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Effects of DETANONOate, a nitric oxide donor, on hemostasis in rabbits: An in vitro and in vivo thrombelastographic analysis
Effects of DETANONOate, a Nitric Oxide Donor, on Hemostasis in Rabbits: An In Vitro and In Vivo Thrombelastographic Analysis Vance G. Nielsen, Brian T. Geary, and Manuel S. Baird Purpose: The purpose of this study was to determine if whole blood thrombelastographic variables (reaction time, K, ␣, and maximum amplitude) would be adversely effected by exposure to the nitric oxide (NO) donor, DETANONOate, in vitro or after alveolar instillation in vivo. Materials and Methods: Conscious rabbits (n ⴝ 10) had blood sampled from ear arteries anticoagulated with sodium citrate. The blood was then incubated with 0, 1, 5, 10, or 20 mmol/L DETANONOate for 30 minutes. Arterial blood from anesthetized rabbits (n ⴝ 4) was obtained and anticoagulated before and 60 minutes after 1 mmol/L DETANONOate (2 mL/kg) was instilled into the right lung. After incubation, all samples were placed in a thrombelastograph and recalcified, with thrombelastographic variables measured for 45 minutes.
Results: In vitro, 10 mmol/L DETANONOate significantly (P .05) increased reaction time, K, and decreased ␣ compared with values observed after incubation with 0, 1, and 5 mmol/L DETANONOate.Twenty mmol/L DETANONOate significantly (P .05) increased reaction time, K, and decreased ␣ and maximum amplitude values compared with all other concentrations. In vivo, DETANONOate administration did not significantly affect thrombelastographic variables. Conclusion: DETANONOate significantly decreased hemostatic function in vitro in a dose-dependent fashion but did not significantly affect hemostatic function in vivo. Copyright © 2000 by W.B. Saunders Company
NHALED NITRIC oxide (NO) has been administered to attenuate pulmonary hypertension1-3 and to improve oxygenation in patients afflicated with the acute respiratory distress syndrome (ARDS).4-6 NO is typically administered to intubated patients with expensive devices that require ancillary personnel to monitor and maintain. Recently, compounds that release NO, the NONOates,7 have been instilled into the lungs of animals to selectively reduce pulmonary hypertension.8-11 NONOates may be aerosolized, have a prolonged residence in the lung8 and may have halflives of approximately 20 hours at physiological pH.7,8 Therefore, the administration of a NONOate could be an attractive alternative method to deliver NO to the lung in the clinical arena. In addition to beneficial effects on pulmonary vasomotor tone, NO has been noted to decrease platelet aggregation in vitro and in vivo via a guanylyl cyclase mechanism.12-17 Exposure to inhaled NO has significantly decreased platelet aggregation
in rats (20 ppm)15 and in humans with ARDS (1 ppm).18 The inhibition of platelet function in these studies was not significantly increased by greater concentrations of NO.15,18 A significant NO-mediated decrease in hemostatic function could play a critical role in the outcome of several patient populations at risk for hemorrhagic complications (eg, postoperative lung transplant patient). Unfortunately, platelet aggregometry is not a commonly available clinical method for assessing the risk of hemorrhagic complications. Unlike aggregometry, thrombelastography is a commonly used method of assessing whole blood hemostatic function at the bedside.19 Thrombelastography has been used to guide transfusion therapy for disorders in coagulation in several settings, such as procedures involving cardiopulmonary bypass20 or organ transplantation.19 Of interest, no investigation of the effects of NO on thrombelastographic variables has been performed to date. The purpose of this study was to determine if the NO donor, DETANONOate, would adversely affect hemostatic function in rabbits as assessed by thrombelastography.
