Anesth Analg 2004;98:668-671
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000101988.62847.0C
ANESTHETIC PHARMACOLOGY
Peroxynitrite Decreases Rabbit Tissue Factor Activity In Vitro
Vance G. Nielsen, MD*, and
John P. Crow, PhD*, ,
Departments of *Anesthesiology,
Pharmacology/Toxicology,
Biochemistry and Molecular Genetics, The Center for Free Radical Biology, The University of Alabama at Birmingham
Address correspondence and reprint requests to Vance G. Nielsen, MD, Associate Professor, Department of Anesthesiology, The University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35249–6810. Address email to vance.nielsen{at}ccc.uab.edu
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Abstract
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Tissue factor (TF) is a primary initiator of physiological coagulation in vivo. Peroxynitrite (OONO-), a molecule formed from nitric oxide (NO) and superoxide (O2 · -), decreases human TF activity in vitro. Coagulopathy has been associated with hepatoenteric ischemia-reperfusion known to involve formation of OONO-. Further, circulating TF activity decreases in rabbits after hepatoenteric ischemia-reperfusion. We hypothesized that exposure of rabbit TF to OONO- would result in a decrease in activity. OONO- generation was performed with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Rabbit brain TF was incubated at 37°C for 90 min with 1) 0 mM SIN-1, 2) 5 mM SIN-1, 3) 5 mM SIN-1 and 2000 U/mL recombinant human superoxide dismutase (hSOD1), or 4) 2000 U/mL hSOD1 (n = 8 per condition). TF activity was assessed by addition of TF samples to human plasma and measuring clot formation kinetics with a thrombelastograph®. TF exposure to SIN-1 resulted in a 48% decrease in activity that was significantly different from the other three conditions (P < 0.001). There were no significant differences between the other conditions. We conclude that rabbit TF is inhibited by OONO-, and further investigation to determine the role of OONO- in coagulopathies associated with hepatoenteric ischemia-reperfusion is warranted.
IMPLICATIONS: Tissue factor (TF) initiates physiological coagulation in vivo. Hepatoenteric ischemia-reperfusion injury is associated with peroxynitrite (OONO-) formation, coagulopathy and decreased TF activity in rabbits. We determined that OONO- decreased rabbit TF activity in vitro via thrombelastography®.
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Introduction
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Tissue factor (TF) plays a pivotal role in the initiation of physiological hemostasis in vivo (1,2). Of interest, hepatoenteric ischemia-reperfusion has been associated with coagulopathy in the setting of hepatic transplantation in humans (3–5) and thoracic aorta occlusion-reperfusion in rabbits (6–8). Although the release of heparin from hepatoenteric mast cells significantly contributes to this coagulopathy (3–8), there is also a significant decrease in coagulation protein function (3–8). In particular, circulating TF activity decreased by 37% after aorta occlusion-reperfusion in rabbits (7). Although hemodilution associated with resuscitation may partially explain this decrease in TF (7), oxidation of TF by radical species formed during reperfusion may also play a role. Specifically, reactive nitrogen species (RNS), peroxynitrite (OONO-) in particular, are formed during and have been found to play a major role in hepatoenteric ischemia-reperfusion injury in several animal models (9–14). Further, recombinant human TF procoagulant activity was decreased by nitration with OONO- in vitro (15). When considered as a whole, these previous investigations support the possibility that OONO- generated during reperfusion could have been responsible for the decrease in TF activity during reperfusion after hepatoenteric ischemia in rabbits (7).
The purpose of the present study was to determine if OONO- generated by 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide (NO) and superoxide (O2 · -), could decrease rabbit TF activity in vitro. Changes in TF activity were assessed by changes in clot kinetics in human plasma using a new thrombelastographic (TEG®) technique.
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Methods
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TF profoundly enhances coagulation function as measured via TEG® (16); thus we reasoned that changes in TF activity could be quantified by changes in TEG® variables. Citrated, lyophilized control plasma (Trinity Biotech, Ventura, CA) was reconstituted according to manufacturer instructions just before experimentation. Rabbit brain TF with an international sensitivity index of 1.07 (Trinity Biotech) was reconstituted with 50 mM potassium phosphate buffer (pH 7.4). Serial dilutions of TF solution were made so that a final concentration of 0.05%, 0.01%, 0.005%, and 0.001% of the stock solution of TF were present in the TEG® reaction solution (n = 4 per concentration). These concentrations of TF were chosen based on those previously reported to result in enhanced coagulation determined by TEG® (16). Plasma (330 µL) was placed with 10 µL of TF solution and 20 µL of 200 mM CaCl2 into a disposable cup in a computer-controlled TEG® (Model 5000; Hemoscope Corp., Niles, IL). The following variables were measured at 37°C: reaction time (R, in min) and angle ( , in degrees). R is defined as the elapsed 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. is the angle formed from R to the inflection point of the TEG® signal as clot strength stabilizes; it is a measure of the speed of clot formation. The thrombelastograph® was programmed to stop measurements after was determined. A detailed description of the methodology of thrombelastography® has been previously presented (16,17).
