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Anesth Analg 2001;93:1106-1110
© 2001 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

Resuscitation with Hextend® Decreases Endogenous Circulating Heparin Activity and Accelerates Clot Initiation After Hemorrhage in the Rabbit

Vance G. Nielsen, MD

Department of Anesthesiology, Divisions of Cardiothoracic Anesthesia and Anesthesiology Research, The University of Alabama at Birmingham, Birmingham, Alabama

Address correspondence and reprint requests to Vance G. Nielsen, MD, Department of Anesthesiology, The University of Alabama at Birmingham, 619 S. 19th St., Birmingham, AL 35249-6810. Address e-mail to vance.nielsen{at}ccc.uab.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Hemorrhagic shock can result in a hypercoagulable state and has been associated with both hemorrhagic and thrombotic complications in the perioperative period. The author hypothesized that hemorrhage and resuscitation could result in a hypercoagulable state via changes in the heparin-antithrombin III anticoagulant mechanism in rabbits. Rabbits sedated with ketamine underwent sham operation (n = 8) or hemorrhage (25 mL/kg blood shed) for 60 min, followed by resuscitation with an equal volume of 5% human albumin (n = 8) or Hextend® (n = 8). Coagulation analysis with the Thrombelastograph® analyzer and determination of endogenous heparin and antithrombin III activity were performed on arterial blood samples obtained before hemorrhage and 30 min after resuscitation. The reaction time significantly decreased by 34% after hemor- rhage and resuscitation with Hextend®, whereas no other significant changes in Thrombelastograph® variables were noted. Antithrombin III activity was significantly less in the Albumin (83% ± 8% of control, mean ± SD) and Hextend® (88% ± 8%) Resuscitated groups compared with the Sham-Operated animals. Of interest, only the Hextend®-Resuscitated animals demonstrated a significant decrease in heparin activity (53.4 ± 13.6 mU/mL before hemorrhage, 42.3 ± 5.6 mU/mL after resuscitation). A Hextend®-mediated decrease of both heparin and antithrombin III activity may explain the acceleration of clot initiation compared with albumin administration after hemorrhage in the rabbit.

IMPLICATIONS: Hemorrhage may result in a hypercoagulable state after resuscitation. Decreases in both endogenous heparin and antithrombin III activity after hemorrhage and Hextend® resuscitation in rabbits resulted in a significantly decreased time to clot coagulation analysis initiation without a significant change in the rate of clot formation or final clot strength.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Perioperative hemorrhage and thrombosis are major sources of morbidity and mortality after major vascular and trauma surgery (1–6). With regard to elective surgical procedures involving <15% loss of estimated blood volume (EBV), intraoperative blood loss treated with crystalloid replacement resulted in significant hypercoagulability, as determined by coagulation analysis with the Thrombelastograph® (TEG®) analyzer (Haemoscope Corp., Niles, IL) (7,8). Trauma patients exposed to massive transfusion are significantly hypocoagulable as assessed by conventional coagulation variables (e.g., activated partial thromboplastin time and factor VIII activity) (9). In contrast, an investigation using both TEG® variables and conventional coagulation variables found that most trauma patients tend to be hypercoagulable, with injury severity score and TEG® variables significantly predictive of need for early transfusion (10). Although these data suggest that thrombotic and hemorrhagic tendencies occur in response to hemodilution coupled with systemic ischemic insults, the etiology of these coagulopathies remains unclear.

Although changes in circulating procoagulants, such as clotting factors and platelets, affect hemostasis after hemorrhage and resuscitation, the effect of alterations in circulating anticoagulant factors such as antithrombin III and endogenous heparin are largely unknown. In the setting of hepatic transplantation (11–13) or thoracic aortic occlusion-reperfusion (14,15), endogenous heparin release contributes to a hypocoagulable state during reperfusion. Consequently, determination of changes in endogenous heparin activity and antithrombin III activity in conjunction with functional determinations of clot function could provide mechanistic insight into the pathogenesis of hemorrhage and resuscitation-induced coagulopathy.