I
From the Department of Anesthesiology, Divisions of Cardiothoracic Anesthesia and Anesthesiology Research, The University of Alabama at Birmingham, Birmingham, AL. Received November 19, 1999. Accepted December 1, 1999. This study was supported in part by the Department of Anesthesiology. Address reprint requests to Vance G. Nielsen, MD, Department of Anesthesiology, The University of Alabama at Birmingham, 619 South 19th St, Birmingham, AL 35249. Copyright © 2000 by W.B. Saunders Company 0883-9441/00/1501-0006$10.00/0 30
MATERIALS AND METHODS The study was approved by the Animal Review Committee of the University of Alabama at Birmingham, AL. All animals received humane care in compliance with the “Principles of Laboratory Care” formulated by the National Society for Medical Research and with the “Guide for the Care and Use of Laboratory Animals” prepared by the National Research Council and the National Academy Press (Revised October 1996, Washington, DC 20418). Journal of Critical Care, Vol 15, No 1 (March), 2000: pp 30-35
NO DONORS AND THROMBELASTOGRAPHY
Animal Preparation and Thrombelastography In vitro studies. Conscious rabbits (n 10)
were briefly restrained (2 minutes) 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 L of 0.129 mmol/L sodium citrate. Anticoagulated blood was subsequently divided into 450-L aliquots placed in capped plastic tubes at room temperature. Fifty microliters of DETANONOate dissolved in 10 mmol/L Na2PO4 buffer (pH 9.0) or buffer alone was added to the 450-L blood samples to yield final DETANONOate concentrations of 0, 1, 5, 10, or 20 mmol/L. The samples were subsequently incubated at 39° C for 30 minutes. Before and after incubation, the blood sample tube was inverted three times, and after incubation 330 L of blood was placed in a disposable cup containing 30 L of 200 mmol/L CaCl2. The cup was then inserted into a computer-controlled thrombelastograph (Model 5000, Haemoscope Corp., Skokie, IL). The proper functioning of the thrombelastograph was confirmed daily with quality control standards purchased from Haemoscope. The following thrombelastographic parameters were measured for each sample over a 45-minute period at 37° C: reaction time (R, min), coagulation time (K, min), angle (, degrees) and maximum amplitude (MA, mm). A detailed description of the method of thrombelastography has been presented in great detail elsewhere in the literature.19 In brief, R is defined as the time from when the blood sample is placed into the thrombelastograph cuvette until initial fibrin formation occurs as noted by a signal of 2 mm amplitude. K, a measure of the speed at which a clot forms with a certain viscoelastic strength, is defined as the time from R until the amplitude of the thrombelastographic signal is 20 mm. is the angle formed from R to the inflection point of the thrombelastographic signal as clot strength stabilizes; it is a measure of the speed of clot formation. Finally, MA is the largest amplitude of the thrombelastographic signal and is a measure of clot strength. In vivo studies. Rabbits were (n 4) initially anesthetized with a bolus of 5 mg/kg ketamine and 1.5 mg/kg xylazine via a marginal ear vein. Anesthesia was maintained with an infusion of 10 mg/kg/hr ketamine and 3 mg/kg/hr xylazine. After tracheotomy and placement of a 3.5 mm outer diameter endotracheal tube, mechanical ventilation with a Harvard Apparatus ventilator was performed with PaCO2 maintained at 32 to 45 mm Hg with FiO2 of 1.0. Pancuronium was administered 0.3 mg/kg/hr IV to facilitate mechanical ventilation. An 18gauge catheter was placed in the right femoral artery for blood sampling and pressure recording on a polygraph (Model 7D, Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer’s solution at 20 mL/kg/hr. Rabbits were subsequently placed in the right lateral decubitus position, with a 30-minute equilibration period following the completion of the surgical preparation and device insertion. Rabbits were next administered 2 mL/kg of 1 mmol/L DETANONOate in 0.9% NaCl over 5 minutes through a 20 G catheter placed into the trachea next to the endotracheal tube. This dose was chosen as the rabbit clears greater than 90% of a crystalloid instillation from the alveolar space in under an hour,21 resulting in a local alveolar concentration of DETANONOate 10 mmol/L. Arterial blood samples were obtained and processed for gas analysis (Model 1640 Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) and thrombelastography after 30 minutes of equilibration and 60
31
minutes after instillation of 1 mmol/L DETANONOate, as mentioned previously. Animals were euthanized with a saturated solution of KCl at the conclusion of experimentation.
