reversal of acute coagulopathy during hypotensive resuscitation

6
Crit Care Med 2012 Vol. 40, No. 8 2417 H emorrhage is responsible for up to 50% of trauma deaths on the battlefield (1, 2) and 30% to 40% of deaths in the civilian population with one third to one half occurring in the prehospital environ- ment (3). Despite significant advances in blood control technologies, there remains an urgent need to promptly resuscitate and stabilize critically wounded soldiers or civilians (4). Two forms of blood loss occur: 1) anatomical bleeding from the site of injury and 2) coagulopathic bleed- ing that arises early as a result of severe trauma and tissue hypoperfusion (5, 6). Coagulopathic bleeding was first reported in the Vietnam War (7) and rediscovered some 40 yrs later by Brohi (6). Several large trials have shown that 24% to 36% of severely injured patients have acute traumatic coagulopathy at admission to the emergency department (mean arrival time 75 mins), and it was associated with a four-fold increase in mortality (6, 8, 9). Acute traumatic coagulopathy is routinely diagnosed from increased plasma acti- vated partial thromboplastin time (aPTT) and prothrombin time (PT), and it is dif- ferent from hypercoagulable disseminated intravascular coagulation, as is often seen in early sepsis (8). Thus in the critically injured, there is an urgent need to: 1) ade- quately resuscitate mean arterial pressure (MAP) to minimize shock and perfuse vital organs and 2) aggressively correct the coagulopathy as early as possible in out- of-hospital and military environments. Previously, we showed that a small intravenous bolus (0.3 mL) of 7.5% NaCl with adenocaine (AL) and magnesium (Mg 2+ ) (without colloids) resuscitated MAP into a hypotensive range in the rat model of severe (40%) to catastrophic (60%) *See also p. 2519. From the Heart Research Laboratory (HLL, LPM, GPD), Department of Physiology and Pharmacology, School of Biomedical Sciences and School of Pharmacy and Molecular Science (NMP), James Cook University, Queensland, Australia. Current address for Dr. Pecheniuk: Institute of Health and Biomedical Innovation, Queensland University of Technology, Queensland, Australia. Dr. Dobson consulted for Hibernation Therapeutics and has equity interest and stock ownership in the company. He is also the inventor of Adenocaine Technology for organ protection and preservation, in- cluding trauma. The remaining authors have not dis- closed any potential conflicts of interest. For information regarding this article, Email: [email protected] Objective: Acute traumatic coagulopathy occurs early in hem- orrhagic trauma and is a major contributor to mortality and mor- bidity. Our aim was to examine the effect of small-volume 7.5% NaCl adenocaine (adenosine and lidocaine, adenocaine) and Mg 2+ on hypotensive resuscitation and coagulopathy in the rat model of severe hemorrhagic shock. Design: Prospective randomized laboratory investigation. Subjects: A total of 68 male Sprague Dawley Rats. Intervention: Post-hemorrhagic shock treatment for acute trau- matic coagulopathy. Measurements and Methods: Nonheparinized male Sprague- Dawley rats (300–450 g, n = 68) were randomly assigned to either: 1) untreated; 2) 7.5% NaCl; 3) 7.5% NaCl adenocaine; 4) 7.5% NaCl Mg 2+ ; or 5) 7.5% NaCl adenocaine/Mg 2+ . Hemorrhagic shock was induced by phlebotomy to mean arterial pressure of 35–40 mm Hg for 20 mins (~40% blood loss), and animals were left in shock for 60 mins. Bolus (0.3 mL) was injected into the femoral vein and hemodynamics monitored. Blood was collected in Na citrate (3.2%) tubes, centrifuged, and the plasma snap frozen in liquid N 2 and stored at −80°C. Coagulation was assessed using activated partial thromboplastin times and prothrombin times. Results: Small-volume 7.5% NaCl adenocaine and 7.5% NaCl adenocaine/Mg 2+ were the only two groups that gradually increased mean arterial pressure 1.6-fold from 38–39 mm Hg to 52 and 64 mm Hg, respectively, at 60 mins (p < .05). Baseline plasma activated partial thromboplastin time was 17 ± 0.5 secs and increased to 63 ± 21 secs after bleeding time, and 217 ± 32 secs after 60-min shock. At 60-min resuscitation, activated par- tial thromboplastin time values for untreated, 7.5% NaCl, 7.5% NaCl/Mg 2+ , and 7.5% NaCl adenocaine rats were 269 ± 31 secs, 262 ± 38 secs, 150 ± 43 secs, and 244 ± 38 secs, respectively. In contrast, activated partial thromboplastin time for 7.5% NaCl adenocaine/Mg 2+ was 24 ± 2 secs (p < .05). Baseline prothrombin time was 28 ± 0.8 secs (n = 8) and followed a similar pattern of correction. Conclusions: Plasma activated partial thromboplastin time and prothrombin time increased over 10-fold during the bleed and shock periods prior to resuscitation, and a small-volume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg 2+ was the only treatment group that raised mean arterial pressure into the permissive range and returned activated partial thromboplastin time and prothrombin time clotting times to baseline at 60 mins. (Crit Care Med 2012; 40: 2417–2422) KEY WORDS: adenocaine; adenosine; coagulopathy; hemorrhagic; hypertonic saline; hypocoagulopathy; lidocaine; magnesium; military; prehospital; resuscitation; shock; small volume; trauma Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0b013e31825334c3 Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg 2+ in the rat model of severe hemorrhagic shock* Hayley L. Letson, BSc; Natalie M. Pecheniuk, PhD; Lebo P. Mhango, BSc; Geoffrey P. Dobson, PhD

