)., Measurements and Main Results: Blood samples were taken at baseline, 15 mins after reaching pH of 7.1, and 15 mins after THAM pH neutralization. Coagulation function was assessed by thrombin generation, prothrombin time, activated partial thromboplastin time, activated clotting time, and thromboelastography (maximum clot formation time [R+K], clotting rapidity [alpha], and clot strength [maximum amplitude]). An additional six pigs (sham group) were infused with THAM, and an equal volume of fluid as the 12 coagulopathic pigs was given to assess effects of THAM and hemodilution. Comparisons were made using a mixed model analysis of variance. No change in any indexes of coagulation was observed in sham pigs. Compared with baseline, acidosis of pH 7.1 decreased base excess from 6.6 +/- 0.5 mM to -12.4 +/- 0.5 mM; reduced fibrinogen levels to 72% +/- 2%, platelet counts to 53% +/- 3%, thrombin generation to 58% +/- 4%, alpha to 84% +/- 2%, and maximum amplitude to 75% +/- 3%; and prolonged prothrombin time to 113% +/- 2%, partial thromboplastin time to 122% +/- 4%, activated clotting time to 124% +/- 3%, and R + K to 119% +/- 3% (all p < .05). THAM infusion corrected pH to 7.40 +/- 0.02 and base excess to 2.6 +/- 0.9 mM (p < .05). However, there were no differences in thrombin generation, prothrombin time, partial thromboplastin time, activated clotting time, R+K, alpha, or maximum amplitude between the groups with or without pH correction., Conclusions: Acidosis impaired coagulation by depleting clotting factors, inhibiting thrombin generation, and affecting clot strength and stability. THAM corrected acid-base deficit but did not acutely reverse the coagulation abnormalities in the model.

Hemorrhagic coagulopathy is a significant complication after traumatic injury, and much of the underlying mechanism remains unclear. We investigated the changes in fibrinogen metabolism and coagulation after a moderate hemorrhage and resuscitation. Pigs of either sex (weight, 40.9+/-0.8 kg) were anesthetized and instrumented with arterial and venous catheters and a thermodilution cardiac output catheter. Pigs were randomized into control (C; n=6), hemorrhage (H; n=6), and hemorrhage and resuscitation (H-LR; n=6) groups. Hemorrhage was induced by bleeding 35% of total blood volume for 30 min in H and H-LR groups. Resuscitation in H-LR group was performed using lactated Ringer's solution (LR) at 3 times the bled volume for 30 min. Fibrinogen metabolism was quantified using a primed constant infusion of 1-13C-phenylalanine (6 h) and d5-phenylalanine (4 h) and subsequent analysis by gas chromatograph-mass spectrometry, together with measurements of hemodynamics (hourly) and coagulation by thromboelastography (at baseline and 4 h after hemorrhage and resuscitation). Hemorrhage caused decreases in arterial pH and base excess, and an increase in arterial lactate content. Fluid resuscitation corrected these changes toward normal levels. Fibrinogen level was unchanged in C and decreased to 76%+/-4% in H and to 73%+/-3% in H-LR (both P<0.05, compared with baseline) after hemorrhage and resuscitation. Fibrinogen breakdown was increased from 3.0+/-0.4 mg kg-1 h-1 in C to 5.4+/-0.6 mg kg-1 h-1 in H and to 5.6+/-0.5 mg kg-1 h-1 in H-LR (both P<0.05, compared with control), but synthesis was unchanged. The clotting reaction time was unchanged in C and shortened to 93%+/-3% in H and to 91%+/-1% in H-LR (both P<0.05, compared with baseline). We conclude that hemorrhagic shock caused accelerated fibrinogen breakdown and coagulation. The LR resuscitation reduced tissue hypoxia indexes but did not affect the changes in fibrinogen metabolism and coagulation from hemorrhage. Thus, effective treatment of hemorrhage should include combining standard-of-care resuscitation with interventions to correct alterations in coagulation.