In vitro documentation of NO release from DETANONOate. To document the release of NO from DETANONOate, arterial blood samples from rabbits (n 3) were incubated with 0, 1, 5, 10, or 20 mmol/L of the NO donor for 60 minutes at 39° C with subsequent determination of methemoglobin with a cooximeter (Model Synthesis, Instrumentation Laboratory, Lexington, MA).
Statistical Analyses All variables are expressed as mean STD. With regard to thrombelastography, it was decided a priori that blood samples that did not clot were to be assigned an R value of 45 minutes, a K value of 45 minutes, an value of 0° and an MA of 0 mm. Analyses of the effects of DETANONOate on thrombelastographic and physiological variables were conducted by analysis of variance (ANOVA). Post hoc analysis was conducted with the Student-Newman-Keuls test. An alpha error of 0.05 was considered significant for these studies. The relationship between the percentage of methemoglobin and DETANONOate concentration present in blood in vitro was determined via linear regression.
RESULTS
Effects of DETANONOate on Thrombelastographic Variables In Vitro Incubation of blood samples with DETANONOate resulted in a dose-dependent decrease in hemostasis (Fig 1). R was significantly increased and was significantly decreased by 10 mmol/L DETANONOate compared with 0, 1, or 5 mmol/L DETANONOate. In turn, 20 mmol/L DETANONOate significantly increased R, decreased and decreased MA compared with all other doses of DETANONOate. Furthermore, 20 mmol/L DETANONOate significantly increased K compared with 0, 1, or 5 mmol/L DETANONOate. Finally, two blood samples exposed to 20 mmol/L DETANONOate displayed no discernable clot. Effects of DETANONOate on Thrombelastographic Variables In Vivo Unlike the changes in hemostasis in vitro, the instillation of DETANONOate did not cause any significant changes in R, K, , or MA (Fig 2). The administration of DETANONOate did not significantly decrease mean arterial pressure or heart rate (Table 1). Similarly, pH and PaCO2 were not significantly affected by DETANONOate administration (Table 1). However, PaO2 was significantly decreased after DETANONOate administration.
32
NIELSEN, GEARY, AND BAIRD
Fig 1. Effects of DETANONOate on thrombelastographic variables in vitro. DETANONOate adversely affected hemostasis in a dose dependent fashion. All values are mean ⴞ STD. *P0.05 vs. 0, 1, and 5 mmol/L DETANONOate. †P.05 vs. all other concentrations.
In Vitro Documentation of NO Release from DETANONOate Incubation of blood samples with DETANONOate resulted in a highly significant linear relationship between the percentage of methemoglobin and the concentration of DETANONOate present (Fig 3). DISCUSSION
This study demonstrates that DETANONOate in vitro decreases whole blood clotting in a dosedependent fashion in rabbits as assessed by thrombelastography. These findings are in sharp contrast to previous studies using platelet aggregometry that demonstrated an apparent “all or none” effect on clotting in response to various concentrations of NO.15,18 One explanation for this difference may be found in the method used to expose blood to NO and the determination of hemostatic function. In the present investigation, blood samples were continuously exposed to a steady concentration of NO released from DETANONOate before and during thrombelastographic measurements. There were no sample preparation procedures (eg, centrifugation;
fractionation of blood into platelet rich or platelet poor plasma) that physically separated components of the coagulation system from the stimulus of DETANONOate-derived NO. Consequently, unlike aggregometry, thrombelastography permits a more rapid assessment of the effects of NO on hemostatic function without the confounding effects of sample processing. As DETANONOate has been demonstrated to release NO at a known rate at physiological pH and temperature,7 the concentration of NO required to significantly affect whole blood hemostasis may be determined. DETANONOate has a half-life of 20 hours at pH 7.4 and 37 °C, resulting in 2.5% of the compound present decaying every hour or 0.04% per minute.7 On decay, each DETANONOate molecule releases two molecules of NO. Consequently, concentrations of 1, 5, 10, and 20 mmol/L DETANONOate may be expected to release 0.8, 4, 8, and 16 mol/L NO per minute. Based on these calculations, our data indicate that NO elaboration of 8 to 16 mol/L per minute is required to significantly affect whole blood hemostasis in vitro in rabbits. Finally, the in vitro measurements of methemoglobin support the concept that
NO DONORS AND THROMBELASTOGRAPHY
33
Fig 2. Effects of DETANONOate on thrombelastographic variables in vivo. Instillation of 1 mmol/L DETANONOate (2 mL/kg) into the right lung did not significantly affect hemostasis. All values are mean ⴞ STD.