OONO- generation by SIN-1 requires O2, and preliminary studies (n = 3) demonstrated that PO2 values in plasma in a closed plastic sample tube at 37°C for 15 min (PO2 = 182 ± 2 mm Hg, mean ± SD) were greater than values in plasma containing 10 mM SIN-1 (PO2 = 13 ± 2 mm Hg) as determined with a blood gas analyzer. We thus placed 500 µL of TF solution and other reactants in covered cell culture plates (Costar 12 well plate; Corning Inc., Corning, NY) with an exposed surface area of 4.9 cm2 and fluid layer thickness of 1.0 mm to prevent O2 depletion. The reaction mixtures consisted of 450 µL of 0.01% TF solution (final concentration, 0.009%) and 50 µL of compounds to be subsequently described. The reaction mixtures were placed in cell culture plates, and the plates were incubated at 37°C for 90 min. SIN-1 (Cayman Chemical, Ann Arbor, MI) was added for a final concentration of 5 mM just before incubation, which would be expected to produce approximately 50 µM OONO- per min at pH 7.4 and 37°C (18). This concentration of SIN-1 was chosen because we were uncertain as to the antioxidant potential of brain extract and wished to maximize the likelihood of a SIN-1 effect. The 4 experimental conditions (n = 8 reactions per condition) were as follows: 1) 0.009% TF; 2) 0.009% TF, 5 mM SIN-1; 3) 0.009% TF, 5 mM SIN-1, 2000 U/mL recombinant human superoxide dismutase (hSOD1); and 4) 0.009% TF, 2000 U/mL hSOD1. hSOD1, produced as previously described (19), was added to some mixtures for a final activity of 2000 U/mL before incubation to prevent OONO- formation by scavenging O2 · - (20).
All variables are expressed as mean ± SD. The correlation of changes in TF activity and TEG® variables for the thrombelastographic TF assay was calculated with commercially available software (Origen 7.0, OrigenLab Corp., Northampton, MA). Analyses of the effects of SIN-1 on R values were conducted with one-way analysis of variance with the Holm-Sidak post hoc test for multiple comparisons. A P value of <0.05 was considered significant.
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Results
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TEG® TF Activity Assay
A pattern of exponential decay accurately depicted the relationship between TF activity and R (Fig. 1), whereas a model of exponential growth best described the relationship between TF activity and (Fig. 2). Although both relationships were significant, the relationship between TF activity and R was strongest and was used to determine TF activities in subsequent experimentation.
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Figure 1. Relationship between tissue factor activity and thrombelastographic variable R. Y = 0.129e(-x/1.83) + 0.001. R2 = 0.99, P < 0.0001.
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Figure 2. Relationship between tissue factor activity and thrombelastographic variable . Y = 6.64 x 10-9e(-x/4.76) – 0.0008. R2 = 0.92, P < 0.0001.
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TF Exposure to OONO-
As shown in Figure 3, exposure to SIN-1 resulted in a significant (P < 0.001) 48% decrease in TF activity compared with samples incubated without SIN-1. Further, the addition of hSOD1 to samples exposed to SIN-1 resulted in TF activities significantly (P < 0.001) greater than samples exposed to SIN-1 without hSOD1 present. Addition of hSOD1 did not result in significant differences in TF activity compared with samples incubated without SIN-1 exposure. The R values (min) for the 4 experimental conditions were as follows: 1) 0.009% TF = 5.0 ± 0.2; 2) 0.009% TF, 5 mM SIN-1 = 6.4 ± 0.3; 3) 0.009% TF, 5 mM SIN-1, 2000 U/mL hSOD1 = 5.3 ± 0.6; and 4) 0.009% TF, 2000 U/mL hSOD1 = 4.9 ± 0.4. R values for condition 2 were significantly greater than the other conditions, and there were no significant differences in R values among conditions 1, 3, and 4.
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Figure 3. Effects of 3-morpholinosydnonimine (SIN-1) on tissue factor (TF) activity. Incubation of TF with 5 mM SIN-1 for 90 min significantly (*P < 0.001) decreased TF activity compared with all other conditions. SOD = 2000 U/mL of recombinant human superoxide dismutase 1.
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Discussion
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The present study demonstrated that OONO- can reduce rabbit TF function in vitro. Of interest, coincubation of SIN-1 with hSOD1 results in SIN-1 functioning as a NO donor (21). Thus, it can also be concluded from our data that NO has no significant effect on TF function at the concentrations of SIN-1 used. One of the primary mechanisms by which OONO- decreases protein function is by nitration of tyrosine residues, forming nitrotyrosine (15). Human TF possesses ten tyrosine residues in the extracellular domain (22), thus making it a prime candidate for OONO--mediated modification (15). In summary, OONO-, but not NO, decreases rabbit TF function.
The in vivo hematological consequences of our observations remain to be elucidated. The interface of vascular endothelium and circulating leukocytes exhibit significant amounts of nitrotyrosine after ischemia-reperfusion (10–14). These data are consistent with OONO- being formed from O2 · - and NO released from leukocytes and endothelial sources during reperfusion. Consequently, given that TF is bound to vascular endothelium, it is conceivable that OONO- may function as a hereto-undescribed endogenous anticoagulant after ischemia-reperfusion. Such a mechanism could, in part, explain coagulopathy associated with hepatic transplantation and major vascular surgery. Further investigation to determine if TF activity is decreased by RNS is warranted in these clinical settings.
In conclusion, OONO- generated by SIN-1 significantly decreased TF function as assessed via a thrombelastographic method. These data serve as the rational basis for determining if decreases in circulating TF activity noted after ischemia-reperfusion (7) may be attributable to nitration. Finally, in future investigation, identification of nitrated TF with decreased function in both laboratory and clinical settings of ischemia-reperfusion and coagulopathy could also justify administration of OONO- scavengers, such as metalloporphyrins (23), to attenuate hemorrhage.
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Acknowledgments
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Supported, in part, by the NINDS division of the National Institutes of Health (RO1 NS40819 to JPC) and the Department of Anesthesiology.
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Accepted for publication October 1, 2003.
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Abstract
Introduction
