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Anesth Analg 2004;98:1312-1317 © 2004 International Anesthesia Research Society doi: 10.1213/01.ANE.0000111105.38836.F6
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Abstract |
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Tissue plasminogen activator (tPA) has a prominent role in physiological fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios (e.g., atherosclerotic disease) known to involve local decreases in tPA activity with concomitant formation of reactive nitrogen species such as peroxynitrite (OONO–), a molecule formed from nitric oxide and superoxide. We hypothesized that exposure of tPA to OONO– would result in a decrease in tPA activity. OONO– was generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Recombinant tPA was incubated at 37°C for 60 min with 0 µM SIN-1; 100 µM SIN-1; 100 µM SIN-1 and 4000 U/mL recombinant human superoxide dismutase; or 4000 U/mL recombinant human superoxide dismutase (n = 8 separate reactions per condition). Changes in tPA activity were assessed by addition of tPA samples to tissue factor-exposed human plasma and measuring clot fibrinolysis with a thrombelastograph®. Exposure to SIN-1 resulted in a decrease in tPA-mediated fibrinolysis (<1% activity of tPA not exposed to SIN-1) that was significantly (P < 0.001) different from the other three conditions. There were no significant differences between the other conditions. We conclude that tPA is inhibited by OONO–, and that OONO– may have a role in clinical thrombotic scenarios.
IMPLICATIONS: Tissue plasminogen activator (tPA) has a prominent role in fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios involving decreases in tPA activity with concomitant formation of the oxidant peroxynitrite. We determined that peroxynitrite decreased tPA activity via thrombelastography®. Peroxynitrite-mediated tPA inactivation may have a role in thrombotic states.
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Introduction |
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Tissue plasminogen activator (tPA) has a prominent role in physiological fibrinolysis in vivo. Indeed, loss of tPA activity in atherosclerotic plaque (1) and in coronary arteries of transplanted hearts (2) may contribute to thrombosis and ischemia. Of interest, tPA activity is decreased by the oxidant species hypochlorous acid in vitro (3), and chlorination of lipoproteins in atherosclerotic plaque has been identified (4). Another prime candidate potentially contributing to thrombosis via inactivation of tPA could be the potent oxidant, peroxynitrite (OONO–), which has been identified in human coronary atherosclerotic plaques (5–7) by formation of nitrotyrosine. Importantly, tPA contains 23 tyrosine residues within the molecule (8), making modification of tPA function via nitration by OONO– very likely. Thus, these data support the possibility that OONO– could diminish tPA function.
The purpose of the present study was to determine if OONO– generated by the release of nitric oxide (NO) and formation of superoxide (O2.–) by 3-morpholinosydnonimine (SIN-1) could decrease tPA activity. Changes in tPA activity were assessed by measurement of clot fibrinolysis in human plasma using a novel in vitro model of hyperfibrinolysis documented via thrombelastograph® (TEG®) analysis.
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Methods |
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TEG® tPA Assay
tPA profoundly enhances clot disintegration as measured via TEG® (9), so it was reasonable to assume that changes in tPA activity could be detected by changes in TEG® variables. Citrated, lyophilized control plasma (Trinity Biotech, Ventura, CA) was reconstituted according to manufacturer instructions just before experimentation. tPA (Genentech, Inc., San Francisco, CA) was reconstituted with deionized water and subsequently diluted with 50 mM potassium phosphate buffer (pH 7.4). Serial dilutions of tPA solution were made so that a final concentration of 0, 125, 250, and 500 U/mL of the stock solution of tPA were present in the TEG® reaction solution (n = 8 per concentration). Plasma (320 µL) was placed with 10 µL of tissue factor (final concentration 0.1% of rabbit brain TF, international sensitivity index of 1.07; Trinity Biotech), 10 µL of tPA, and 20 µL of 200 mM CaCl2 into a disposable cup in a computer-controlled TEG® (model 5000; Haemoscope Corp., Niles, IL). The concentration of TF described was used because this concentration of TF reliably initiated clot formation in <2 min. The following variables were determined at 37°C: reaction time (R, minutes), angle (
, degrees), amplitude (A, millimeter), and shear elastic modulus (G, dyne/square centimeter). R is defined as the time from when the blood sample is placed into the TEG® cuvette 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 and is reflective of the kinetics of clot formation. A is the amplitude of the TEG® signal and is a measure of clot strength. G (dyne/square centimeter) is a measure of clot strength calculated from A as follows: G = (5000 x A)/(100 – A). The relationship between A and G is curvilinear. As A varies from 0 to 100, G concordantly varies from 0 to infinity. Given this relationship, it is conceptually and statistically important to express clot strength as G (10). Consequently, whereas A was determined, G was reported. The TEG® sample was observed for 15 min, with the maximum clot strength determined after coagulation commenced designated as Gmax and the minimum clot strength noted after Gmax designated as Gmin. The difference between Gmax and Gmin was defined as G
. Figure 1 displays a typical fibrinolytic thrombelastogram with the aforementioned variables depicted. Thus, the effect of tPA-mediated plasmin generation on TF-initiated clot formation would be expected to manifest as a dose-dependent decrease of both Gmax and Gmin whereas G would likely increase. A detailed description of other aspects of the methodology of thrombelastography has been previously presented (10,11).
