Anesth Analg 2004;99:21-26
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000116962.93953.70
CARDIOVASCULAR ANESTHESIA
Peroxynitrite Decreases Hemostasis in Human Plasma In Vitro
Vance G. Nielsen, MD*,
John P. Crow, PhD
,
Ashish Mogal, MD*,
Fen Zhou, MD*, and
Dale A. Parks, PhD*,
,
Departments of *Anesthesiology,
Physiology and Biophysics, and
Pediatrics, The Center for Free Radical Biology, The University of Alabama at Birmingham, Birmingham, Alabama, and the
Departments of Pharmacology and Toxicology, The University of Arkansas for Medical Sciences, Little Rock, Arkansas
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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Coagulopathy has been associated with clinical scenarios that involve reactive nitrogen species such as peroxynitrite (OONO–). Further, OONO– decreases tissue factor and fibrinogen function in vitro. Thus, we hypothesized that exposure of plasma to the OONO– generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide, would result in a decrease in hemostatic function via diminished coagulation protein activity. Hemostatic function of plasma exposed to SIN-1 (0, 1, 5, and 10 mM for 60 min at 37°C) was assessed with thrombelastography, activated partial thromboplastin time, and prothrombin time in the presence or absence of superoxide dismutase (SOD) or an OONO– scavenger. SIN-1 exposure resulted in a significant (P < 0.05), dose-dependent decrease in plasma hemostatic function and concurrent significant (P < 0.05) decreases in activities of factor VII, factor VIII complex, and factor X. Fibrinogen concentration was not affected by SIN-1. Antithrombin and protein C activity also decreased significantly (P < 0.05). Coincubation with SOD or an OONO– scavenger significantly (P < 0.05) attenuated SIN-1 mediated changes in hemostasis and procoagulant/anticoagulant activity. We conclude that OONO– may decrease hemostatic function in human plasma by nitration of key procoagulants and that OONO– may play a significant role in hemorrhagic states.
IMPLICATIONS: Coagulopathy has been associated with clinical scenarios involving reactive nitrogen species such as peroxynitrite. It was determined that exposure of human plasma to peroxynitrite generated by 3-morpholinosydnonimine resulted in hypocoagulability measured by prothrombin time, activated partial thromboplastin time, and thrombelastography. Peroxynitrite may play a significant role in hemorrhagic states.
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Introduction
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Reactive nitrogen species, and peroxynitrite (OONO–) in particular, play a major role in hepatoenteric ischemia-reperfusion injury in animal models (1,2). In addition, hepatoenteric ischemia-reperfusion has been associated with coagulopathy in the setting of human hepatic transplantation (3,4) and thoracic aorta occlusion-reperfusion in rabbits (5). Although the release of heparin from hepatoenteric mast cells significantly contributes to this coagulopathy (3–5), there is also a significant decrease in coagulation protein function (3–5). Oxidant species such as singlet oxygen have been found in vitro to directly inactivate factor V, factor VIII, and factor X (6). Further, fibrinogen has been found to be nitrated in vitro by OONO– (7,8) and nitrated fibrinogen has been detected in vivo in the plasma of patients with acute respiratory distress syndrome (8). Nitration has been found to both enhance (8) and diminish (7) the capacity for fibrinogen to be cleaved by thrombin. In sum, these previous investigations support the possibility that OONO– could either enhance or diminish hemostatic factor function.
The purpose of the present study was to determine if 3-morpholinosydnonimine (SIN-1), a molecule that releases nitric oxide and forms superoxide (O2 · –) to generate OONO–, could decrease hemostatic function in human plasma as determined by thrombelastography (TEG®), activated partial thromboplastin time (aPTT), and prothrombin time (PT). Further, the effects of OONO– on key procoagulants (factor VII [FVII], factor VIII complex [FVIII:C], and factor X [FX]) and anticoagulants (antithrombin [AT] and protein C [PC]) were also assessed. Finally, nitration of tyrosines contained in FX and PC by OONO– was determined by western blot analysis.
