Anesth Analg 2001;92:320-323
© 2001 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
Nitric Oxide Decreases Coagulation Protein Function in Rabbits as Assessed by Thromboelastography
Vance G. Nielsen, MD
Department of Anesthesiology, Division of Cardiothoracic Anesthesia, 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. Address e-mail to vance.nielsen{at}ccc.uab.edu
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Abstract
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Nitric oxide (NO) is administered via infusion of donors such as nitroglycerin or in inhaled form for treatment of ischemia and pulmonary hypertension, respectively. In rabbits, the NO donor, DETANONOate, decreases whole blood clotting function as assessed by thromboelastographic variables (R, reaction time; 
, angle; and G, a measure of clot strength). I hypothesized that DETANONOate-derived NO would adversely affect coagulation protein and platelet function. Blood obtained from ear arteries of conscious rabbits (n = 8) anticoagulated with sodium citrate. The blood was then incubated with 0 or 10mM DETANONOate for 30 min. After incubation and recalcification, thromboelastography was performed for 60 min under four conditions: 1) 0mM DETANONOate, 2) 0mM DETANONOate with platelet inhibition with cytochalasin D, 3) 10mM DETANONOate, and 4) 10mM DETANONOate with platelet inhibition. DETANONOate significantly (P < 0.05) increased R and decreased and G in samples with or without platelet inhibition, compared with samples not exposed to DETANONOate. Lastly, the percentage of total G (GT) attributable to platelet function (GP) was significantly more in the absence of DETANONOate (GP = 92.3% ± 1.6%; mean ± SD) than after exposure to DETANONOate (GP = 90.2% ± 2.3%). DETANONOate-derived NO significantly decreased coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.
Implications: In rabbit whole blood, nitric oxide (NO) decreases hemostatic function by decreasing both coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.
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Introduction
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Nitric oxide (NO) is often administered in the perioperative period as an NO donor (e.g., nitroglycerin) or as an inhalant. In addition to the administration of NO via infusion of nitroglycerin or nitroprusside for treatment of angina or hypertension, inhaled NO has also been administered for the treatment of pulmonary hypertension (1–3) and acute respiratory distress syndrome (ARDS) (4–6). Also of interest, NO decreases platelet aggregation in vitro and in vivo via a guanylyl cyclase mechanism (7–12). Further, exposure to inhaled NO significantly decreases platelet aggregation in rats (10) and in humans with ARDS (13). The inhibition of platelet function in these studies was not significantly increased by the administration of larger concentrations of NO (10,13). However, it has been recently demonstrated in rabbits that exposure to NO derived from the donor DETANONOate in vitro decreases hemostatic function of whole blood in a dose-dependent fashion as assessed by thromboelastography (14). One important finding of this investigation was that some of the blood samples exposed to the largest concentration of DETANONOate had no discernible clot (14). This finding was significant, because even in the absence of platelet function, rabbit blood will still clot via coagulation protein activity (15). Consequently, these data indirectly demonstrated that NO might affect the function of coagulation proteins in whole blood. Given the vast clinical administration of NO in parenteral and inhaled forms and incidence of pathologic conditions involving excess NO production (e.g., sepsis), the identification of a platelet-independent anticoagulant effect of NO would be of great importance.
This study tested the hypothesis that exposure of whole rabbit blood to NO via DETANONOate would decrease the contribution of both coagulation protein and platelet function to clot initiation, rate of formation, and final clot strength.
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Methods
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This study was approved by our animal review committee. Conscious rabbits (n = 8) were briefly (<2 min) restrained and had 2.7 mL of blood aseptically drawn from central ear arteries. The blood samples were immediately anticoagulated in a glass tube containing 300 µL of 0.129mM sodium citrate. Anticoagulated blood was subsequently divided into 450-µL aliquots placed in capped plastic tubes at room temperature. Fifty microliters of DETANONOate dissolved in 10mM Na2PO4 buffer (pH 9.0) or buffer alone was added to the 450-µL blood samples to yield final DETANONOate concentrations of 0 or 10mM. This concentration of DETANONOate was used because it was the smallest concentration that significantly decreased clotting function in the rabbit (14). The samples were subsequently incubated at 39°C for 30 min. After incubation, samples were exposed to one of the following four thromboelastographic conditions: 1) 320 µL of blood with 10 µL of 0.9% NaCl and 30 µL of 200mM CaCl2, 2) 320 µL of blood with 10 µL of cytochalasin D (final concentration 10µM) and 30 µL of CaCl2, 3) 320 µL of blood exposed to DETANONOate with 10 µL of NaCl and 30 µL of CaCl2, or 4) 320 µL of blood exposed to DETANONOate with 10 µL of cytochalasin D and 30 µL of CaCl2. Cytochalasin D (10µM) inhibits microtubule formation (and glycoprotein IIb/IIIa activation) in platelets, resulting in a thromboelastographic signature caused only by coagulation proteins in whole blood (15). All samples were placed in thromboelastograph cups containing heparinase I (from Flavobacterium heparinum, 2.0 IU per cup), because the rabbit has variable endogenous heparinoid activity (16). Thromboelastographic analyses were performed with two computer-controlled Thrombelastographs® (Model 5000; Haemoscope Corp., Skokie, IL), each with two channels, for a total of four thromboelastograms generated per rabbit. The proper functioning of the Thrombelastograph was confirmed daily with quality control standards purchased from Haemoscope. The following thromboelastographic variables were mea- sured for each sample for a 1-h period at 39°C (the normal temperature of the rabbit): reaction time (R [min]), angle ( [degrees]), maximum amplitude (MA [mm]), and shear elastic modulus (G [dyne/cm2]). A detailed description of the methodology of thromboelastography has been presented in great detail elsewhere (17,18). G is a measure of clot strength (18) calculated from MA as follows: G = (5000 x MA)/(100 - MA). While MA was determined, G was reported. The contribution of platelets to G (GP) was defined by the total G of whole blood not exposed to cytochalasin D (GT) minus the G of blood exposed to cytochalasin D, which is attributable to the soluble components of the coagulation pathway (GSC) (15,18).
