Kaohsiung Journal of Medical Sciences (2015) 31, 115e122

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ORIGINAL ARTICLE Investigation of possible prophylactic, renoprotective, and cardioprotective effects of thromboprophylactic drugs against ischemiaereperfusion injury

Sinan Demirtas a,*, Oguz Karahan a, Suleyman Yazıcı b, Orkut Guclu a, Ahmet Calıskan a, Orhan Tezcan a, Ibrahim Kaplan c, Celal Yavuz a a Medical School of Dicle University, Department of Cardiovascular Surgery, Diyarbakir, Turkey b Istanbul Bilim University, Sisli Florence Nightingale Hospital, Department of Cardiovascular Surgery, Istanbul, Turkey c Medical School of Dicle University, Department of Biochemistry, Diyarbakir, Turkey

Received 28 April 2014; accepted 3 October 2014 Available online 13 January 2015

KEYWORDS Abstract The aim of this study was to investigate whether and antiaggregant Antiaggregant drugs; agents have protective effects against oxidative damage induced by peripheral ischemia Anticoagulant drugs; ereperfusion (I/R). Groups were created as follows: control group, I/R group (sham group), Cardioprotection; I/R plus acetylsalicylic acid (Group I), I/Rþclopidogrel (Group II), I/Rþrivaroxaban (Group Ischemiaereperfusion III), I/Rþbemiparin sodium (Group IV), and I/Rþenoxaparin sodium (Group V). In Groups I, injury; II, III, IV, and V, drugs were administered daily for 1 week before I/R creation. Peripheral I/ Renoprotection R was induced in the I/R groups by clamping the right femoral artery. The rats were sacrificed 1 hour after reperfusion. Nitrogen oxide levels, malondialdehyde (MDA) levels, paraoxonase-1 (PON1) activity, and prolidase activity were evaluated in both cardiac and renal tissues. There was no significant difference in nitrogen oxide levels between the groups. However, cardiac and renal MDA were significantly higher and PON1 activity was markedly lower in the I/R groups compared with the control group (p < 0.05). Although elevated prolidase activity was detected in both the cardiac and renal tissue of the I/R groups, only the sham group and Group V had significantly higher renal prolidase activity (p < 0.05). Group V had significantly higher cardiac MDA, PON1, prolidase levels, and renal prolidase activity compared with the sham group (p < 0.05). Significant improvement in renal MDA levels was only observed in Group III, and marked improvement was observed in the cardiac MDA levels of Group II when compared with

Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Medical School of Dicle University, Department of Cardiovascular Surgery, Diyarbakir, Turkey. E-mail address: [email protected] (S. Demirtas). http://dx.doi.org/10.1016/j.kjms.2014.12.005 1607-551X/Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved. 116 S. Demirtas et al.

the sham group (p < 0.05). Thromboprophylactic agents appear to provide partial or prominent protection against I/R injury. Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.