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Methods
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Citrated, lyophilized control plasma (Trinity Biotech, Ventura, CA) was reconstituted according to manufacturer instructions just before experimentation. The reaction mixtures consisted of 450 µL of plasma and 50 µL of compounds to be subsequently described. OONO– generation by SIN-1 consumes O2, and preliminary studies demonstrated that plasma could rapidly (<15 min) become anaerobic if reacted in a closed plastic sample tube (data not shown). In previous investigations, plasma was vigorously mixed at frequent intervals to avoid this experimental design problem (8). However, mixing could also affect hemostasis; therefore the 500 µL of plasma and other reactants were placed 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 were subsequently incubated at 37°C for 1–2 h in room air. SIN-1 (Cayman Chemical, Ann Arbor, MI) was added for a final concentration of 0, 1, 5, or 10 mM just before incubation, which would be expected to produce approximately 0, 10, 50, and 100 µM OONO– per minute (9). Recombinant human superoxide dismutase (hSOD1), produced as previously described (10), was added to some mixtures for final activities of 2 x 104, 2 x 103, or 2 x 102 U/mL before incubation to scavenge O2 · – and prevent OONO– formation (11). Other samples had the catalytic antioxidant, 5,10,15,20-tetrakis-4-carboxyphenyl porphyrin (FeTCPP), added for final concentrations of 20, 4, or 0.8 µM. The OONO–-scavenging rate constant for FeTCPP in 100 mM potassium phosphate buffer at 37°C was 1.65 x 106 M–1s–1, determined spectrophotometrically as previously described (12).
After incubation, plasma was removed from the cell culture plates for hemostatic and coagulation factor analyses. Plasma (340 µL) was placed with 20 µL of 200 mM CaCl2 into a disposable cup in a computer-controlled thrombelastograph® (Model 5000; Haemoscope Corp., Niles, IL). The following variables were measured over 45 min at 37°C: reaction time (R, minutes), angle (
, degrees), maximum amplitude (MA, mm), and shear elastic modulus (G, dyne/cm2). A detailed description of the methodology of TEG® has been previously described (13,14). After incubation, aPTT, PT, and fibrinogen concentration were determined with an analyzer (Hemochron Response®, International Technidyne Corporation, Edison, NJ). Spectrophotometric determination of plasma activity of the following coagulation proteins was performed: FVII, VIII:C, FX, AT, and PC. FVII, VIII:C, FX, and PC kits were obtained from DiaPharma (West Chester, OH). AT activity was determined with an assay from Trinity Biotech. All samples were assayed in duplicate in these spectrophotometric determinations. Coincubation of SIN-1 with hSOD1 (and not FeTCPP) was performed in experiments involving chromogenic assays as these assays involve measurement of the release of p-nitroaniline, the maximal absorption wavelength of which is 405 nm. FeTCPP significantly absorbs at 409 nm, thus likely interfering with chromogenic-based assays. Plasma values obtained in all assays were compared to human reference plasma (Trinity Biotech) values or standards provided in the kit.