DETANONOate releases NO in a linear, dosedependent fashion. Unlike the in vitro results, we were unable to demonstrate hemostatic aberrations after administration of DETANONOate into the lung in vivo. One likely explanation is that insufficient DETANONOate was administered to adversely affect hemostasis. Of interest, the dose of DETANONOate administered was 20-fold that reported to significantly modify chronic pulmonary hypertension in rats.8 Another reason why DETANONOate administration may not have affected hemostasis is that the NO donor may have been rapidly absorbed from the alveolar space and eliminated from the circulation. This is improbable as, unlike alveolar ad-
ministration, intravenous administration of DETANONOate is associated with marked hypotension and an increase in the concentration of NO metabolites (NO2 and NO3) in plasma.8 No hemodynamic changes were observed in this study after pulmonary administration of DETANONOate. These data raise the clinically attractive possibility that administration of NO via DETANONOate may locally modulate the pulmonary vasculature without adversely affecting systemic hemostasis or hemodynamics. In summary, this study demonstrated that incubation of whole blood with DETANONOate in vitro decreased hemostasis in a dose-dependent fashion. However, a concentration of DETANONOate in the alveolar space far in excess of
Table 1. In Vivo Hemodynamic and Arterial Blood Gas Data Time
30 minutes EQ 60 minutes after instillation of DETANONOate
MAP (mm Hg)
HR (beats/min)
pH
PaCO2 (mm Hg)
PaO2 (mm Hg)
80 13 72 16
158 15 188 15
7.49 0.03 7.48 0.04
36.9 4.6 38.0 1.2
425 790* 162 110*
All values are expressed as the mean STD. Abbreviations: MAP, mean arterial pressure; HR, heart rate. *P 0.05 vs. value at 30 minutes EQ.
34
NIELSEN, GEARY, AND BAIRD
Fig 3. Association of methemoglobin formation and DETANONOate concentration. Incubation of whole blood for 60 minutes with various concentrations of DETANONOate resulted in a linear increase in methemoglobin. Dotted lines represent 95% confidence intervals.
that required to attenuate pulmonary hypertension did not affect hemostasis as assessed by thrombelastography. Additionally, thrombelastography may provide a bedside assessment of the effects of NO
(inhaled or intravenous) on hemostasis. Finally, this study serves as the rational basis for investigations to determine the effects of inhaled NO gas and instilled NO donors on thrombelastography.
REFERENCES 1. Ziegler JW, Ivy DD, Wiggins JW, et al: Effects of dipyridamole and inhaled nitric oxide in pediatric patients with pulmonary hypertension. Am J Respir Crit Care Med 158:13881395, 1998 2. Ricciardi MJ, Knight BP, Martinez FJ, et al: Inhaled nitric oxide in primary hypertension: A safe and effective agent for predicting response to nifedipine. J Am Coll Cardiol 32:1068-1073, 1998 3. Hoehn T, Krause M, Hentschel R: High-frequency ventilation augments the effect of inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Eur Respir J 11:234238, 1998 4. Iotti GA, Olivei MC, Palo A, et al: Acute effects of inhaled nitric oxide in adult respiratory distress syndrome. Eur Respir J 12:1164-1171, 1998 5. Mathisen DJ, Kuo EY, Hahn C, et al: Inhaled nitric oxide for adult respiratory distress syndrome after pulmonary resection. Ann Thorac Surg 66:1894-1902, 1998 6. Germann P, Poschl G, Leitner C, et al: Additive effect of nitric oxide inhalation on the oxygenation benefit of the prone position in the adult respiratory distress syndrome. Anesthesiology 89:1401-1406, 1998 7. Keefer LK, Nims RW, Davies KM, et al: “NONOates” (1Substituted Diazen-1-ium-1,2-diolated) as nitric oxide donors: Convenient nitric oxide dosage forms. Meth Enzymol 268:281293, 1996