All variables are expressed as mean ± SD. Analyses of the effects of DETANONOate on thromboelastographic variables in the presence or absence of platelet inhibition with cytochalasin D were conducted with paired t-tests. An error of <0.05 was considered significant.
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Results
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Blood samples not exposed to cytochalasin D that were incubated with DETANONOate exhibited a significant (P < 0.05) increase in R, decrease in , and decrease in GT compared with samples not incubated with DETANONOate, as depicted in Table 1. Of further interest, incubation with DETANONOate resulted in a significant (P < 0.05) increase in R, decrease in , and decrease in GSC in samples exposed to cytochalasin D compared with samples without DETANONOate. Finally, GP as a percentage of GT was significantly decreased in samples incubated with DETANONOate compared with those not exposed to DETANONOate.
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Discussion
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This study is the first to demonstrate that NO modulates clotting by decreasing coagulation protein function as well as platelet function. Specifically, NO prolongs R predominantly by decreasing coagulation protein function. This conclusion is drawn from the 10–12-minute increase in R after DETANONOate exposure in the presence or absence of platelet inhibition (Table 1). In further support of the concept of NO-mediated decreases in coagulation protein function, was decreased by 27°–30° after exposure to DETANONOate in the presence or absence of platelet inhibition. Last, DETANONOate exposure decreased GP by 45% (6015 dyne/cm2), whereas GSC was decreased by 30% (340 dyne/cm2). Therefore, although most of the effect of NO on G is platelet mediated, the decrease in the speed of initiation of clotting (R) and speed of clot formation () is caused by NO-mediated coagulation protein dysfunction.
This study did not determine the molecular mechanisms responsible for NO-mediated coagulation protein function. Of interest, there is a paucity of data that ascertain whether there are interactions between individual coagulation factors and NO. NO is known to nitrosylate protein sulfhydryl groups, and polynitrosylation of tissue-type plasminogen activator decreases enzymatic activity (19). Further, posttranslational disulfide bond formation is critical to the function of the catalytic domain of serine proteases, which include coagulation factors (20). In addition, factors VII, IX, and X have a disulfide bond connecting the amino terminal half with the carboxy terminal half of the protein so that after activation, the two portions of the molecule do not dissociate (20). Although there may be other mechanisms by which NO decreases coagulation factor activity (e.g., peroxynitrite-mediated events), nitrosylation of critical sulfhydryl groups is very likely.
DETANONOate releases NO at a known rate at physiologic pH and temperature (21). DETANONOate has a half-life of 20 hours at pH 7.4 and 37°C, resulting in 2.5% of the compound present decaying each hour, or 0.04% per minute (21). Upon decay, each DETANONOate molecule releases two molecules of NO. Consequently, a concentration of 10 millimolar DETANONOate will release eight micromolar NO per minute. Although this may seem to be an excessive amount of NO required to affect coagulation, a significant amount of NO released from DETANONOate is scavenged by hemoglobin in whole blood. Further, the amount of methemoglobin formed over 30 minutes at 39°C by 10 millimolar DETANONOate would be calculated to be approximately 2% in rabbit blood, on the basis of previous work (14). A 2% or more level of methemoglobin can often be encountered clinically in settings when NO is administered (22–24). Additionally, it has been advocated that treatment is not necessary if the methemoglobin is <5% (24). Therefore, it may be possible that significant, unrecognized coagulation protein dysfunction might occur in the clinical realm.
In summary, this study demonstrated that exposure of whole blood to NO released from DETANONOate increased R and decreased primarily through a coagulation protein-dependent mechanism, whereas G was decreased primarily through a platelet-dependent mechanism. These NO-mediated effects on coagulation may occur in an occult fashion in the presence of mildly increased methemoglobin. The administration of either inhaled NO or infused NO donor occurs in several clinical settings (e.g., cardiac surgery or ARDS). Consequently, this study serves as the rational basis for future clinical and basic science investigations of NO-mediated coagulation protein and platelet dysfunction in situations involving either NO administration or pathologic increases in NO production.
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Acknowledgments
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Supported in part by the Department of Anesthesiology.
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References
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Accepted for publication October 20, 2000.
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Abstract
Introduction