Background: Coagulopathy is an important contributor to morbidity and mortality in trauma patients. Acidosis contributes to coagulopathy. Acidosis can be neutralized with intravascular bicarbonate, but it is unclear if the coagulation defect is rapidly reversed. The effects of acidosis and bicarbonate neutralization on coagulation function were investigated in vivo., Methods: Acidosis was induced in 12 pigs by infusing 0.2 mol/L HCl to pH 7.1. Pigs were then infused with either LR to maintain a pH of 7.1 (A-LR, n = 6) or 0.3 mol/L bicarbonate to a pH of 7.4 (A-Bi, n = 6). Blood samples were taken at baseline, 15 minutes after acidosis induction, and 15 minutes after bicarbonate neutralization. Coagulation function was assessed by prothrombin time (PT), partial thromboplastin time (PTT), thrombin generation, initial clot formation time (R), clotting rapidity (alpha), and clot strength (MA)., Results: Compared with baseline values, acidosis reduced fibrinogen concentration to 66% +/- 2% in A-LR and to 71% +/- 3% in A-Bi, and decreased platelet counts to 49% +/- 4% in A-LR and to 53% +/- 4% in A-Bi. Thrombin generation decreased to 60% +/- 4% in A-LR and to 53% +/- 7% in A-Bi. Acidosis prolonged PT and PTT about 20% and decreased alpha and MA. After pH neutralization, fibrinogen and platelet levels remained depleted and no reversal of acidosis-induced changes in thrombin generation, PT, PTT, alpha, and MA were observed., Conclusion: Acidosis impaired coagulation by depleting fibrinogen and platelets and by inhibiting clotting kinetics. The deficit associated with acidosis was not reversed with bicarbonate pH neutralization.

Hemorrhagic coagulopathy is involved in the morbidity and mortality of trauma patients. Nonetheless, many aspects of the mechanisms underlying this disorder are poorly understood. We have therefore investigated changes in fibrinogen metabolism and coagulation function after a moderate hemorrhagic shock, using a new stable isotope approach. Twelve pigs were randomly divided into the control (C) and hemorrhage (H) groups. Hemorrhage was induced by bleeding 35% total blood volume over a 30-min period. A primed constant infusion of [1-(13)C]phenylalanine (Phe), d5-phenylalanine, and alpha-[1-(13)C]-ketoisocaproate (KIC) was given to quantify fibrinogen synthesis and breakdown, together with measurements of circulating liver enzyme activities and coagulation function. Mean arterial pressure was decreased by hemorrhage from 89 +/- 4 mmHg in C to 47 +/- 4 mmHg in H (P < 0.05), followed by a rebound to 68 +/- 5 mmHg afterward. Fibrinogen fractional synthesis rate increased from 2.7 +/- 0.2%/h in C to 4.2 +/- 0.4%/h in H by Phe (P < 0.05) and from 3.1 +/- 0.4%/h in C to 4.4 +/- 0.5%/h in H by KIC (P < 0.05). Fibrinogen fractional breakdown rate increased from 3.6 +/- 1.0%/h in C to 12.9 +/- 1.8%/h in H (P < 0.05). The absolute breakdown rate accelerated from 3.0 +/- 0.4 mg x kg(-1) x h(-1) in C to 5.4 +/- 0.6 mg x kg(-1) x h(-1) in H (P < 0.05), but the absolute synthesis rate remained unchanged. These metabolic changes were accompanied by a reduction in blood clotting time to 92.7 +/- 1.6% of the baseline value by hemorrhage (P < 0.05). No changes were found in liver enzyme activities. We conclude that the observed changes in coagulation after hemorrhagic shock are mechanistically related to the acute acceleration of fibrinogen degradation.

Background: Clinical coagulopathy occurs frequently in the presence of acidosis and hypothermia. The purpose of this study was to determine the relative contributions of acidosis and hypothermia to coagulopathy, as measured by current standard bedside and clinical laboratory analyses (i.e., bleeding time and prothrombin time). In addition, we investigated possible mechanisms of these effects using a modified prothrombin time test, thromboelastography, and thrombin kinetics analyses. An improved understanding of coagulopathy should facilitate hemorrhage control., Methods: Twenty-four pigs were randomly allocated into normal (pH, 7.4; 39 degrees C), acidotic (pH, 7.1; 39 degrees C), hypothermic (pH, 7.4; 32 degrees C), and acidotic and hypothermic (pH, 7.1; 32 degrees C) combined groups. Acidosis was induced by the infusion of 0.2N hydrochloric acid in lactated Ringer's solution. Hypothermia was induced by using a blanket with circulating water at 4 degrees C. Development of a clinical coagulopathy was defined as a significant increase in splenic bleeding time. Measurements were compared before (pre) and 10 minutes after (post) the target condition was achieved., Results: Acidosis, hypothermia, or both caused the development of coagulopathy, as indicated by 47%, 57%, and 72% increases in splenic bleeding time (p < 0.05, pre vs. post). Plasma fibrinogen concentration was decreased by 18% and 17% in the acidotic and combined groups, respectively, but not in the hypothermic group. Hypothermia caused a delay in the onset of thrombin generation, whereas acidosis primarily caused a decrease in thrombin generation rates. At 4 minutes' quench time, thrombin generation in the acidotic, hypothermic, and combined groups were 47.0%, 12.5%, and 5.7%, respectively, of the value in the control group. There were no changes in serum tumor necrosis factor-alpha and interleukin-6 in any group during the study., Conclusion: Acidosis and hypothermia cause a clinical coagulopathy with different thrombin generation kinetics. These results confirm the need to prevent or correct hypothermia and acidosis and indicate the need for improved techniques to monitor coagulopathy in the trauma population.