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Reversal of Acute Coagulopathy During Hypotensive Resuscitation

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Page 1: Reversal of Acute Coagulopathy During Hypotensive Resuscitation

Crit Care Med 2012 Vol. 40, No. 8 2417

Hemorrhage is responsible for up to 50% of trauma deaths on the battlefield (1, 2) and 30% to 40% of deaths in the

civilian population with one third to one half occurring in the prehospital environ-ment (3). Despite significant advances in blood control technologies, there remains an urgent need to promptly resuscitate and stabilize critically wounded soldiers or civilians (4). Two forms of blood loss

occur: 1) anatomical bleeding from the site of injury and 2) coagulopathic bleed-ing that arises early as a result of severe trauma and tissue hypoperfusion (5, 6). Coagulopathic bleeding was first reported in the Vietnam War (7) and rediscovered some 40 yrs later by Brohi (6). Several large trials have shown that 24% to 36% of severely injured patients have acute traumatic coagulopathy at admission to the emergency department (mean arrival

time 75 mins), and it was associated with a four-fold increase in mortality (6, 8, 9). Acute traumatic coagulopathy is routinely diagnosed from increased plasma acti-vated partial thromboplastin time (aPTT) and prothrombin time (PT), and it is dif-ferent from hypercoagulable disseminated intravascular coagulation, as is often seen in early sepsis (8). Thus in the critically injured, there is an urgent need to: 1) ade-quately resuscitate mean arterial pressure (MAP) to minimize shock and perfuse vital organs and 2) aggressively correct the coagulopathy as early as possible in out-of-hospital and military environments.

Previously, we showed that a small intravenous bolus (0.3 mL) of 7.5% NaCl with adenocaine (AL) and magnesium (Mg2+) (without colloids) resuscitated MAP into a hypotensive range in the rat model of severe (40%) to catastrophic (60%)

*See also p. 2519.From the Heart Research Laboratory (HLL, LPM,

GPD), Department of Physiology and Pharmacology, School of Biomedical Sciences and School of Pharmacy and Molecular Science (NMP), James Cook University, Queensland, Australia.

Current address for Dr. Pecheniuk: Institute of Health and Biomedical Innovation, Queensland University of Technology, Queensland, Australia.