8. Hampl V, Tristani-Fiorouzi M, Hutsell TC, et al: Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension. Cardiovasc Res 31:55-62, 1996 9. Schutte H, Grimminger F, Otterbein J, et al: Efficiency of aerosolized nitric oxide donor drugs to achieve sustained pulmonary vasodilation. J Pharmacol Exp Ther 282:985-994, 1997 10. Brilli RJ, Krafte-Jacobs B, Smith DJ, et al: Intratracheal instillation of a novel NO/nucleophile adduct selectively reduces pulmonary hypertension. J Appl Physiol 83:1968-1975, 1997 11. Brilli RJ, Krafte-Jacobs B, Smith DJ, et al: Aerosolization of novel nitric oxide donors selectively reduce pulmonary hypertension. Crit Care Med 26:1390-1396, 1998 12. Weber A, Strobach H, Schror K: Direct inhibition of platelet function by organic nitrates via nitric oxide formation. Eur J Pharmacol 247:29-37, 1993 13. Moro MA, Russell RJ, Cellek S, et al: cGMP mediates the vascular and platelet actions of nitric oxide: Confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci USA 93:1480-1485, 1996 14. Aoki H, Inoue M, Mizobe T, et al: Platelet function is inhibited by nitric oxide liberation during nitrogycerin-induced anaesthesia. Br J Anesth 79:476-481, 1997 15. Nong Z, Hoylaerts M, Van Pelt N, et al: Nitric oxide inhalations inhibits platelet aggregation and platelet-mediated pulmonary thrombosis in rats. Circ Res 81:865-869, 1997
NO DONORS AND THROMBELASTOGRAPHY
16. Brune B, Hanstein K: Rapid reversibility of nitric oxide induced platelet inhibition. Thrombosis Res 90:83-91, 1998 17. Gries A, Bode C, Peter K, et al: Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo. Circulation 97:1481-1487, 1998 18. Samama CM, Diaby M, Fellahi J, et al: Inhibition of platelet aggregation by inhaled nitric oxide in patients with acute respiratory distress syndrome. Anesthesiology 83:56-65, 1995
35
19. Caprini JA, Traverso CI, Walenga JM, et al: Thromboelastography. Semin Thromb Hemost 21:1-93, 1995 (suppl) 20. Shore-Lesserson L, Manspeizer HE, DePerio M, et al: Thromboelastography-guided transfusion algorithm reduces transfusions in complex cardiac surgery. Anesth Analg 88:312319, 1999 21. Smerdira N, Gates L, Hastings R, et al: Alveolar and lung liquid clearance in anesthetized rabbits. J Appl Physiol 70:18271835, 1991
Results: In vitro, 10 mmol/L DETANONOate significantly (P .05) increased reaction time, K, and decreased ␣ compared with values observed after incubation with 0, 1, and 5 mmol/L DETANONOate.Twenty mmol/L DETANONOate significantly (P .05) increased reaction time, K, and decreased ␣ and maximum amplitude values compared with all other concentrations. In vivo, DETANONOate administration did not significantly affect thrombelastographic variables. Conclusion: DETANONOate significantly decreased hemostatic function in vitro in a dose-dependent fashion but did not significantly affect hemostatic function in vivo. Copyright © 2000 by W.B. Saunders Company
NHALED NITRIC oxide (NO) has been administered to attenuate pulmonary hypertension1-3 and to improve oxygenation in patients afflicated with the acute respiratory distress syndrome (ARDS).4-6 NO is typically administered to intubated patients with expensive devices that require ancillary personnel to monitor and maintain. Recently, compounds that release NO, the NONOates,7 have been instilled into the lungs of animals to selectively reduce pulmonary hypertension.8-11 NONOates may be aerosolized, have a prolonged residence in the lung8 and may have halflives of approximately 20 hours at physiological pH.7,8 Therefore, the administration of a NONOate could be an attractive alternative method to deliver NO to the lung in the clinical arena. In addition to beneficial effects on pulmonary vasomotor tone, NO has been noted to decrease platelet aggregation in vitro and in vivo via a guanylyl cyclase mechanism.12-17 Exposure to inhaled NO has significantly decreased platelet aggregation
in rats (20 ppm)15 and in humans with ARDS (1 ppm).18 The inhibition of platelet function in these studies was not significantly increased by greater concentrations of NO.15,18 A significant NO-mediated decrease in hemostatic function could play a critical role in the outcome of several patient populations at risk for hemorrhagic complications (eg, postoperative lung transplant patient). Unfortunately, platelet aggregometry is not a commonly available clinical method for assessing the risk of hemorrhagic complications. Unlike aggregometry, thrombelastography is a commonly used method of assessing whole blood hemostatic function at the bedside.19 Thrombelastography has been used to guide transfusion therapy for disorders in coagulation in several settings, such as procedures involving cardiopulmonary bypass20 or organ transplantation.19 Of interest, no investigation of the effects of NO on thrombelastographic variables has been performed to date. The purpose of this study was to determine if the NO donor, DETANONOate, would adversely affect hemostatic function in rabbits as assessed by thrombelastography.