Lyophilized PC and FX (Enzyme Research Laboratories, South Bend, IN) in 20 mM Tris-HCl/0.1 M NaCl/1 mM benzamidine buffer, pH 7.4, were diluted to 2 mg/mL. The concentration of commercially available OONO– (Cayman Chemical, Ann Arbor, MI) was determined spectrometrically (
= 302 nm, 
= 1.67 mM–1 · s–1). This formulation of OONO– has a small nitrite content (
1%) and negligible residual hydrogen peroxide. OONO– was diluted to 10 mM in 0.3 M NaOH and then added to aliquots of PC or FX while continually mixing. Samples were allowed to incubate for 15 min at room temperature. After incubation, an equal volume of lysis buffer (0.625 M Tris-HCl, 1.37 M glycerol, 10% SDS, 0.7 M 2-ß mercaptoethanol, 0.1% bromophenol blue) was added and samples incubated at 100°C for 5 min. The final protein concentration was 1 mg/mL. Approximately 75 µg protein was separated on 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes (Amersham-Pharmacia, Piscataway, NJ). The membrane was blocked in 5% nonfat dry milk dissolved in Tris-buffered saline and incubated for 60 min at room temperature with 2 µg/mL rabbit anti-nitrotyrosine polyclonal antibody (Cayman Chemical), washed, and then incubated for 60 min with horseradish-peroxidase-conjugated goat, anti-rabbit immunoglobulin G (NEN; Life Science Products, Boston, MA). Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Supersignal; Pierce Biochemicals, Rockford, IL). Protein bands were quantified by densitometric scanning of bands using a Fluorchem gel documentation system (Alpha Innotech, San Leandro, CA). Controls consisted of the addition of decomposed OONO–, prepared by incubating OONO– for 10 min at 100°C. An additional control for antibody specificity consisted of the addition of 10 mM nitrotyrosine (Sigma Chemical, St. Louis, MO).
All variables are expressed as mean ± SD. Analyses of the effects of SIN-1 in the presence or absence of hSOD1 or FeTCPP on all variables were conducted with one-way analysis of variance (ANOVA) with the Holm-Sidak post hoc test for multiple comparisons. Regression analysis was performed to determine the significance of the linear response of tyrosine nitration of FX and PC by OONO–. A P value of <0.05 was considered significant.
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Results
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Hemostatic data are presented in Tables 1–3. With regard to TEG®-derived data, exposure to SIN-1 resulted in a significant, dose-dependent increase in R and decrease in , MA, and G (Table 1). In addition to a dose-dependent effect, a time-dependent, SIN-1-mediated decrease in hemostatic function was observed, as plasma incubated for 2 h with 2.5 mM SIN-1 did not have significantly different hemostatic function than plasma incubated with 5 mM SIN-1 for 1 h (Table 1). Incubation with hSOD1 prevented a 10 mM SIN-1-mediated increase in R and decrease in until only 200 U/mL hSOD1 was present (Table 2). Similarly, the OONO–-scavenging metalloporphyrin FeTCPP prevented an increase in R mediated by 10 mM SIN-1 until only 0.8 µM FeTCPP was present (Table 3). However, concentrations of 4 and 0.8 µM FeTCPP were unable to prevent SIN-1 mediated decreases in MA and G.
With regard to PT data, incubation of plasma with 0, 5, and 10 mM SIN-1 resulted in values of 11.3 ± 0.0, 12.2 ± 0.3, and 13.3 ± 0.3 s, all significantly different from one another. Coincubation of 10 mM SIN-1 with 20 µM FeTCPP resulted in a PT of 12.2 ± 0.2 s, a value that was significantly more than samples exposed to 0 mM SIN-1 but significantly less than samples exposed to 10 mM SIN-1. With regard to aPTT data, incubation of plasma with 0, 5, and 10 mM SIN-1 resulted in values of 30.5 ± 2.6, 40.9 ± 1.3, and 53.8 ± 3.0 s, all significantly different from one another. Coincubation of 10 mM SIN-1 with 20 µM FeTCPP resulted in an aPTT of 35.9 ± 4.3 s, a value that was significantly more than samples exposed to 0 mM SIN-1 but significantly less than samples exposed to 5 mM or 10 mM SIN-1. Finally, exposure of plasma to either 0 or 10 mM SIN-1 resulted in fibrinogen concentrations of 184 ± 46 and 185 ± 43 mg/dl, respectively, that were not significantly different from one another.
Hematological data are displayed in Table 4. FVII activity was significantly decreased by 10 mM SIN-1, and coincubation with hSOD1 abrogated SIN-1-mediated effects. Both VIII:C and FX activities demonstrated a SIN-1-mediated, dose-dependent decrease in function that was abrogated by hSOD1. Similarly AT activity demonstrated a decrease in function after exposure to either 5 or 10 mM SIN-1 that was attenuated by hSOD1. PC activity significantly decreased after exposure to each tested concentration of SIN-1—the only analyte to do so. Further, although coincubation with hSOD1 significantly increased PC activity to values of plasma samples incubated with 10 mM SIN-1, these values were still significantly less than samples exposed to 0, 1, and 5 mM SIN-1.