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SIN-1-Mediated Decreases in tPA Activity
Reaction mixtures consisted of a total of 50 µL of 50 mM potassium phosphate buffer (pH 7.4) containing 18,000 U/mL tPA (final activity of 500 U/mL tPA in the TEG® cup for assay) to which was added SIN-1 (Cayman Chemical, Ann Arbor, MI) for a final concentration of 0, 25, 50, and 100 µM. Each condition was represented by eight separate reactions. The volume of additives equaled 1% of the total reaction mixture volume. Reaction mixtures were incubated at 37°C for 60 min. After incubation, 10 µL of reaction mixture was added to plasma, 1% TF, and 200 mM CaCl2 in a TEG® cuvette as described previously to assess changes in tPA function. Concentrations of 0, 25, 50, and 100 µM SIN-1 would be expected to produce, respectively, 0, 0.25, 0.50, and 1 µM OONO– per minute at pH 7.4 and 37°C (12).
Exposure of tPA to SIN-1 and Recombinant Human Superoxide Dismutase (hSOD1)
hSOD1, produced as previously described (13), was added to reaction mixtures to prevent OONO– formation by scavenging O2.– by SIN-1 (14). Reaction mixtures consisted of a total of 50 µL of 50 mM potassium phosphate buffer (pH 7.4) containing 18,000 U/mL tPA (final activity of 500 U/mL tPA in the TEG® cup for assay) to which was added: 1) no additives, 2) 100 µM SIN-1, 3) 100 µM SIN-1 and 4000 U/mL hSOD1, and 4) 4000 U/mL hSOD1. Each condition was represented by eight separate reactions. The volume of additives equaled 2% of the total reaction mixture volume. Reaction mixtures were incubated at 37°C for 60 min. After incubation, 10 µL of reaction mixture was added to plasma, 0.1% TF, and 200 mM CaCl2 in a TEG® cuvette as described previously to assess changes in tPA function. The concentration of SIN-1 added would be expected to produce approximately 1 µM OONO– per minute at pH 7.4 and 37°C (12).
Exposure of tPA to SIN-1 and 5,10,15,20-Tetrakis-4-carboxyphenyl Porphyrin (FeTCPP)
The metalloporphyrin FeTCPP, an OONO– scavenger, was added to reaction mixtures to confirm that OONO–, not O2.–, was responsible for decreases in tPA activity mediated by SIN-1 exposure. The OONO–-scavenging rate constant for FeTCPP in 100 mM potassium phosphate buffer at 37°C is 1.65 x 106 M–1s–1 determined spectrophotometrically as previously described (15). Reaction mixtures consisted of a total of 50 µL of 50 mM potassium phosphate buffer (pH 7.4) containing 18,000 U/mL tPA (final activity of 500 U/mL tPA in the TEG® cup for assay) to which was added: 1) 100 µM SIN-1, 2) 100 µM SIN-1 and 100 µM FeTCPP, 3) 100 µM SIN-1 and 200 µM FeTCPP, and 4) 0 µM SIN-1 and 200 µM FeTCPP. Each condition was represented by eight separate reactions. The volume of additives equaled 11% of the total reaction mixture volume. Reaction mixtures were incubated at 37°C for 60 min. After incubation, 10 µL of reaction mixture was added to plasma, 0.1% TF, and 200 mM CaCl2 in a TEG® cuvette as described previously to assess changes in tPA function.
Western Blot Analysis of tPA Nitration by OONO–
Lyophilized tPA (Genentech) in phosphate buffer at pH 7.3 was 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 low nitrite content (approximately 1%) and negligible residual hydrogen peroxide. OONO– was diluted to 10 mM in 0.3 M NaOH and then added to aliquots of tPA 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% sodium dodecyl sulfate, 0.7 M 2-ß mercaptoethanol, 0.1% bromophenol blue) was added and samples were incubated at 100°C for 5 min. The final protein concentration was 1 mg/mL. Approximately 75 µg of protein was separated on 10% sodium dodecyl sulfate-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 Western blot conditions were represented by three separate reactions.
Given the unequal variance in thrombelastographic variable values between experimental data sets, all variables are expressed as median and 1st–3rd quartiles. Analyses of the effects of different doses of tPA and on Gmax, Gmin, and G were conducted with Kruskal-Wallis one-way analysis of variance with the Student-Newman-Keuls post hoc test for multiple comparisons. Similar analyses were conducted to compare the effects of different doses of SIN-1 and hSOD1 or FeTCPP in the presence or absence of SIN-1 on tPA activity as assessed by changes in Gmax, Gmin, and G. The correlation of changes in tPA activity and nitrotyrosine content with increasing concentrations of OONO– was calculated with commercially available software (Origin 7.0; OriginLab Corp., Northampton, MA). A P value < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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TEG® tPA Assay
Clot Gmax and Gmin were significantly decreased by tPA in a dose-dependent manner (Table 1). G
of clots were also significantly decreased by tPA, although there was no significant difference between values obtained by exposure to 250 or 500 U/mL. Based on these data, subsequent experiments used 500 U/mL tPA, because maximum clot dissolution was observed at this activity.