Dr. Dobson consulted for Hibernation Therapeutics and has equity interest and stock ownership in the

company. He is also the inventor of Adenocaine Technology for organ protection and preservation, in-cluding trauma. The remaining authors have not dis-closed any potential conflicts of interest.

For information regarding this article, Email: [email protected]

Objective: Acute traumatic coagulopathy occurs early in hem-orrhagic trauma and is a major contributor to mortality and mor-bidity. Our aim was to examine the effect of small-volume 7.5% NaCl adenocaine (adenosine and lidocaine, adenocaine) and Mg2+ on hypotensive resuscitation and coagulopathy in the rat model of severe hemorrhagic shock.

Design: Prospective randomized laboratory investigation.Subjects: A total of 68 male Sprague Dawley Rats.Intervention: Post-hemorrhagic shock treatment for acute trau-

matic coagulopathy.Measurements and Methods: Nonheparinized male Sprague-

Dawley rats (300–450 g, n = 68) were randomly assigned to either: 1) untreated; 2) 7.5% NaCl; 3) 7.5% NaCl adenocaine; 4) 7.5% NaCl Mg2+; or 5) 7.5% NaCl adenocaine/Mg2+. Hemorrhagic shock was induced by phlebotomy to mean arterial pressure of 35–40 mm Hg for 20 mins (~40% blood loss), and animals were left in shock for 60 mins. Bolus (0.3 mL) was injected into the femoral vein and hemodynamics monitored. Blood was collected in Na citrate (3.2%) tubes, centrifuged, and the plasma snap frozen in liquid N2 and stored at −80°C. Coagulation was assessed using activated partial thromboplastin times and prothrombin times.

Results: Small-volume 7.5% NaCl adenocaine and 7.5% NaCl adenocaine/Mg2+ were the only two groups that gradually

increased mean arterial pressure 1.6-fold from 38–39 mm Hg to 52 and 64 mm Hg, respectively, at 60 mins (p < .05). Baseline plasma activated partial thromboplastin time was 17 ± 0.5 secs and increased to 63 ± 21 secs after bleeding time, and 217 ± 32 secs after 60-min shock. At 60-min resuscitation, activated par-tial thromboplastin time values for untreated, 7.5% NaCl, 7.5% NaCl/Mg2+, and 7.5% NaCl adenocaine rats were 269 ± 31 secs, 262 ± 38 secs, 150 ± 43 secs, and 244 ± 38 secs, respectively. In contrast, activated partial thromboplastin time for 7.5% NaCl adenocaine/Mg2+ was 24 ± 2 secs (p < .05). Baseline prothrombin time was 28 ± 0.8 secs (n = 8) and followed a similar pattern of correction.

Conclusions: Plasma activated partial thromboplastin time and prothrombin time increased over 10-fold during the bleed and shock periods prior to resuscitation, and a small-volume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg2+ was the only treatment group that raised mean arterial pressure into the permissive range and returned activated partial thromboplastin time and prothrombin time clotting times to baseline at 60 mins. (Crit Care Med 2012; 40: 2417–2422)

Key Words: adenocaine; adenosine; coagulopathy; hemorrhagic; hypertonic saline; hypocoagulopathy; lidocaine; magnesium; military; prehospital; resuscitation; shock; small volume; trauma

Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins

DOI: 10.1097/CCM.0b013e31825334c3

Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg2+ in the rat model of severe hemorrhagic shock*

Hayley L. Letson, BSc; Natalie M. Pecheniuk, PhD; Lebo P. Mhango, BSc; Geoffrey P. Dobson, PhD