Introduction for this study. The rats were obtained from the Laboratory Animal Production Unit of the university. All rats were can locally obstruct distal flow and cause distal maintained in standard humidity (50 5%) and temperature ischemia or disrupt distant organ perfusion via emboli [1]. (22 2 C) controlled cages with a 12-hour light/dark cycle Therefore, thrombosis prophylaxis regimens for prevention for 1 week prior to the start of the study. of ischemic complications in high-risk populations are frequently investigated [2]. Thrombosis and embolic events Study protocol can occur despite sufficient management and even when appropriate therapy is administered [3]. The rats were randomly divided into seven equal groups. All Treatment for both acute embolic and thrombotic occlusion operations were performed simultaneously on sham and focuses on reperfusion of the ischemic limb. Reducing the study groups for sample standardization. All rats were adverse outcomes of the ischemic injury is also a main anesthetized with ketamine (Ketalar; Pfizer, Ta¨by, target of treatment [1]. Furthermore, the duration of Sweden) at a dose of 130 mg/kg and xylazine (Rompun; ischemic exposure is an important determinant of tissue Bayer, Gothenburg, Sweden) at a dose of 20 mg/kg via an recovery and of the undesired effects of reperfusion [4,5]. intraperitoneal line. Maintenance of anesthesia was pro- Therefore, receiving prophylaxis prior to thrombosis- vided with ketamine hydrochloride (50 mg/kg). induced ischemia is important. Antiaggregant and anticoagulant agents can be used for Experimental ischemia/reperfusion injury model thromboprophylaxis [6]. Previous investigations of anti- preparation aggregant agents have claimed that these agents modulate The right femoral artery was clamped for 6 hours and then vascular smooth muscle and induce apoptosis, as well as the clamp was removed to create reperfusion. Two millili- inhibit platelet adhesion and aggregation [7]. Previous re- ters of blood samples were obtained from inferior vena ports have also identified antioxidant effects of anti- cava at the 1st hour of reperfusion. The rats were sacrificed aggregant drugs [8]. Similarly, antithrombotic agents have 1 hour after reperfusion, and cardiac tissue samples and cellular effects, on growth factor regulation and cell left kidneys were obtained. adhesion, in addition to preventing new thrombosis for- Eight of the rats were placed in the control group at the mation [9,10]. Previous studies have also mentioned beginning of the study, and 2 mL of blood samples from possible antioxidant features of antithrombotic agents inferior vena cava, cardiac tissue samples, and left kidneys [10,11]. were obtained for determining the baseline nitrogen oxide The purpose of the present study was to investigate and (NOx) levels, malondialdehyde (MDA) levels, and compare the possible cardiac and renal protective effects paraoxonase-1 (PON1) and prolidase activity in blood, of thromboprophylactic agents against ische- renal, and cardiac tissue without any intervention. The miaereperfusion (I/R) injury in an experimental peripheral remaining rats were separated for experimental I/R injury I/R model. induction. The six peripheral I/R injury groups of eight animals Materials and methods each were as follows: Sham group: I/R only. Group I (n Z 8): Acetylsalicylic acid (ASA, Coraspin; All animal procedures were approved by the Animal Bayer, Leverkusen, Germany) was administered orally via Research Committee of Dicle University and were per- gavage at a dose of 30 mg/kg/day beginning 1 week prior to formed in accordance with the Animal Welfare Act and the the start of study. I/R was induced after 1 week of drug Guide for the Care and Use of Laboratory Animals pre- administration. pared by the ethics committee. The principal investigator, Group II (n Z 8): bisulfate (Planor; Koc¸ak animal laboratory staff, and the primate handling staff Farma, Tekirdag, Turkey) was administered orally via were present for all procedures. Study design and protocol gavage at a dose of 1 mg/kg/day beginning 1 week prior to was created according to our previous experimental the start of the study. I/R was induced after 1 week of drug modeling [12]. administration. Group III (n Z 8): (Xarelto; Bayer Schering Animals Pharma AG, Wuppertal, Germany) was administered orally via gavage at a dose of 3 mg/kg/day beginning 1 week prior Fifty-six male SpragueeDawley rats (aged 8e12 weeks) to the start of the study. I/R was induced after 1 week of weighing 230 30 g (mean standard deviation) were used drug administration. Thrombosis prophylaxis and I/R injury 117