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SIN-1-Mediated Decreases in tPA Activity
Incubation of tPA with SIN-1 at concentrations of 25, 50, and 100 µM significantly increased Gmax in clots compared with those exposed to tPA incubated with 0 µM SIN-1 (Table 2). Clot Gmin significantly, dose-dependently increased when tPA was incubated with progressively increased SIN-1 concentrations. Finally, 50 and 100 µM SIN-1 incubations with tPA resulted in clot G
values significantly smaller than those obtained after incubation with 0 and 25 µM SIN-1.
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Exposure of tPA to SIN-1 and hSOD1
Incubation of tPA with SIN-1 and hSOD1 resulted in clots with significantly lesser Gmax, Gmin, and G
values compared with tPA incubated with SIN-1 alone (Table 3). Incubation with hSOD1 had no significant effect on Gmax, Gmin, and G values.
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Exposure of tPA to SIN-1 and FeTCPP
Compared with tPA incubated with SIN-1, tPA coincubated with SIN-1 and 100 µM FeTCPP resulted in clots with no significant difference in Gmax, Gmin, and G
values (Table 4). However, incubation of tPA with SIN-1 and 200 µM FeTCPP resulted in clots with significantly lesser Gmin and greater G values compared with clots exposed to tPA incubated with SIN-1 alone. There were no significant differences in Gmax, Gmin, and G values in clots exposed to tPA incubated with SIN-1 and 200 µM FeTCPP or tPA incubated with 200 µM FeTCPP exclusively.
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With regard to R and
values, there were no significant differences among groups within all the aforementioned sets of experiments. Rather, R and values were dependent on the concentration of TF activity present. R and values for all samples exposed to 0.1% TF were, respectively, R = 1.4 ± 0.2 min and = 77.7° ± 1.5°. R and values for all samples exposed to 1% TF were, respectively, R = 0.4 ± 0.1 min and = 81.3° ± 1.0°.
Western Blot Analysis of tPA Nitration by OONO–
Western blot analysis revealed that tPA had a dose-dependent increase in nitrotyrosine content in response to exposure to progressively larger OONO– concentrations (Fig. 2A). Exposure to decomposed OONO– or preincubation with nitrotyrosine (Fig. 2, A and B) resulted in background level chemiluminescence. The increase in nitrotyrosine content of tPA was significantly related to the concentration of OONO– present, best described with a Boltzmann equation (Fig. 2C).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study demonstrated that OONO– is capable of reducing tPA function in vitro. Of interest, coincubation of SIN-1 with hSOD1 results in SIN-1 functioning as a NO donor (16). Thus, it can also be concluded from our data that NO has no significant effect on tPA 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 (17–19). As previously mentioned, tPA contains 23 tyrosine residues; 2 tyrosines are contained in the fibrin-binding "finger" domain and 9 tyrosines are found in the serine protease domain (8). Concordantly, our Western blot data confirm that OONO– does increase tPA nitrotyrosine content. In summary, OONO–, but not NO, decreases tPA function and increases nitrotyrosine content.
The in vivo hematological consequences of our observations remain to be elucidated. The interface of vascular endothelium and macrophages has been noted to exhibit significant amounts of nitrotyrosine in atherosclerotic plaque (6,7). These data are consistent with OONO– being formed from O2.– and NO released from macrophage and endothelial sources during chronic inflammation associated with atherosclerosis. Consequently, given that tPA is released from vascular endothelium stimulated by thrombin formation, it is conceivable that OONO–-mediated inactivation of tPA may decrease clot lysis, contributing to a thrombotic state. However, OONO– also has been found to decrease human TF activity in vitro (19). Thus, OONO– is capable of slowing TF-mediated clot initiation and decreasing tPA-mediated clot disintegration. Whether TF or tPA is more vulnerable to nitration by OONO– remains unknown, and the location of OONO– generation (endothelial surface or within forming clot) during thrombosis has yet to be clearly elucidated. Consequently, further investigation to determine if tPA activity is decreased by reactive nitrogen species via nitrotyrosine formation in atherosclerotic plaque is warranted.
In conclusion, OONO– generated by SIN-1 significantly decreased tPA function as assessed via a thrombelastographic method. Furthermore, Western blot analyses demonstrate nitrotyrosine formation after exposure to OONO–. These data serve as the rational basis for determining if decreases in regional tPA activity noted in atherosclerotic plaque (1,2) may be attributed to nitration. Finally, in future investigations, identification of nitrated tPA or TF with decreased function in clinically encountered scenarios such as arteriosclerosis could justify administration of OONO– scavengers, such as metalloporphyrins (15), to restore normal hemostasis.
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Acknowledgments |
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This investigation was supported in part by the NINDS division of the National Institutes of Health (RO1 NS40819 [JPC]; R01 AA/DK11589 and R01 HL70071 [DAP]) and the Department of Anesthesiology.
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