Jdivya

10.1097/CCM.0b013e31825334c3

40

8

Jdivya

0

Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins

2012

Critical Care Medicine

Page 2: Reversal of Acute Coagulopathy During Hypotensive Resuscitation

2418 Crit Care Med 2012 Vol. 40, No. 8

blood loss and hemorrhagic shock (10, 11). The AL/Mg2+ concept, at high con-centrations, is currently used in cardiac surgery as the world’s first low-potassium, polarizing cardioplegia (12), an idea that was borrowed from the “tricks” of natural hibernators (13), and at lower concentra-tions, it is used to resuscitate the heart (12), and has potent antiarrhythmic, antiischemic (14, 15), and anti-inflamma-tory (16) properties. Given the intimate connection between inflammation and coagulation, and because adenosine has been shown to inhibit tissue factor (17) and thrombin-linked platelet aggregation (18, 19), lidocaine has been reported to reduce adenosine diphosphate–induced P-selectin and platelet-neutrophil aggre-gation (20) and deep vein thrombosis (21), and Mg2+ can inhibit platelet-dependent thrombosis in patients with coronary heart disease (22) and reduce clotting by accelerating the reactions between plasma protease inhibitors, antithrombin III, α-antitrypsin and α-2-macroglobulin, and factor Xa (23), we investigated the effect of a small bolus of 7.5% NaCl, 7.5% NaCl AL, and 7.5% NaCl AL/ Mg2+ on raising MAP in nonheparinized animals following 40% blood loss, and their effect on coagulation (aPTT and PT) during bleeding, 60 mins of shock, and 60-min resuscitation.

MATERIALS AND METHODS

Animals and Reagents. Male Sprague-Dawley rats (300–450 g) were fed ad libitum with free access to water and housed in a

12-hr light-dark cycle. Animals were anes-thetized with an intraperitoneal injection of 100 mg/kg sodium thiopentone (Thiobarb) (Ethics approval number A1148). Anesthetic was administered as required throughout the protocol. The study conforms to the Guide for Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animals were not heparinized as this interferes with clotting time measurements. Adenosine (A9251 >99% purity) and all other chemicals were obtained from Sigma Chemical Company (Castle Hill, NSW, Australia). Lidocaine hy-drochloride was purchased as a 2% solution (ilium) from the local Lyppards in Townsville, Queensland.

Surgical Protocol. The surgical procedure has been described by Letson and Dobson (10) and others (24). A tracheotomy was performed and the animals were artificially ventilated at 90–100 strokes per min on hu-midified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, MA). Rectal temperature was monitored. The left femoral vein and artery were cannulated us-ing PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab), and the right femoral artery was cannulated for blood letting. All cannulae contained citrate-phosphate-dextrose solution (0.14/mL). Nonheparinized rats were stabi-lized for 10 mins prior to blood withdrawal. Any animal that was difficult to anesthetize, experienced arrhythmias during preparation, stabilization, or within the shock period, or was hemodynamically unstable prior to phle-botomy was excluded from the study.

Experimental Design. Nonheparinized rats (n = 68) were randomly assigned to one of eight groups: 1) baseline blood (n = 10); 2) 20-min

bleed (n = 8); 3) 60-min shock (n = 8); 4) untreat-ed (n = 8); 5) 7.5% NaCl (n = 7); 6) 7.5% NaCl AL (n = 10); 7) 7.5% NaCl Mg2+ (n = 9); and 8) 7.5% NaCl AL/Mg2+ (n = 8). To avoid anemia, three separate groups were required for blood withdrawal and then sacrificed at baseline, 20-min bleed, and 60-min shock, and five separate treatment groups for continuous hemodynamic measurements until 60-min resuscitation (blood removed for clotting times at this point) (Fig. 1). The concentrations of drugs/ions in 0.3 mL 7.5% NaCl bolus were MgSO4 (2.5 mM), AL (1 mM adenosine and 3 mM lidocaine-HCl), or combined AL/Mg2+ (Fig. 1).