Group IV (n Z 8): Bemiparin sodium (Hibor 3500 IU/ liberated p-nitrophenol at 405 nm and 37C [15]. PON1 0.2 mL; Laboratorios Farmaceuticos Rovi SA, Spain) was activity was expressed as U/L for blood samples and U/g administered intraperitoneally at a dose of 250 U/kg/day protein for tissue extracts. beginning 1 week prior to the start of the study. I/R was induced after 1 week of drug administration. Statistical analysis Group V (n Z 8): (Oksapar 2000 ANTI-  XA IU/0.2 mL; Koc¸ak Farma, Tekirdag, Turkey) was admin- All statistical analyses were performed using SPSS software istered intraperitoneally at a dose of 250 U/kg/day begin- version 15.0 (SPSS Inc., Chicago, IL, USA). All results are ning 1 week prior to the start of the study. I/R was induced expressed as mean standard deviation. Continuous vari- after 1 week of drug administration. ables were compared using one-way analysis of variance (ANOVA). The control group, sham group, and other study groups were compared using the Dunnett test for posthoc Laboratory analysis analysis. A p-value 0.05 was considered statistically significant. Blood samples (2 mL) were obtained from vena cava for evaluating NOx, MDA, PON1, and prolidase levels in plasma. Samples were centrifuged at 4042 g (4000 RPM at 22.6 cm) Results at þ4C for 10 minutes to obtain plasma. Thereafter, plasmas were transferred into Eppendorf tubes. After ani- The blood NOx, MDA, PON1, and prolidase levels were mals were sacrificed, hearts and left kidneys from all 12.1 3.2mM, 21.4 19.3mM, 103.5 89.3 U/L, groups were dissected at 4C and quickly frozen in liquid 102.2 111.1 U/g in control group, respectively. Changes nitrogen. Tissue was homogenized in 9 mL of 0.25M sucrose in NOx and PON1 levels in sham and study groups were and 10mM EDTA (pH 7.4) using a Teflon homogenizer to found to be insignificant. However, the MDA levels were obtain a 10% suspension, and centrifuged at 3800 g for 30 significantly increased in the sham group (155.6 88.5mM) minutes. Tissue extracts and blood samples were stored at when compared with baseline levels in control group 70C until further analysis. (p > 0.05). Moreover, the decrement of serum MDA levels in drug groups (Group I, II, III, IV, V) was statistically signifi- NOx measurement cant compared with the sham group (p < 0.001). Addition- Nitrogen oxide (NOx) levels, an available measure of ally, the prolidase levels were significantly increased in the platelet-derived nitric oxide,were detected using the Gri- sham group (1748.2 1148.8 U/g) when compared with the ess reagent method. The method is based on a modified control group (p < 0.05). Prolidase levels were significantly cadmium reaction as described in Yavuz et al [4]. A cali- decreased in Group II (313.4 241.7 U/g), Group III bration curve was provided by adding the Griess reagent (513.2 488.0 U/g) and Group IV (491.1 376.3 U/g) into 0mM, 20mM, 40mM, 60mM, 80mM, and 100 mM solu- compared with the sham group. The serum values of tions of sodium nitrite and reading the optical density (OD) oxidative markers are compared between groups in Table 1. at 545 nm on a spectrophotometer (Bio-tek Instruments The baseline cardiac levels of biochemical oxidative Inc., Winooski, VT, USA). NO concentrations were calcu- markers in the control group were as follows: NOx, lated as mmol/l for blood samples and mM/g protein for 11.05 0.12mM/g protein; MDA, 84.12 8.98mM/g protein; tissue extracts. PON1, 71.83 8.91 U/g protein; prolidase, 265.62 37.19 U/g protein. The values for renal tissue MDA measurement were as follows: NOx, 12.04 3.99mM/g protein; MDA, Changes in tissue lipid peroxidation levels were determined 7.52 2.21mM/g protein; PON1, 101.35 7.12 U/g protein; by measuring malondialdehyde (MDA) levels, determined by and prolidase, 14.40 1.58 U/g protein. the quantity of thiobarbituric acid reactive products. NOx values for the sham group were not significantly Advanced glycation end products and MDA levels were different: 9.09 4.94mM/g protein in cardiac tissue and determined according to the method described by Ohkawa 13.56 3.82mM/g protein in renal tissue (p > 0.05). The I/R et al [13]. MDA values were expressed as mM for blood groups treated with thromboprophylactic regimens had samples and mM/g protein for tissue extracts. insignificantly higher values compared with the sham group (ANOVA, F Z 1.98, p Z 0.11). Cardiac NOx levels were as Prolidase measurement follows: 25.68 15.76mM/g protein in the group treated Prolidase levels were determined spectrophotometrically with acetylsalicylic acid (Group I), 9.40 4.36mM/g protein based on measurement of proline, a prolidase product, in the group treated with clopidogrel bisulfate (Group II), using the method described by Myara et al. [14]. Proline 27.84 16.70mM/g protein in the group treated with levels (expressed as U/L for blood samples and U/g protein rivaroxaban (Group III), 14.29 15.86mM/g protein in the for tissue extracts) were detected by measuring the group treated with bemiparin sodium (Group IV), and absorbance at 515 nm. 12.61 5.80mM/g protein in the group treated with enox- aparin sodium (Group V). Conversely, the treated groups PON1 measurement had insignificantly lower renal NOx levels: 11.62 1.99mM/ PON1 levels were determined spectrophotometrically using g protein, 11.99 1.14mM/g protein, 8.49 1.92mM/g the modified Eckerson method. The hydrolysis rate of par- protein, 11.33 0.70mM/g protein, and 12.59 1.53mM/g aoxon (0.0-diethyl-0-p-nitrophenylphosphate; Sigma protein for Groups I, II, III, IV, and V, respectively, Chemical Co., London, UK) was determined by measuring compared with the sham group (ANOVA, F Z 1.63, 118 S. Demirtas et al.