I
From the Department of Anesthesiology, Divisions of Cardiothoracic Anesthesia and Anesthesiology Research, The University of Alabama at Birmingham, Birmingham, AL. Received November 19, 1999. Accepted December 1, 1999. This study was supported in part by the Department of Anesthesiology. Address reprint requests to Vance G. Nielsen, MD, Department of Anesthesiology, The University of Alabama at Birmingham, 619 South 19th St, Birmingham, AL 35249. Copyright © 2000 by W.B. Saunders Company 0883-9441/00/1501-0006$10.00/0 30
MATERIALS AND METHODS The study was approved by the Animal Review Committee of the University of Alabama at Birmingham, AL. All animals received humane care in compliance with the “Principles of Laboratory Care” formulated by the National Society for Medical Research and with the “Guide for the Care and Use of Laboratory Animals” prepared by the National Research Council and the National Academy Press (Revised October 1996, Washington, DC 20418). Journal of Critical Care, Vol 15, No 1 (March), 2000: pp 30-35
NO DONORS AND THROMBELASTOGRAPHY
Animal Preparation and Thrombelastography In vitro studies. Conscious rabbits (n 10)
were briefly restrained (2 minutes) 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 L of 0.129 mmol/L sodium citrate. Anticoagulated blood was subsequently divided into 450-L aliquots placed in capped plastic tubes at room temperature. Fifty microliters of DETANONOate dissolved in 10 mmol/L Na2PO4 buffer (pH 9.0) or buffer alone was added to the 450-L blood samples to yield final DETANONOate concentrations of 0, 1, 5, 10, or 20 mmol/L. The samples were subsequently incubated at 39° C for 30 minutes. Before and after incubation, the blood sample tube was inverted three times, and after incubation 330 L of blood was placed in a disposable cup containing 30 L of 200 mmol/L CaCl2. The cup was then inserted into a computer-controlled thrombelastograph (Model 5000, Haemoscope Corp., Skokie, IL). The proper functioning of the thrombelastograph was confirmed daily with quality control standards purchased from Haemoscope. The following thrombelastographic parameters were measured for each sample over a 45-minute period at 37° C: reaction time (R, min), coagulation time (K, min), angle (, degrees) and maximum amplitude (MA, mm). A detailed description of the method of thrombelastography has been presented in great detail elsewhere in the literature.19 In brief, R is defined as the time from when the blood sample is placed into the thrombelastograph cuvette until initial fibrin formation occurs as noted by a signal of 2 mm amplitude. K, a measure of the speed at which a clot forms with a certain viscoelastic strength, is defined as the time from R until the amplitude of the thrombelastographic signal is 20 mm. is the angle formed from R to the inflection point of the thrombelastographic signal as clot strength stabilizes; it is a measure of the speed of clot formation. Finally, MA is the largest amplitude of the thrombelastographic signal and is a measure of clot strength. In vivo studies. Rabbits were (n 4) initially anesthetized with a bolus of 5 mg/kg ketamine and 1.5 mg/kg xylazine via a marginal ear vein. Anesthesia was maintained with an infusion of 10 mg/kg/hr ketamine and 3 mg/kg/hr xylazine. After tracheotomy and placement of a 3.5 mm outer diameter endotracheal tube, mechanical ventilation with a Harvard Apparatus ventilator was performed with PaCO2 maintained at 32 to 45 mm Hg with FiO2 of 1.0. Pancuronium was administered 0.3 mg/kg/hr IV to facilitate mechanical ventilation. An 