Western blot analyses revealed that FX and PC had a dose-dependent increase in nitrotyrosine content in response to exposure to progressively larger OONO– concentrations (Fig. 1A and 1C). Exposure to decomposed OONO– or preincubation with excess nitrotyrosine (Fig. 1B and 1D) resulted in background level chemiluminescence. The increase in nitrotyrosine content of FX and PC was significantly and linearly related to the concentration of OONO– present (Fig. 2).
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Figure 1. The effect of peroxynitrite on extent of nitration of Factor X and Protein C. Factor X (A) and Protein C (C) were incubated with peroxynitrite (62.5, 125, 250, or 500 µM) for 15 min. Aliquots of the reacted protein were separated on 10% SDS-polyacrylamide gel and nitrated product identified by rabbit anti-nitrotyrosine polyclonal antibody. Decomposed (Dec) peroxynitrite was used as a control for nonspecific nitration. As a control for antibody specificity, peroxynitrite-treated Factor X (B) and Protein C (D) were preincubated with 3-NT before addition of primary antibody.
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Figure 2. Dose dependent effect of peroxynitrite on the extent of nitration of Factor X and Protein C. Factor X (top panel) or Protein C (bottom panel) were reacted with various concentrations of peroxynitrite and the western blot images (n = 3 for each condition) digitized using a Fluorchem gel documentation system (Alpha Innotech, San Leandro, CA). The dotted line represents the linear regression curve. Decomposed (Dec) peroxynitrite controls were included for specificity of the nitration.
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Discussion
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OONO– generated from SIN-1 significantly decreased hemostatic function in human plasma as assessed by TEG®, PT, and aPTT. This interpretation was supported by attenuation of SIN-1-mediated decreases in hemostasis by decreasing OONO– formation with hSOD1 or directly scavenging OONO– with FeTCPP. Further, SIN-1 mediated decreases in procoagulant function (e.g., FX) were also attenuated by hSOD1, supporting the contention that OONO– modified enzymatic activity. Importantly, fibrinogen function was not affected by SIN-1 at the largest dose used; thus, unlike previous studies (7,8), nitration of fibrinogen appears neither to enhance nor diminish interactions with thrombin in human plasma. Of interest, both AT and PC activity were decreased by SIN-1, with at least partial restoration of function during coincubation with hSOD1. Thus, a decrease in procoagulant and not an increase in anticoagulant function may account for SIN-1 mediated decreases in hemostasis. In further support of our hypothesis, OONO–-mediated nitration likely contributed to procoagulant and anticoagulant dysfunction, as dose-dependent increases in nitrotyrosine formation in FX and PC were observed after exposure to OONO–. In sum, these data implicate nitration of procoagulants by OONO– as a mechanism responsible for decreased hemostatic function in human plasma exposed to SIN-1.
Although the concentrations of OONO– required to affect hemostatic and hematological variables in normal human plasma in the present study may not be encountered acutely in clinical settings, smaller concentrations of OONO– present for a prolonged period of time after situations such as hepatoenteric reperfusion or during sepsis could degrade procoagulants. Indeed, given the short half-life and great reactivity of OONO–, no clinical study has been able to quantify OONO– concentrations except indirectly (and qualitatively) via nitrotyrosine formation. It has already been documented that fibrinogen is nitrated in the setting of acute respiratory distress syndrome (8), thus demonstrating that OONO– does react with coagulation proteins in vivo. An important experimental limitation of OONO– exposure in the present study is that, unlike circulating plasma in vivo, plasma in vitro at 37°C will likely lose procoagulant activity after a few hours, preventing one from exposing plasma to smaller concentrations of SIN-1 for longer periods of time. Nevertheless, the concentrations of both SIN-1 and OONO– used in the present study were similar to previous investigations (7,8,15,16).