Table 1 The evaluation and comparison of oxidative markers in blood samples and posthoc analysis between groups. n Z 8 for each group NOx (mM) MDA (mM) Prolidase (U/g) PON1 (U/L) Posthoc analysis Control group 12.1 3.2 21.4 19.3 102.2 111.1 103.5 89.3 p* # U Sham group 9.2 1.7 155.6 88.5 1748.2 1148.8 117.2 99.9 Sham vs. control < 0.001 0.001 Acetylsalicylic 14.3 5.9 54.5 48.1 712.0 512.2 83.6 49.2 Sham vs. Group I 0.006 0.069 acid (Group I) Clopidogrel (Group II) 12.6 4.4 42.6 35.5 313.4 241.7 97.0 79.1 Sham vs. Group II 0.000 0.012 Rivaroxaban (Group III) 19.2 9.5 31.6 33.5 513.2 488.0 109.6 69.7 Sham vs. Group III 0.002 0.036 Bemiparin (Group IV) 16.8 4.6 39.5 27.2 491.1 376.3 77.9 57.5 Sham vs. Group IV 0.001 0.028 Enoxaparin (Group V) 18.4 7.1 45.2 30.9 701.5 617.4 86.8 72.6 Sham vs. Group V 0.005 0.062 p (One-way ANOVA) 0.612 <0.001 0.042 0.089 #: p for MDA U : p for prolidase *p < 0.05 is significant MDA Z malondialdehyde; NOx Z nitrogen oxide; PON1 Z paraoxonase 1. p Z 0.18). The NOx levels of the different groups are the control group (p < 0.001). The treatment groups had compared in Fig. 1. slightly higher values compared with the sham group MDA levels were significantly higher in the sham group (p > 0.05). For the treatment groups, cardiac PON1 levels (243.56 9.60mM/g protein in cardiac tissue and were as follows: 33.30 3.22 U/g protein, 28.09 2.62 U/ 19.76 4.99mM/g protein in renal tissue) compared with gprotein,39.42 0.41 U/g protein, 37.61 6.21 U/g the controls (p < 0.001). In contrast, groups treated with protein, and 31.20 3.01 U/g protein. Renal PON1 levels thromboprophylactic agents had lower cardiac MDA levels: were detected as 16.35 2.23 U/g protein, 170.69 29.85mM/g protein, 151.67 45.01mM/g protein, 16.61 1.62 U/g protein, 16.68 3.45 U/g protein, 162.25 26.89mM/g, protein 172.95 30.89mM/g protein, 16.57 2.68 U/g protein, and 15.52 1.59 U/g protein for and 149.65 33.42mM/g protein for Groups I, II, III, IV, and Groups I, II, III, IV, and V, respectively. PON1 levels are V, respectively (ANOVA, F Z 10.44, p < 0.001). Renal MDA compared in Fig. 3. levels were as follows: 17.72 3.09mM/g protein, The I/R study groups had markedly different prolidase 11.69 2.25mM/g protein, 14.66 3.52mM/g protein, values from the controls in both cardiac and renal tissue 7.42 1.28mM/g protein, and 13.73 0.90mM/g protein for (ANOVA, F Z 5.22, p Z 0.002 and F Z 146.47, p < 0.001, the five treatment groups, respectively (ANOVA, F Z 33.84, respectively). Prolidase levels were significantly higher in p < 0.001). The MDA values of the different groups are the sham group samples, 5149.54 3386.47 U/g protein summarized in Fig. 2. (p Z 0.001) in cardiac tissue and 95.62 3.96 U/g protein I/R groups had significantly lower PON1 levels in cardiac (p Z 0.006) in renal tissue. The drug treatment groups had (ANOVA, F Z 76.71, p < 0.001) and renal tissue (ANOVA, a range of lower prolidase values compared with the sham F Z 316.19, p < 0.001) compared with the control group. group. Prolidase values were as follows in Groups I, II, III, Lower PON1 levels were observed in the sham group IV, and V, respectively: 804.87 205.54 U/g protein, (23.46 2.39 U/g protein in cardiac tissue and 1155.97 1.51 U/g protein, 1020.27 910.46 U/g protein, 13.59 4.15 U/g protein in renal tissue) compared with 1159.53 1389.40 U/g protein, and 349.54 3386.47 U/g