18gauge catheter was placed in the right femoral artery for blood sampling and pressure recording on a polygraph (Model 7D, Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer’s solution at 20 mL/kg/hr. Rabbits were subsequently placed in the right lateral decubitus position, with a 30-minute equilibration period following the completion of the surgical preparation and device insertion. Rabbits were next administered 2 mL/kg of 1 mmol/L DETANONOate in 0.9% NaCl over 5 minutes through a 20 G catheter placed into the trachea next to the endotracheal tube. This dose was chosen as the rabbit clears greater than 90% of a crystalloid instillation from the alveolar space in under an hour,21 resulting in a local alveolar concentration of DETANONOate 10 mmol/L. Arterial blood samples were obtained and processed for gas analysis (Model 1640 Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) and thrombelastography after 30 minutes of equilibration and 60
31
minutes after instillation of 1 mmol/L DETANONOate, as mentioned previously. Animals were euthanized with a saturated solution of KCl at the conclusion of experimentation.
In vitro documentation of NO release from DETANONOate. To document the release of NO from DETANONOate, arterial blood samples from rabbits (n 3) were incubated with 0, 1, 5, 10, or 20 mmol/L of the NO donor for 60 minutes at 39° C with subsequent determination of methemoglobin with a cooximeter (Model Synthesis, Instrumentation Laboratory, Lexington, MA).
Statistical Analyses All variables are expressed as mean STD. With regard to thrombelastography, it was decided a priori that blood samples that did not clot were to be assigned an R value of 45 minutes, a K value of 45 minutes, an value of 0° and an MA of 0 mm. Analyses of the effects of DETANONOate on thrombelastographic and physiological variables were conducted by analysis of variance (ANOVA). Post hoc analysis was conducted with the Student-Newman-Keuls test. An alpha error of 0.05 was considered significant for these studies. The relationship between the percentage of methemoglobin and DETANONOate concentration present in blood in vitro was determined via linear regression.
RESULTS
Effects of DETANONOate on Thrombelastographic Variables In Vitro Incubation of blood samples with DETANONOate resulted in a dose-dependent decrease in hemostasis (Fig 1). R was significantly increased and was significantly decreased by 10 mmol/L DETANONOate compared with 0, 1, or 5 mmol/L DETANONOate. In turn, 20 mmol/L DETANONOate significantly increased R, decreased and decreased MA compared with all other doses of DETANONOate. Furthermore, 20 mmol/L DETANONOate significantly increased K compared with 0, 1, or 5 mmol/L DETANONOate. Finally, two blood samples exposed to 20 mmol/L DETANONOate displayed no discernable clot. Effects of DETANONOate on Thrombelastographic Variables In Vivo Unlike the changes in hemostasis in vitro, the instillation of DETANONOate did not cause any significant changes in R, K, , or MA (Fig 2). The administration of DETANONOate did not significantly decrease mean arterial pressure or heart rate (Table 1). Similarly, pH and PaCO2 were not significantly affected by DETANONOate administration (Table 1). However, PaO2 was significantly decreased after DETANONOate administration.
32
NIELSEN, GEARY, AND BAIRD
Fig 1. Effects of DETANONOate on thrombelastographic variables in vitro. DETANONOate adversely affected hemostasis in a dose dependent fashion. All values are mean ⴞ STD. *P0.05 vs. 0, 1, and 5 mmol/L DETANONOate. †P.05 vs. all other concentrations.