Shock Protocol. Hemorrhagic shock was induced by withdrawing arterial blood to an MAP of 35–40 mm Hg and phlebotomy was continued for 20 mins. The average shed volume was 11.1 ± 0.2 mL (n = 68) (range 7.5–14.8 mL) and represented an average blood loss of 41 ± 0.4% (range 34% to 50%) and was similar to our study using heparinized rats (10). Rats were left in shock for 60 mins with blood withdrawal or infusion to ensure that MAP remained between 35 and 40 mm Hg. At the end of shock, rats were injected with 0.3 mL treatment bolus (~3% to 4% of shed vol-ume) into the femoral vein over a 10-sec peri-od. Hemodynamic parameters were monitored throughout the study including heart rate, sys-tolic pressure, diastolic pressure, and MAP (10).

aPTT and PT Time. Arterial blood was col-lected from untreated and treated rats in 3.2% sodium citrate tubes. Whole blood was cen-trifuged at room temperature and the plasma removed, snap frozen in liquid nitrogen, and stored at −80°C until use. aPTT was deter-mined by incubating 50 μL plasma with 50 μL of an aPTT reagent (TriniCLOT aPTT; Trinity Biotech, Ireland) for 5 mins at 37°C. The clot-ting process was initiated by the addition of

Figure 1. A schematic of the pressure controlled in vivo rat protocol of hemorrhagic shock. Shed blood volume was taken over a 20 min period to maintain mean arterial blood pressure (BP) of 35 to 40 mm Hg (~40% blood loss), and the rat remained in shock for a period of 60 mins prior to resuscitation. Rectal temperature fell from 37°C to 34°C during the bleed and remained at this temperature throughout. MAP, mean arterial pressure; HR, heart rate; ECG, electrocardiography; PT, prothrombin time; aPTT, activated partial thromboplastin time.

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Crit Care Med 2012 Vol. 40, No. 8 2419

50 μL of 30 mM CaCl2 and clotting time re-corded on a microcoagulometer (Amelung KC4A; Trinity Biotech). Following 10-min in-cubation of 50 μL plasma, PT was determined by the addition of 100 μL of thromboplastin re-agent containing CaCl2 (TriniCLOT PT; Trinity Biotech) and clotting time recorded (Amelung KC4A). All measurements were performed in triplicate.

Statistical Analysis. All values were ex-pressed as mean ± sem. Clotting times were evaluated using a one-way analysis of variance for comparison to baseline. A homogeneity of variance test was performed to determine whether a Tukey’s honestly significant dif-ference or Dunnett’s post hoc test was used. Hemodynamic data were analyzed using re-peated measures analysis of variance, with the Greenhouse-Geisser correction applied if the condition of sphericity was not met. Statistical significance was defined as p < .05.

RESULTS

Hemodynamics. Hemodynamics at baseline, end of the 20-min bleed, and end of the 60-min shock period were not significantly different among the groups. Two of the eight untreated animals died at 38 ± 0.6 min resuscitation, which was similar to three deaths in our earlier study using “heparinized” rats (10). No other deaths occurred. MAP, systolic pres-sure, and diastolic pressure are shown in Fig. 2A–C. Untreated rats could not main-tain MAP, systolic pressure, or diastolic pressure during 60-min resuscitation. In direct contrast, all other groups increased MAP at 5 mins from ~36 to 45 mm Hg (Fig. 2A). After 10 mins, the 7.5% NaCl Mg2+ group failed to further increase MAP, which continued for the remainder of resuscitation. The three groups that con-tinued to increase MAP after 10 mins were 7.5% NaCl alone, and AL and AL/Mg2+ groups. After 15 mins, the MAP of the 7.5% NaCl alone group began to decrease toward preresuscitation values at a rate of ~0.3 mm Hg per min (Fig. 2A). The 7.5% NaCl AL and 7.5% NaCl AL/Mg2+-treated rats continued to increase MAP from 15 to 60 mins, and after 30 mins MAP was significantly higher than any group (Fig. 2A). While 7.5% NaCl AL/Mg2+ group con-sistently generated a higher MAP (13% to 20% higher) than the AL group, these differences were not significant (Fig. 2A). Arterial systolic and diastolic pressure profiles are shown in Figures 2B and C, and there were similar changes and sig-nificant differences as for MAP among the groups during resuscitation. Heart rate was expressed as a percentage of preresus-citation values and is shown in Figure 2D.