Figure 1. (A) Nitrogen oxide levels in cardiac tissue extracts. (B) Nitrogen oxide levels in renal tissue extracts. Baseline values were obtained from the control group; Sham: I/R only; Group I: I/R group treated with acetylsalicylic acid; Group II: I/R group treated with clopidogrel; Group III: I/R group treated with rivaroxaban; Group IV: I/R group treated with bemiparin; Group V: I/R group treated with enoxaparin. p < 0.05 was considered statistically significant. Thrombosis prophylaxis and I/R injury 119

Figure 2. (A) Malondialdehyde levels in cardiac tissue extracts. (B) Malondialdehyde levels in renal tissue extracts. Baseline values were obtained from the control group; Sham: I/R only (p Z sham vs. baseline); Group I: I/R group treated with acetylsa- licylic acid (p Z sham vs. Group I); Group II: I/R group treated with clopidogrel (p Z sham vs. Group II); Group III: I/R group treated with rivaroxaban (p Z sham vs. Group III); Group IV: I/R group treated with bemiparin (p Z sham vs. group IV); Group V: I/R group treated with enoxaparin (p Z sham vs. Group V). protein in cardiac tissue and 75.64 4.29 U/g protein, levels of the drug treatment groups were lower than those 73.58 4.20 U/g protein, 58.79 3.74 U/g protein, of the sham group (p < 0.05). After thromboprophylaxis, all 70.42 4.88 U/g protein, and 20.56 5.84 U/g protein in of the drug groups had lower MDA levels compared with the renal tissue. Prolidase levels are compared between groups sham group. However, the decrease was statistically insig- in Fig. 4. nificant (p > 0.05). As a result of I/R, the highest cardiac and renal prolidase values occurred in the sham group. The drug treatment Discussion groups had decreased cardiac and renal prolidase levels compared with the sham group. Furthermore, only cardiac This study demonstrated that I/R induces oxidative stress in prolidase levels differed significantly between the sham cardiac and renal tissues. The sham group had higher car- group and the drug treatment groups (all except the diac and renal prolidase and MDA levels and lower cardiac enoxaparin sodium group). Renal prolidase levels were and renal PON1 levels compared with the baseline levels of markedly lower, almost at baseline values, for the group the control group. No significant differences in NOx values treated with enoxaparin sodium (p Z 0.001). To our were observed. However, all of the I/R drug treatment knowledge, this is the first study to compare the cardiac groups except the rivaroxaban group had slightly higher and renal protective effects of antiaggregants and antico- NOx levels. Differences between the sham group and the agulants (including the new oral anticoagulant rivaroxaban) drug groups were insignificant. Cardiac and renal MDA in an experimental I/R model.

Figure 3. (A) PON1 levels in cardiac tissue extracts; (B) PON1 levels in renal tissue extracts. Baseline values were obtained from the control group; Sham: I/R only (p Z sham vs. baseline); Group I: I/R group treated with acetylsalicylic acid (p Z sham vs. Group I); Group II: I/R group treated with clopidogrel (p Z sham vs. Group II); Group III: I/R group treated with rivaroxaban (p Z sham vs. Group III); Group IV: I/R group treated with bemiparin (p Z sham vs. Group IV); Group V: I/R group treated with enoxaparin (p Z sham vs. Group V). 120 S. Demirtas et al.

Figure 4. (A) Prolidase levels in cardiac tissue extracts. (B) Prolidase levels in renal tissue extracts. Baseline values were ob- tained from the control group; Sham: I/R only (p Z sham vs. baseline); Group I: I/R group treated with acetylsalicylic acid (p Z sham vs. Group I); Group II: I/R group treated with clopidogrel (p Z sham vs. Group II); Group III: I/R group treated with rivaroxaban (p Z sham vs. Group III); Group IV: I/R group treated with bemiparin (p Z sham vs. Group IV); Group V: I/R group treated with enoxaparin (p Z sham vs. Group V).