In Vitro Documentation of NO Release from DETANONOate Incubation of blood samples with DETANONOate resulted in a highly significant linear relationship between the percentage of methemoglobin and the concentration of DETANONOate present (Fig 3). DISCUSSION
This study demonstrates that DETANONOate in vitro decreases whole blood clotting in a dosedependent fashion in rabbits as assessed by thrombelastography. These findings are in sharp contrast to previous studies using platelet aggregometry that demonstrated an apparent “all or none” effect on clotting in response to various concentrations of NO.15,18 One explanation for this difference may be found in the method used to expose blood to NO and the determination of hemostatic function. In the present investigation, blood samples were continuously exposed to a steady concentration of NO released from DETANONOate before and during thrombelastographic measurements. There were no sample preparation procedures (eg, centrifugation;
fractionation of blood into platelet rich or platelet poor plasma) that physically separated components of the coagulation system from the stimulus of DETANONOate-derived NO. Consequently, unlike aggregometry, thrombelastography permits a more rapid assessment of the effects of NO on hemostatic function without the confounding effects of sample processing. As DETANONOate has been demonstrated to release NO at a known rate at physiological pH and temperature,7 the concentration of NO required to significantly affect whole blood hemostasis may be determined. DETANONOate has a half-life of 20 hours at pH 7.4 and 37 °C, resulting in 2.5% of the compound present decaying every hour or 0.04% per minute.7 On decay, each DETANONOate molecule releases two molecules of NO. Consequently, concentrations of 1, 5, 10, and 20 mmol/L DETANONOate may be expected to release 0.8, 4, 8, and 16 mol/L NO per minute. Based on these calculations, our data indicate that NO elaboration of 8 to 16 mol/L per minute is required to significantly affect whole blood hemostasis in vitro in rabbits. Finally, the in vitro measurements of methemoglobin support the concept that
NO DONORS AND THROMBELASTOGRAPHY
33
Fig 2. Effects of DETANONOate on thrombelastographic variables in vivo. Instillation of 1 mmol/L DETANONOate (2 mL/kg) into the right lung did not significantly affect hemostasis. All values are mean ⴞ STD.
DETANONOate releases NO in a linear, dosedependent fashion. Unlike the in vitro results, we were unable to demonstrate hemostatic aberrations after administration of DETANONOate into the lung in vivo. One likely explanation is that insufficient DETANONOate was administered to adversely affect hemostasis. Of interest, the dose of DETANONOate administered was 20-fold that reported to significantly modify chronic pulmonary hypertension in rats.8 Another reason why DETANONOate administration may not have affected hemostasis is that the NO donor may have been rapidly absorbed from the alveolar space and eliminated from the circulation. This is improbable as, unlike alveolar ad-
ministration, intravenous administration of DETANONOate is associated with marked hypotension and an increase in the concentration of NO metabolites (NO2 and NO3) in plasma.8 No hemodynamic changes were observed in this study after pulmonary administration of DETANONOate. These data raise the clinically attractive possibility that administration of NO via DETANONOate may locally modulate the pulmonary vasculature without adversely affecting systemic hemostasis or hemodynamics. In summary, this study demonstrated that incubation of whole blood with DETANONOate in vitro decreased hemostasis in a dose-dependent fashion. However, a concentration of DETANONOate in the alveolar space far in excess of
Table 1. In Vivo Hemodynamic and Arterial Blood Gas Data Time
30 minutes EQ 60 minutes after instillation of DETANONOate
MAP (mm Hg)
HR (beats/min)
pH
PaCO2 (mm Hg)
PaO2 (mm Hg)
80 13 72 16
158 15 188 15
7.49 0.03 7.48 0.04
36.9 4.6 38.0 1.2
425 790* 162 110*
All values are expressed as the mean STD. Abbreviations: MAP, mean arterial pressure; HR, heart rate. *P 0.05 vs. value at 30 minutes EQ.
34
NIELSEN, GEARY, AND BAIRD
Fig 3. Association of methemoglobin formation and DETANONOate concentration. Incubation of whole blood for 60 minutes with various concentrations of DETANONOate resulted in a linear increase in methemoglobin. Dotted lines represent 95% confidence intervals.
that required to attenuate pulmonary hypertension did not affect hemostasis as assessed by thrombelastography. Additionally, thrombelastography may provide a bedside assessment of the effects of NO
(inhaled or intravenous) on hemostasis. Finally, this study serves as the rational basis for investigations to determine the effects of inhaled NO gas and instilled NO donors on thrombelastography.
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NO DONORS AND THROMBELASTOGRAPHY
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