Thus, the purpose of this study was to determine whether hemorrhage and resuscitation resulted in a hypercoagulable state via modulation of the heparin-antithrombin III anticoagulant system in a minimally sedated rabbit model. Further, this study proposed to determine whether there were significant coagulation differences after hemorrhage resulting from resuscitation with 5% human albumin in 0.9% NaCl versus resuscitation with a 6% hydroxyethyl starch solution. Changes in endogenous heparin activity, antithrombin III activity, and TEG® variables served as end points to determine the extent and possible etiology of hemorrhage and resuscitation-induced coagulopathy.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study was approved by our animal review committee. Male New Zealand White rabbits (Myrtle’s Rabbits, Thompson Station, TN) weighing 1.8–2.8 kg (n = 24) were briefly restrained (<2 min) and had 22-gauge catheters placed in the central ear artery and the marginal ear vein and were left unrestrained on a padded surgical table. The author continuously attended the animals throughout experimentation. After a 5-min resting period, baseline mean arterial pressure (MAP) and heart rate (HR) were recorded with a Grass Model 7D polygraph (Grass Instruments, Quincy, MA). MAP and HR were subsequently continuously recorded for the duration of the experiment. Rabbits were randomly assigned to the Sham-Operated group, the Hemorrhaged group resuscitated with 5% human albumin (Baxter Healthcare Corporation, Glendale, CA), or the Hemorrhaged group resuscitated with a 6% hetastarch solution (Hextend®; Abbott Laboratories, Chicago, IL; average molecular weight, 450 kd) (n = 8 per group). After baseline blood samples (described subsequently) were obtained, all rabbits were administered ketamine 10 mg/kg IV immediately followed by a ketamine infusion of 10 mg · kg-1 · h-1for 10 min. No further intervention occurred in the Sham-Operated group. In the Hemorrhage groups, 25 mL/kg (~36% of EBV) of blood (less the volume of blood obtained at baseline) was removed over a 10-min period. Sixty minutes later, rabbits in the Hemorrhaged groups were administered 25 mL/kg of either 5% human albumin or hetastarch solution that was warmed to 39°C in a water bath. Thirty minutes later, additional blood samples were obtained, and the animals were subsequently killed with an overdose of pentobarbital (100 mg/kg IV).

Arterial blood samples (7 mL) were obtained at baseline and at the conclusion of the experiments for arterial blood gas analysis, Ca2+ concentration, platelet count, coagulation analysis, and determination of heparin and antithrombin III activity. The arterial pH, PaCO2, PaO2, and Ca2+ concentrations of arterial blood samples were determined at 37°C by using a blood gas analyzer (model 1306; Instrumentation Laboratory, Lexington, MA). Hematocrit and platelet concentrations were concurrently determined with a Sysmex K-800 device (TOA Medical Electronics Co., Ltd., Tokyo, Japan). Coagulation analyses were performed with two computer-controlled TEG® machines (model 5000), each with two channels. All blood samples were placed in 360-µL standard disposable plastic cups or cups that contained heparinase I (from Flavobacterium heparinum, 2.0 IU per cup). Cups containing 2 IU of heparinase will digest up to 6 IU/mL of heparin activity, which is more than sufficient to neutralize the circulating heparin activity encountered in the rabbit (14,15). The proper functioning of the TEG® analyzer was confirmed daily with quality control standards purchased from Haemoscope. The following coagulation analysis variables were measured for each sample over a 1-h period at 39°C (the normal temperature of the rabbit): reaction time (R; min), angle ({alpha}; degrees), maximum amplitude (MA; mm) and shear elastic modulus (G, dynes/cm2). A detailed description of the methodology of coagulation analysis has been presented in great detail elsewhere (16,17). In brief, R is defined as the time from when the blood sample is placed into the TEG® cup until initial fibrin formation occurs, as noted by a signal of 2 mm amplitude; {alpha} is the angle formed from R to the inflection point of the TEG® signal as clot strength stabilizes; it is a measure of the kinetics of clot formation. MA is the largest amplitude of the TEG® signal and is a measure of clot strength. Finally, G is a measure of clot strength (16) calculated from MA as follows: G = (5000 x MA)/(100 - MA). The relationship between MA and G is curvilinear; as MA varies from 0 to 100, G concordantly varies from 0 to infinity. Consequently, whereas MA was determined, G was reported. Lastly, the remaining volume of all blood samples were anticoagulated with 129 mM sodium citrate (9 volumes of blood to 1 volume of sodium citrate). These samples were centrifuged at 2500g for 15 min, with the plasma then removed and centrifuged a second time at 2500g for 15 min to obtain platelet-poor plasma. Plasma samples were immediately stored at -85°C before heparin activity determination via changes in anti-Xa activity (18,19) and determination of antithrombin III activity (20) by using commercially available kits (Biopool International, Ventura, CA) using a modified chromogenic assay.

All variables are expressed as mean ± SD. Analyses of the effects of hemorrhage and resuscitation on all variables were conducted by analysis of variance with repeated measures. The Student-Newman-Keuls test was used for post hoc comparisons. An {alpha} error of <0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Hemodynamic data are displayed in Table 1. Hemorrhage resulted in a significantly decreased MAP and increased HR compared with baseline and sham operated values. After resuscitation, there was no significant difference in MAP among the three groups, but both resuscitated groups had a significantly faster HR compared with the Sham-Operated group.


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  		Table 1. Hemodynamic Data
 
Arterial blood gas, Ca+2, hematocrit, and platelet concentration data are listed in Table 2. There were no significant changes in arterial pH, PaO2, or Ca2+ concentration among the three groups. PaCO2 significantly increased in all three groups, and hematocrit significantly decreased in all three groups, with the resuscitated groups noted to have significantly smaller hematocrit values compared with the Sham group. Finally, the platelet concentration significantly decreased in both resuscitated groups.