NOx, the main actor in cGMP-dependent vasodilatation, treated with clopidogrel in a study by Taher and Nassir [26]. plays a role in many molecular, cellular, and physiological Additionally, clopidogrel treatment was found to reduce events, such as the metabolism of proteins, lipids, and oxidative stress in patients with type 2 diabetes [26]. Deepa nucleic acids [4]. In particular, NOx is reported to be a and Varalakshmi studied the possible protective effects of critical mediator, and thus, an important signaling mole- LMWHs on cardiac and renal oxidative changes induced by cule for cardiovascular health [4,16]. Oxidative stress, experimental atherogenesis, and they reported that LMWHs inflammation, endothelial dysfunction, and apoptotic reduced lipid peroxidation and demonstrated protective events have been associated with a lack of nitric oxide effects against oxidative damage [27]. Similar to previous activity [4,17]. ASA, which inhibits cyclooxygenase, was reports, we found that both cardiac and renal MDA levels previously found to inhibit the inducible form of nitric oxide were reduced in I/R-induced rats treated with thrombo- and its products of oxidation in infracted heart muscle in prophylactic agents. In particular, renal MDA levels were situ by Kimura et al [18]. Conversely, de la Cruz et al re- reduced to baseline values in I/R-induced rats treated with ported that ASA stimulates NOx production from neutro- bemiparin sodium (p < 0.01). phils and elevates plasma nitrite/nitrate levels [19]. In our PON1 is a liver enzyme that hydrolyzes various types of study, administration of ASA caused insignificant elevation substrates, such as arylesters, phosphate esters, and lac- in cardiac NOx levels and a minimal decrease in renal NOx tones [28]. Decreased PON1 levels were reported during levels (p > 0.05). Clopidogrel, a platelet ADP receptor oxidative stress by Cebeci et al [29]. Jaichander et al sug- antagonist, was found to increase endothelial nitric oxide gested that the antiatherosclerotic properties of ASA might bioavailability and demonstrated vasoprotective effects in be mediated by induction of PON1 [28]. Tselepis et al patients with symptomatic coronary artery disease [20]. claimed that PON1 enzyme activity is inversely related to Kanko et al reported reducing the effects of clopidogrel on clopidogrel efficacy as a determinant of antiplatelet ac- plasma, liver, and lung NO in an experimental I/R model tivity [30]. Topsakal et al reported that enoxaparin sodium [21]. We detected insignificant changes in cardiac and renal leads to alterations in PON1 activity during acute oxidative NOx levels in the group treated with clopidogrel. Higher stress in an experimental model of spinal cord injury [31]. cardiac NO levels were reported in rats treated with topical However, in our study, significant differences in cardiac and low molecular weight (LMWHs) by He et al [22]. renal PON1 activity were not detected in I/R groups who Lakshmi et al reported that LMWHs improve wound healing received thromboprophylactic treatment compared with by reducing nitric oxide synthetase levels [23]. In the cur- the sham group. rent study, we did not find any significant changes in groups Prolidase is an essential cytosolic enzyme that has been treated with either oral or injectable factor Xa inhibitor demonstrated to play an important role in collagen meta- (rivaroxaban or enoxaparin sodium and bemiparin sodium, bolism [32]. Higher prolidase levels during oxidative stress respectively). have been previously reported [32]. Miltyk et al claimed MDA is a product of lipid peroxidation in processes that nonsteroidal anti-inflammatory drugs inhibit prolidase associated with oxidative stress [24]. Ulubas‚ et al reported activity in cultured human skin fibroblasts [33]. In addition, that MDA levels were reduced with ASA treatment in ASA was determined to be an inhibitor of collagen synthesis cisplatin-induced oxidative stress [25]. Kanko et al studied by inhibiting prolidase activity [34]. In particular, renal the protective effects of clopidogrel on oxidant damage in tissue is the most prolidase-rich structure of the human a rat ischemia reperfusion model, and they reported body [35]. Thus, we investigated cardiac and renal tissue increased MDA levels in rats treated clopidogrel [21]. Con- levels of prolidase in an experimental I/R model with and trastingly, MDA levels decreased in diabetic patients without thromboprophylactic treatment. Both cardiac and Thrombosis prophylaxis and I/R injury 121 renal prolidase levels were significantly higher in the I/R [10] Manduteanu I, Dragomir E, Voinea M, Capraru M, group that did not receive thromboprophylactic treatment Simionescu M. Enoxaparin reduces H2O2-induced activation of (sham group) compared with the baseline values (control human endothelial cells by a mechanism involving cell adhe- group). I/R groups that received thromboprophylactic sion molecules and nuclear transcription factors. Pharma- e treatment had dramatically lower cardiac prolidase values cology 2007;79:154 62. [11] Zupan Z, Pilipovic K, Dangubic B, FrkovicV, SusticA, Zupan G. compared with the sham group. The most distinct differ- Effects of enoxaparin in the rat hippocampus following trau- ence in cardiac prolidase levels was observed in the group matic brain injury. Prog Neuropsychopharmacol Biol Psychia- treated with enoxaparin sodium, in which cardiac values try 2011;35:1846e56. were almost baseline values. Furthermore, only the enox- [12] Caliskan A, Yavuz C, Karahan O, Yazici S, Guclu O, Demirtas S, aparin sodium group had