Commonly encountered comorbidities and surgical procedures may render patients vulnerable to OONO–-mediated changes in hemostasis. Liver transplant recipients (17,18) and vascular surgery patients (19,20) have been demonstrated to have significant decreases in circulating and/or tissue antioxidant defenses. Further, liver transplant recipients (21,22) and vascular surgery patients (23) have also been demonstrated to have significant decreases in several coagulation proteins. These patients also endure additional losses of circulating antioxidants and procoagulants via resuscitation and consequent hemodilution in the perioperative period (17,20,21). With regard to oxidant stress, hepatoenteric ischemia-reperfusion, demonstrated to result in OONO– formation in animal models (1,2), occurs in the conduct of major vascular and orthotopic liver transplantation surgery. Of interest, our western blot analyses demonstrate that purified FX and PC are readily nitrated with a relatively small concentration of OONO–, consistent with observations that OONO– decreases function and increases nitration of recombinant human tissue factor (16). Thus, if circulating antioxidant and procoagulant activities are critically decreased, it is conceivable that a compromised patient undergoing a significant hepatoenteric ischemic insult may be vulnerable to hemorrhage mediated in part by OONO– formed in vivo.
A focus of future investigation is the determination of the differential vulnerability of specific components of the procoagulant and anticoagulant systems to OONO–. The present study demonstrated that the effects of SIN-1 on the intrinsic coagulation system (measured by aPTT and TEG®) were more than on the extrinsic coagulation system (determined via PT). However, although SIN-1-mediated decreases in FVII activity and increase in PT values were modest in the present study, when coupled with potential OONO–-mediated decreases in endothelial tissue factor activity (16) in vivo, a more profound effect on clinical hemorrhage may be possible. It remains to be resolved whether specific procoagulants are differentially compromised by OONO– or if several procoagulants are modestly compromised with the sum effect being hypocoagulability.
Another important subject is the determination of the mechanism of OONO– decreases in enzymatic function of specific proteins. For example, tyrosine is important for heparin and heparan sulfate binding (24) and the heparin-binding site of AT contains tyrosine (25). Thus, AT activity could be decreased not only by OONO–-mediated modification of tyrosines that affect serine protease binding but also by nitrotyrosine formation in the heparin-binding, activity enhancing site of AT. Finally, the method of OONO– exposure may differentially affect specific proteins in the procoagulant and anticoagulant systems. PC demonstrated a SIN-1-dependent, OONO–-independent decrease in function as determined by only limited return of function in the presence of SOD (Table 4). In contrast, direct exposure of PC to OONO– resulted in a linear increase in nitrotyrosine (Figs. 1 and 2). We interpret these data to indicate that a SIN-1 metabolite is likely responsible for OONO–-independent decreases in PC function. In sum, the specific mechanisms by which OONO– modulates procoagulant and anticoagulant protein function remain to be elucidated.
In conclusion, the hemostatic response of human plasma to OONO– exposure is a decrease in hemostatic function likely mediated by decreases in activities of coagulation factors such as FVII, VIII:C, and FX. Nitration of fibrinogen appears to have no hemostatic effects in plasma, and exposure of AT and PC to OONO– probably did not further decrease hemostatic function, as anticoagulant activity was concordantly decreased by OONO–. Preclinical investigation involving animal models of coagulopathy wherein OONO– likely plays a role (e.g., liver ischemia-reperfusion) are required to determine the efficacy of OONO– scavengers in improving hemostasis. If nitration of key procoagulants by OONO– is found to play a significant role in such animal models, further human investigations in settings such as liver transplantation and major vascular surgery are warranted.
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Acknowledgments
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Supported, in part, by the National Institutes of Health (RO1 NS40819 to JPC and R01 AA/DK11589 and R01 HL70071 to DAP) and the Department of Anesthesiology, The University of Alabama at Birmingham.
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Accepted for publication December 17, 2003.
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Abstract
Introduction