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.

We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.

We assessed the approach of using intracellular free amino acid enrichment as precursor enrichment for calculating the fractional synthetic rate of using the stable isotope tracer incorporation technique. We assumed that the true rate of protein synthesis was reflected by the rate of tracer incorporation over time divided by the plateau enrichment in protein. Isolated human fibroblasts and myocytes were cultured in medium supplemented with [(15)N]glycine, [(15)N]proline, and [d(5)]phenylalanine. Culture medium and cells were collected daily from d 1 to 5. A portion of cells harvested on d 5 was subcultured for an additional 3 passages to d 20. Protein enrichments in both cell types reached a plateau after 20 d of cell culture. In fibroblasts, the true protein synthesis rates were 0.74, 0.85, and 0.86%/h, using protein plateau enrichments of [(15)N]glycine, [(15)N]proline, and [d(5)]phenylalanine as precursor enrichments, respectively. When the corresponding intracellular free amino acid enrichments were used, protein synthesis rates were 0.76, 0.79, and 0.76%/h, respectively. Similarly, in myocytes, the true protein synthesis rates were 0.98 and 1.14%/h by protein plateau enrichments of [(15)N]glycine and [d(5)]phenylalanine, respectively. The synthesis rates were 0.94 and 1.01%/h by the corresponding intracellular enrichments, respectively. Extracellular amino acid enrichments resulted in underestimation of protein synthesis by a variable amount. We conclude that the intracellular free amino acid enrichment is an optimal surrogate for precursor enrichment to quantify protein synthesis.

We have quantified DNA synthetic rates from different pathways in cultured cells using a new stable isotope technique. Human fibroblasts and myocytes were grown in culture media supplemented with [U-(13)C(6)]glucose and [(15)N]glycine. The cells were sampled daily from day 1 to day 5. A portion of the cells harvested at day 5 was subcultured for an additional 3 passages to reach isotopic plateau. In both cell types total DNA fractional synthetic rate (FSR) was found to agree closely with the rate of cell proliferation determined by cell counting (FSR = 0.94%. h(-1) v 0.92%. h(-1) for DNA synthesis and cell count, respectively, in myocytes and 0.85%. h(-1) v 0.91%. h(-1) for DNA synthesis and cell count, respectively, in fibroblasts). In fibroblasts the deoxyribonucleoside salvage pathway accounted for over 70% of total DNA synthesis. In myocytes the deoxyribonucleoside salvage pathway was minimal, whereas the de novo base synthesis pathway accounted for almost 80% of total DNA synthesis. We conclude that the contributions of various pathways to DNA synthesis are highly dependent on cell type. This new stable isotope technique can be modified for application in in vivo studies.

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-(13)C(3)]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 +/- 3.3% and 0.078 +/- 0.009 micromol x min(-1) x g(-1). Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 +/- 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 +/- 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.

Objectives: We investigated the effect of various dietary fats on pulmonary surfactant composition and lung function changes that occur before and after endotoxin infusion in pigs., Methods: Eighteen pigs were assigned to three groups (n = 6 per group) to receive a diet of protein (20% of calories), carbohydrate (20% of calories), and fat (40% of calories). In one group the fat content consisted entirely of palmitic acid. In the second group, fat came from Intralipid, which provided predominantly linoleic acid. The third group was fed fish oil. Pigs were maintained on these diets for 21 d before the experiment. Cardiovascular and pulmonary functions were determined on day 22. Pigs then were infused with endotoxin (80 mg. kg(-1). min(-1)) until the pulmonary arterial pressure reached a pressure similar to that found in trauma victims (45 to 50 mmHg). Cardiovascular and pulmonary function tests were then repeated, the animals killed, and the lungs removed for study., Results: Compliance was reduced in the linoleate and fish-oil groups compared with the palmitate group before and after endotoxin. Compliance changes in pigs fed the linoleate and fish-oil diets were consistent with significant increases in lung wet:dry weight ratios, increased CO(2) retention, histologic evidence of vascular congestion, intra-alveolar edema, and alveolar septa thickening. Changes in surfactant phosphatidylcholine composition between groups were consistent with the notion that increased unsaturated fatty acids could affect surfactant function., Conclusions: We concluded that the common practice of providing calories in the form of polyunsaturated fatty acids to critically ill patients carries the risk of being detrimental to lung function.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine., ((c) 2002 Elsevier Science (USA).)