Figure 2. A, Effects of four small-volume hypertonic saline resuscitation fluids on recovery of mean arterial pressure (MAP) following 60 mins of severe hemorrhagic shock in the rat in vivo. Untreated (◆), 7.5% NaCl (◼), 7.5% NaCl Mg2+ (×), 7.5% NaCl AL (∆), 7.5% NaCl AL/Mg2+ (•). Values are Mean ± sem. B, Effects of four small-volume hypertonic saline resuscitation fluids on recovery of arterial systolic pressure (SP) following 60 mins of severe hemorrhagic shock in the rat in vivo (symbols as in A). Values are Mean ± sem. C, Effects of four small-volume hypertonic saline resuscitation fluids on re-covery of arterial diastolic pressure (DP) following 60 mins of severe hemorrhagic shock in the rat in vivo. (symbols as in A). Values are Mean ± sem. D, Effects of four small-volume hypertonic saline resuscitation fluids on recovery of heart rate (HR) following 60 mins of severe hemorrhagic shock in the rat in vivo. Values are expressed as a percentage of preresuscitation (shock) value (symbols as in A). Values are Mean ± sem. *p < .05 compared with untreated group; #p < .05 compared with untreated and 7.5% NaCl groups; ‡p < .05 compared with untreated, 7.5% NaCl, and 7.5% NaCl Mg2+ groups; †p < .05 compared with untreated and 7.5% NaCl Mg2+ groups.

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2420 Crit Care Med 2012 Vol. 40, No. 8

With the exception of untreated animals, all groups maintained or increased their heart rate during 60-min resuscitation with the largest increases seen in Mg2+- and AL/Mg2+-treated rats (Fig. 2D).

aPTT and PT Times. Baseline mean aPTT was 17 ± 0.5 secs (n = 10), and was similar to published rat values of 16.5 ± 0.4 secs (n = 23) (25). Baseline aPTT values increased significantly to 63 ± 21 and 107 ± 33 secs after blood withdrawal (20 mins) (n = 8) and over ten times baseline after 60-min shock at 217 ± 32 secs (n = 8) (Fig.  3A). During this time, there was no change in rectal temperature. Sixty minutes after 0.3 mL bolus resuscitation, the aPTT for the untreated group was 269 ± 31 secs (n = 8), 7.5% NaCl group was 262 ± 38 secs (n = 7), 7.5% NaCl AL group was 244 ± 38 secs (n = 10), 7.5% NaCl Mg2+ group was 150 ± 42 secs (n = 9), and 7.5% NaCl AL/Mg2+ group was 24 ± 33 secs (n = 8) (Fig. 3A). Baseline PT times were 28 ± 0.8 secs (n = 10) and close to published rat values of 27 ± 0.4 secs (n = 23) (25). PT increased signifi-cantly from 28 to 107 ± 33 secs after blood withdrawal (20 mins) (n = 8) and nearly ten times baseline after 60-min shock before treatment (278 ± 11 secs, n = 8) (Fig. 3B). Sixty minutes after 0.3 mL bolus, PT value was 201 ± 41 secs for the untreated group (n = 8), 261 ± 39 secs for the 7.5% NaCl group (n = 7), 193 ± 44 secs for the 7.5% NaCl AL group (n = 10), 182 ± 47 secs for the 7.5% NaCl Mg2+ group (n = 9), and 33 ± 1.0 secs for the 7.5% NaCl AL/ Mg2+ group (n = 8) (Fig. 3B).

DISCUSSION

Despite significant advances in blood control technologies, there remains an urgent need to promptly but gently resus-citate MAP and stabilize exsanguinating civilians and military personnel as close to the incident as possible (26–28). In addition to controlling blood loss and gradually restoring cardiovascular func-tion, correcting hypocoagulopathy and blunting the inflammatory cascade are critical to reduce mortality and morbid-ity. We report in the nonheparinized rat model of severe hemorrhagic shock that hypocoagulopathy occurs very early dur-ing bleeding and shock, and a small-vol-ume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg2+ raised MAP into the permissive range and returned aPTT and PT clotting times to baseline at 60 mins.