statistically significantly lower et al. Factor-Xa inhibitors protect against systemic oxidant prolidase levels. damage induced by peripheral-ischemia reperfusion. In conclusion, thromboprophylaxis with antiaggregant J Thromb 2014;37:464e8. and antithrombotic agents is important for preventing [13] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal ischemic disorders. Furthermore, thrombotic and embolic tissues by thiobarbituric acid reaction. Anal Biochem 1979;95: e complications can occur even when thromboprophylactic 351 8. [14] Myara I, Charpentier C, Lemonnier A. Optimal conditions for treatment is administered. Nevertheless, thromboprophy- prolidase assay by proline colorimetric determination: appli- laxis with antiaggregant or anticoagulant agents seems to cation to iminodipeptiduria. Clin Chim Acta 1982;125: have protective efficacy against both renal and cardiac 193e205. oxidative damage in I/R conditions. [15] Eckerson HW, Romson J, Wyte C, La Du BN. The human serum The major limitation of this study was the animal model. paraoxonase polymorphism: identification of phenotypes by The limitation concerns the creation of isolated I/R without their response to salts. Am J Hum Genet 1983;35:214e27. thrombotic tendency. Thrombosis may lead to different [16] Keskin D, Kızıltunc¸ A. Time dependent changes in serum nitric results in different study groups with thrombotic tendency. oxide levels after long bone fracture. Tohoku J Exp Med 2007; 213:283e9. [17] Levine AB, Punihaole D, Levine TB. Characterization of the Funding role of nitric oxide and its clinical applications. Cardiology 2012;122:55e68. No financial support was received for the research and/or [18] Kimura A, Roseto J, Suh KY, Cohen AM, Bing RJ. Effect of authorship of this article. acetylsalicylic acid on nitric oxide production in infarcted heart in situ. Biochem Biophys Res Commun 1998;251:874e8. [19] de la Cruz JP, Guerrero A, Gonza´lez-Correa JA, Ma´rquez E, References Nacle I, Sa´nchez de la Cuesta F. Effects of two preparations of 75-mg extended-release on platelet aggregation, [1] O’Connell JB, Quin˜ones-Baldrich WJ. Proper evaluation and prostanoids and nitric oxide production in humans. Eur J Clin management of acute embolic versus thrombotic limb Pharmacol 2002;57:775e80. ischemia. Semin Vasc Surg 2009;22:10e6. [20] Heitzer T, Rudolph V, Schwedhelm E, Karstens M, Sydow K, [2] Millar JA, Lett JE, Bagley LJ, Densie IK. Eligibility for medical Ortak M, et al. Clopidogrel improves systemic endothelial ni- thromboprophylaxis based on risk-factor weights, and clinical tric oxide bioavailability in patients with coronary artery thrombotic event rates. Med J Aust 2012;196:457e61. disease: evidence for antioxidant and antiinflammatory ef- [3] Hignett M, Spence JEH, Claman P. Internal jugular vein fects. Arterioscler Thromb Vasc Biol 2006;26:1648e52. thrombosis: a late complication of ovarian hyperstimulation [21] Kanko M, Ozden M, Maral H, Acil C. Effect of clopidogrel on syndrome despite mini-dose prophylaxis. Hum Reprod nitric oxide levels in an ischemia reperfusion model. J Car- 1995;10:3121e3. diovasc Pharmacol 2006;48:797e801. [4] Yavuz C, Yazici S, Karahan O, Demirtas S, Caliskan A, Guclu O, [22] He YF, Wang YR, Li LF, Wang SH, Ye JW. Effect of low mo- et al. Serum nitric oxide level could be a predictive biomarker lecular heparin sodium cream on the content of serum nitric for detection of critical ischaemia duration. Biomarkers 2013; oxide in random pattern skin flap: experiment with rats. 18:116e20. Zhonghua Yi Xue Za Zhi 2007;87:3092e4 [Article in Chinese]. [5] Caliskan A, Yavuz C, Karahan O, Demirtas S, Yazici S, Guclu O, [23] Lakshmi RT, Priyanka T, Meenakshi J, Mathangi KR, et al. Serum ischaemia-modified albumin level is an irrelevant Jeyaraman V, Babu M. Low molecular weight heparin medi- predictive factor for ischaemic duration in mesenteric ated regulation of nitric oxide synthase during burn wound ischaemia. Perfusion 2014;29:226e30. healing. Ann Burns Fire Disasters 2011;24:24e9. [6] Carolei A, Sacco S, Marini C. Antiaggregant therapy and/or [24] Kowalczuk K, Stryjecka-Zimmer M. The influence of oxidative anticoagulant therapy in the cerebrovascular patient. Hae- stress on the level of malondialdehyde (MDA) in different matologica 2001;86:36e9. areas of the rabbit brain. Ann Univ Mariae Curie Sklodowska [7] Akyu¨rek S, Yildiz F, Cengiz M, Onal C, Yildiz O, Genc M, et al. Med 2002;57:160e4. Importance of timing of antiaggregant treatment in the pre- [25] Ulubas‚ B, Cimen MY, Apa DD, Saritas‚ E, Mus‚lu N, Cimen OB. vention of radiation induced enteropathy. Med Hypotheses The protective effects of acetylsalicylic acid on free radical 2005;65:736e9. production in cisplatin induced nephrotoxicity: an experi- [8] Hu H, Batteux F, Che´reau C, Kavian N, Marut W, Gobeaux C, mental rat model. Drug Chem Toxicol 2003;26:259e70. et al. Clopidogrel protects from cell apoptosis and oxidative [26] Taher MA, Nassir ES. Beneficial effects of clopidogrel on gly- damage in a mouse model of renal ischaemia-reperfusion cemic indices and oxidative stress in patients with type 2 injury. J Pathol 2011;225:265e75. diabetes. Saudi Pharm J 2011;19:107e13. [9] Dogan OT, Polat ZA, Karahan O, Epozturk K, Altun A, Akkurt I, [27] Deepa PR, Varalakshmi P. Salubrious effect of low molecular et al. Antiangiogenic activities of bemiparin sodium, enox- weight heparin on atherogenic diet-induced cardiac, hepatic aparin sodium, and . and renal lipid peroxidation and collapse of antioxidant de- Thromb Res 2011;128:e29e32. fences. Mol Cell Biochem 2003;254:111e6. 122 S. Demirtas et al.