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  		Table 2. Arterial Blood Gas, Ca+2, Hematocrit, and Platelet Concentration Data
 
TEG®-derived data, heparin activity, and antithrombin III activity obtained from the three groups are contained in Table 3. After hemorrhage and resuscitation, rabbits in the group administered hetastarch solution had significantly shorter R values in TEG® samples without heparinase compared with baseline values and compared with Sham-Operated animals. In the presence of heparinase, animals resuscitated with albumin or hetastarch solution were noted to have shorter R values compared with baseline and Sham group values. Animals resuscitated with albumin had significantly greater {alpha} values in the presence of heparinase after resuscitation compared with Sham-Operated and Hetastarch Solution-Resuscitated rabbits. Animals administered hetastarch solution had significantly decreased G values compared with baseline and Sham-Operated group values in the presence of heparinase. With regard to endogenous heparin activity, only the Hetastarch Solution-Treated group demonstrated a significant decrease in activity compared with baseline and Sham-Operated group values. Finally, both resuscitated groups demonstrated significantly decreased antithrombin III activity after hemorrhage compared with Sham-Operated rabbits.


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  		Table 3. Thrombelastograph® Variables R (min), {alpha} (degrees), and G (dynes/cm2); Endogenous Heparin Activity (mU/mL); and Antithrombin III Activity (% baseline activity)
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Hemorrhage and resuscitation in a minimally sedated rabbit model resulted in little perturbation of hemostasis, as determined by unmodified TEG® analysis. However, animals resuscitated with hetastarch solution did have a 34% decrease in R values compared with baseline or Sham-Operated group R values, and this can be interpreted as an acceleration in clot formation or initiation. Data generated during heparinase-modified TEG® analysis demonstrated similar decreases in R values in both resuscitated groups compared with baseline or sham-operated R values. The decrease in R values observed during heparinase-modified TEG® analysis can be explained in part by the significantly decreased antithrombin III activity found in the resuscitated groups, because in the absence of its cofactor heparin, antithrombin III activity can decrease by two or more orders of magnitude (21). Similarly, the significantly decreased endogenous heparin activity after hemorrhage and resuscitation with hetastarch solution coupled with a decrease in antithrombin III activity may explain R values that are less than those of Sham-Operated animals. Endogenous heparin is present in humans (22–25) and other mammals (14,15,26) and is probably secreted by hepatoenteric mast cells after ischemia-reperfusion injury (11–15). One possible scenario is that circulating endogenous heparin activity increases during hemorrhage and decreases during reperfusion, as has been seen during hepatic transplantation (11–13). Given that hetastarch and pentastarch solutions decrease hepatoenteric ischemia-reperfusion injury (27,28), it may be possible that hetastarch solution administration decreased mast cell degranulation upon reperfusion, or perhaps better preserved heparin-clearing mechanisms compared with albumin administration after hemorrhage in this study. In summary, the initiation of clot formation (R time) seems to be modulated by changes in the heparin-antithrombin III anticoagulant system in the rabbit model presented.

In addition to anticoagulant-mediated changes in hemostasis, heparinase-modified TEG® analysis demonstrated an albumin-mediated increase in the rate of clot formation ({alpha}) after resuscitation compared with the two other groups. Further, the overall final clot strength (G) of the Hetastarch Solution-Resuscitated group was less than baseline or sham-operated values, whereas Albumin-Resuscitated group G values were not significantly different from baseline or Sham-Operated group values. One possible explanation for these data is that hemorrhage and resuscitation with albumin results in the elaboration of an unidentified thrombotic mediator that enhances clot formation and overall strength. However, hetastarch administration to humans decreases factor VIII activity compared with albumin (29), perhaps explaining the hetastarch solution-mediated changes in {alpha} and G after hemorrhage and resuscitation in the rabbit. However, there were no significant differences in {alpha} and G values after replacement of 75% of the EBV with either human albumin or hetastarch solution in rabbits (30), whereas only 50% dilution of EBV occurred in this study, on the basis of changes in hematocrit (Table 2). Thus, rather than inhibiting endogenous procoagulant factors, hetastarch solution administration may attenuate the elaboration of thrombotic mediators after hemorrhage, just as a pentastarch solution attenuated thrombotic mediator release after hepatoenteric ischemia-reperfusion in the rabbit (15).

In conclusion, diminution of the activity of the heparin-antithrombin III anticoagulant system was responsible for coagulation changes in rabbits after hemorrhage and resuscitation. Remarkably, the administration of a hetastarch solution resulted in an acceleration of clot initiation compared with albumin administration after hemorrhage because of a decrease in endogenous circulating heparin activity. Further study of the role of endogenous anticoagulant systems in the evolution of coagulopathy encountered in shock states is warranted, as well as additionally defining the role played by resuscitative fluids in the modulation of both anticoagulant and thrombotic mediator activity.


   Acknowledgments
 
Supported in part by a grant from BioTime, Inc., Berkeley, CA, Abbott, Chicago, IL, and the Department of Anesthesiology.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication June 18, 2001.




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This Article
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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press