Hemodynamics. Untreated, 7.5% NaCl, and 7.5% NaCl Mg2+-treated rats failed to significantly increase MAP

from their preresuscitation values after 15 mins (Fig 2A). The inability of 7.5% NaCl to sustain MAP after 10 mins with or without Mg2+ appears to be related to the lower osmotic load from the small intravenous bolus volume (~1 mL/kg), compared with the more commonly used 4 mL/kg, and a more rapid return of water into the extravascular compartment (10). In contrast, 7.5% NaCl AL and 7.5% NaCl AL/Mg2+-treated rats significantly raised MAP from shock values of 38–39 mm Hg to 52 ± 5 and 64 ± 6 mm Hg, respec-tively (p < .05) (Fig. 2A–C). MAP for the 7.5% NaCl AL/Mg2+ group at 60 mins was not significantly different from our ear-lier study using heparinized rats (MAP increased from 38 ± 0.5 mm Hg to 60 ± 3 mm Hg (10)) and indicates that hepa-rin has little or no effect on small-volume

7.5% NaCl AL/Mg2+ resuscitation poten-tial in this model. Interestingly, the abil-ity of small-volume 7.5% NaCl AL/Mg2+ to resuscitate MAP, systolic pressure, and diastolic pressure was up to 20% higher than AL group alone, but these differ-ences were not significant. Diastolic res-cue in the AL/Mg2+ and AL groups was also significantly higher than all other groups and may indicate: 1) improved blood per-fusion to the compromised heart and/or 2) may reflect higher peripheral vascular tone (10, 11).

Hypocoagulopathy begins early dur-ing phlebotomy and shock. Plasma aPTT and PT clotting times increased 3.8-fold immediately after blood withdrawal (20 mins), and 10- to 13-fold from baseline after 60-min shock (Fig. 3A and B). The early appearance of hypocoagulopathy

Figure 3. Effects of four small-volume hypertonic saline resuscitation fluids on plasma clotting times at different stages of the protocol from baseline to 60-min resuscitation. A, Activated partial thrombo-plastin time (aPTT), and B, prothrombin time (PT). #p< .05 compared with all groups except bleed and 7.5% NaCl AL/Mg2+ groups; *p < .05 compared with shock, untreated, 7.5% NaCl, and 7.5% NaCl AL groups; ‡p < .05 compared with all groups except baseline and bleed groups; †p < .05 compared with shock group.

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Crit Care Med 2012 Vol. 40, No. 8 2421

in our study supports the small human observational study of Floccard and col-leagues (8) who showed abnormal coagu-lopathy in 56% of the 45 patients at the site of the accident through to admis-sions. These large increases in aPTT or PT clotting times following shock and before treatment were not due to hypothermia because despite an early 1°C fall in rec-tal temperature, no further decreases occurred during 60-min shock (Fig. 1). We conclude that accidental hypother-mia was not associated with 10- to 13-fold increases in aPTT or PT clotting times after shock in our rat model. Similarly, the coagulopathy was not due to hemo-dilution of clotting factors as no intrave-nous bolus had been administered. The data suggest therefore that early hypoco-agulopathy was related to: 1) trauma of the surgery; 2) severe blood loss; and/or 3) the shock state itself involving regional and global tissue hypoperfusion.