[28] Jaichander P, Selvarajan K, Garelnabi M, Parthasarathy S. In- oxidative stress in an experimental model of spinal cord duction of paraoxonase 1 and apolipoprotein A-I gene injury. Acta Neurochir (Wien) 2002;144:1021e31. expression by aspirin. J Lipid Res 2008;49:2142e8. [32] Hilali N, Vural M, Camuzcuoglu H, Camuzcuoglu A, Aksoy N. [29] Cebeci E, Alibaz-Oner F, Usta M, Yurdakul S, Erguney M. Increased prolidase activity and oxidative stress in PCOS. Clin Evaluation of oxidative stress, the activities of paraoxonase Endocrinol (Oxf) 2013;79:105e10. and arylesterase in patients with subclinical hypothyroidism. [33] Miltyk W, Karna E, Pałka J. Inhibition of prolidase activity by J Investig Med 2012;60:23e8. non-steroid antiinflammatory drugs in cultured human skin [30] Tselepis AD, Tsoumani ME, Kalantzi KI, Dimitriou AA, Tellis CC, fibroblasts. Pol J Pharmacol 1996;48:609e13. Goudevenos IA. Influence of high-density lipoprotein and [34] Surazynski A, Pałka J, Wołczynski S. Acetylsalicylic acid- paraoxonase-1 on platelet reactivity in patients with acute dependent inhibition of collagen biosynthesis and beta1- coronary syndromes receiving clopidogrel therapy. J Thromb integrin signaling in cultured fibroblasts. Med Sci Monit 2004; Haemost 2011;9:2371e8. 10:BR175e9. [31] Topsakal C, Kilic N, Erol FS, Kaplan M, Akdemir I, Tiftikci M, [35] Evrenkaya TR, Atasoyu EM, Kara M, Unver S, Gultepe M. The et al. Medroxyprogesterone acetate, enoxaparin and pentox- role of prolidase activity in the diagnosis of uremic bone dis- yfylline cause alterations in lipid peroxidation, paraoxonase ease. Ren Fail 2006;28:271e4. (PON1) activities and homocysteine levels in the acute