Reversal of Hypocoagulopathy with 7.5% NaCl AL and Mg2+. In contrast to adenosine, lidocaine, and Mg2+ alone hav-ing a number of anticoagulant properties (17–23), one of the standout and unex-pected results of the present study was a near full reversal of aPTT and PT clotting times to baseline with 7.5% NaCl AL/Mg2+ at 60-min resuscitation (Fig. 3A and B). The reversal may in part have arisen from improved MAP resuscitation and/or improved tissue perfusion. However, 7.5% NaCl AL significantly raised MAP (admit-tedly not as high as the AL/Mg2+ group) but did not correct the coagulopathy. There-fore, the correction appears to be a direct effect of the combination of 7.5% NaCl AL/Mg2+. Unfortunately, we did not withdraw blood at earlier time points to establish the timing of the reversal of clotting times. This reversal at 60 mins was in direct con-trast to any group (Fig. 3A and B), which all failed to clot plasma after 5 mins of reac-tion time. The underlying mechanisms for a near full restoration of aPTT and PT clot-ting times after small-volume 7.5% NaCl AL/Mg2+ resuscitation are not known. It is curious how both the aPTT and PT clot-ting times were restored, as it implicates a combined effect of 7.5% NaCl AL/Mg2+ on a region of commonality shared by the contact (intrinsic) and tissue factor (TF) (extrinsic) clotting pathways, respectively. One common point is activation of factor X, which activates prothrombin to throm-bin, and subsequently leads to the conver-sion of fibrinogen to fibrin. Recently, Shaz and colleagues (29) reported a decrease in common and TF- factor VII pathway

factor activities, and a decreased inhibition of the coagulation cascade (antithrom-bin and protein C activities) in trauma patients with coagulopathy compared with matched control patients.

Thus in our study, increased thrombin availability may arise from one or more of the following: 1) a shift in the thrombin-antithrombin pathway to produce throm-bin within both the contact activation and TF pathways; 2) down-regulation of the protein C pathway; and/or 3) increasing thrombin-activated fibrinolysis inhibitor leading to a hypofibrinolytic state (30). In addition to down-regulating the protein C pathway, reducing the availability/activ-ity of activated protein C may indirectly reduce fibrinolysis and assist to correct coagulation. Activation of the protein C pathway has been suggested by Brohi and colleagues (31) as a possible mechanism for early hypocoagulopathy in patients following trauma-related blood loss. Lastly, a reversal of PT clotting time may also arise by increasing TF availability by down-regulating TF pathway inhibitor and favoring blood coagulation. In sum-mary, small-volume 7.5% NaCl AL/Mg2+ fluid may enhance the natural blood clot-ting system by increasing the availability of thrombin, TAFI, and fibrinogen. Fur-ther work is required to study these pos-sibilities in our model.

Methodological Considerations: Coag-ulation reactions in vivo occur on specific cell surfaces such as activated platelets and vascular endothelial cells rather than on phospholipid vesicles, as they do in aPTT or PT clotting timed assays (32). The plasma-based (platelet poor) aPTT and PT assays are routine single-point tests to assess the kinetics of clot for-mation and the risk of patient bleeding. Both tests detect the time to formation of a threshold amount of fibrin, and they do not provide much information about global coagulation because of the high concentrations of activators used (33). Thrombelastography on citrated whole blood may offer some advantages over aPTT and PT assays in our study (24, 34), and it would be interesting to investigate the different sensitivities and specificities of the various coagulation methods in the rat model of hemorrhagic shock.

Possible Clinical and Military Impli-cations. Currently, emergency first-responder teams and combat medics have a limited range of pharmacological options for rescuing and stabilizing civil-ians or soldiers following massive hemor-rhage in the prehospital arena (28). It is

possible that the restorative properties of small-volume IV bolus of 7.5% NaCl with AL/Mg2+ to slowly increase MAP into the permissive hypotensive range (sys-tolic pressure 60–80 mm Hg) and cor-recting aPTT and PT may provide a new first-responder or tactical damage con-trol resuscitation therapy for buying or extending biological/physiological time in out-of-hospital or far-forward military environments. It may also have applica-tions in cardiac surgery or other surgeries that have coagulation imbalances.

CONCLUSIONS

Plasma aPTT and PT increased over 10-fold during the bleed and shock peri-ods prior to resuscitation, and a small-volume (~1mL/kg) intravenous bolus of 7.5% NaCl AL/Mg2+ was the only treat-ment group that raised MAP into the per-missive range and returned aPTT and PT clotting times to baseline at 60 mins.

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