Effect of Pretreatment with Omega-3 Polyunsaturated Fatty Acids (pufas) on Hematological Parameters and Platelets Aggregation in Patients during Elective Coronary Artery Bypass Grafting

Efekat omega-3 polinezasiüenih masnih kiselina na hematološke parametre i agregaciju trombocita kod elektivne revaskularizacije srca Abstract Bacground/Aim. Using omega-3 polyunsaturated fatty acids (PUFAs) in coronary artery bypass graft surgery (CABG) could provide protection against ischemic-reperfusion damage, prevention of postoperative arrhyth-mia and attenuation of inflammatory response. However, omega-3 PUFAs inhibit cyclooxygenase (and thus decrease the synthesis of thromboxane A2 from arachidonic acid in platelets), which leads to decreased platelet aggregation. In cardiac surgery it is necessary to achieve a balance between inhibition and full platelets function. It is as well as important to closely follow hematological parameters, impaired by CABG itself. Therefore, the aim of the study was to establish the effects of pretreatment with omega-PUFAs on hematological parameters and plateletes aggre-gation in patients with elective CABG. Methods. This prospective, randomized, placebo-controlled, single-center trial was performed on parallel groups. The patients (n = 40) undergoing elective CABG were randomized receiving preoperative intravenous omega-3 PUFAs (Omegaven ® 10%) infusion (the PUFAs group) or the same volume of 0.9% saline solution infusion (the control group). Infusion was given a day before surgery and repeated four hours before starting extracorporeal circulation (CPB) via the peripheral vein at single doses of 100 mL (25 mL/h). Platelet function analysis was performed using multiple electrode aggregometry (MEA, multiplate-analyzer) before starting CPB and 2 h postoperatively for the patients of both groups. Results. There were no clinically relevant differences in baseline characteristics between the groups. He-matological parameters were not significantly different between the groups pre-, intra-and postoperatively. During the first 24 h after surgery, the loss of blood was similar in the PUFAs and the control group (680 ± 274 mL and 608 ± 210 mL, respectively; p = 0.356). Postopera-tively, platelet aggregation was not significantly different between the PUFAs and the control group in adenosine diphosphate (ADP) test (39 ± 11 and 42 ± 15, respectively ; p = 0.701), arachidonic acid (ASPI) test (64 ± 24 and 70 ± 27, respectively; p = 0.525) and trombin receptor activating peptide (TRAP) test (68 ± 25 and 75 ± 26, respectively; p = 0.396), while their aggregation in collagen (COL) test was statistically significantly lower in the PU-FAs related to the control group (32 ± 15 and 47 ± 20, respectively ; p = 0.009). Conclusion. Acute pretreatment with omega-3 PUFAs insignificantly affected the activity of platelets and did not influence postoperative blood loss.ne gubitke krvi.

function analysis was performed using multiple electrode aggregometry (MEA, multiplate-analyzer) before starting CPB and 2 h postoperatively for the patients of both groups.Results.There were no clinically relevant differences in baseline characteristics between the groups.Hematological parameters were not significantly different between the groups pre-, intra-and postoperatively.During the first 24 h after surgery, the loss of blood was similar in the PUFAs and the control group (680 ± 274 mL and 608 ± 210 mL, respectively; p = 0.356).Postoperatively, platelet aggregation was not significantly different between the PUFAs and the control group in adenosine diphosphate (ADP) test (39 ± 11 and 42 ± 15, respectively; p = 0.701), arachidonic acid (ASPI) test (64 ± 24 and 70 ± 27, respectively; p = 0.525) and trombin receptor-activating peptide (TRAP) test (68 ± 25 and 75 ± 26, respectively; p = 0.396), while their aggregation in collagen (COL) test was statistically significantly lower in the PU-FAs related to the control group (32 ± 15 and 47 ± 20, respectively; p = 0.009).Conclusion.Acute pretreatment with omega-3 PUFAs insignificantly affected the activity of platelets and did not influence postoperative blood loss.

Introduction
Bleeding is a common complication of cardiac surgery with cardiopulmonary bypass (CPB), which can require transfusion of blood products [1][2][3] and in 3% ± 6% of cases mediastinal re-exploration 4 .Among causes of excessive bleeding, platelet dysfunction is considered to be the most important in the early postoperative period.During coronary artery bypass graft (CABG) surgery there is the opposition between the benefit of platelet inhibition to reduce the risk of pre-operative infarction and postoperative occlusion of anastomosed coronary arteries, and the need to maintain full platelet function for optimal hemostasis in surgical incisions.
Previous studies have demonstrated beneficial effects of omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in platelet functions 5 .Whereas the predominant product of arachidonic acid (AA) in platelets, thromboxane A 2 (TXA 2 ) is a platelet agonist, the corresponding product of EPA, thromboxane A 3 (TXA 3 ), is virtually biologically inert.In contrast, both prostaglandin I 2 derived from AA, and prostaglandin I 3 derived from EPA, are potent vasodilators and platelet inhibitors 6 .Supplementation of omega-3 PUFAs resulted in a shift towards the production of more favorable eicosanoids that inhibit platelet aggregability 7 .
Omega-3 PUFAs are commonly considered to have antithrombotic effects, based on increased bleeding times at very high doses (e.g. 15 g/day) 8 .Conversely, in human trials, omega-3 PUFA consumption has no consistent effects on platelet aggregation or coagulation factors 9,10 .No excess clinical bleeding risk has been seen in randomized clinical trials of fish or fish oil consumption, including people undergoing surgery or percutaneous intervention and/or also taking aspirin or warfarin 8,11 .
We have recently shown that preoperative administration of omega-3 PUFAs has cardioprotective effect in pa-tients with CPB manifested in increased oxygen extraction and lactate uptake with simultaneous decrease of serum troponin I and creatinin kinase myocardial band (MB) levels 12 .In the frame of the same study, we followed the effect of omega-3 PUFAs on their influence on postsurgical patients platelet aggregation.According to the previous findings of the other authors 11,13 that omega-3 PUFAs do not affect bleeding in the same or similar categories of patients we assumed that they will not provoke bleeding surpassing the risk expected in the control group of patients.
The aim of the study was to establish the relationship between hematological parameters and the activity of plateletes in patients with elective CABG pretreated by 3-omega PUFAs.

Methods
This prospective, randomized, placebo-controlled study was performed on parallel groups.Study enrollment occurred between August 2010 and September 2011.The study protocol was approved by the Ethical Committee of Military Medical Academy, and all the patients gave written informed consent.
Forty patients scheduled to undergo their first on-pump CABG surgery were included in the study.For recruitment, patients needed to be older than 18 years of age, in normal sinus rhythm, and in stable hemodynamic conditions before surgery.Patients were excluded in cases of emergency CABG, redo CABG, combined CABG and any other cardiac procedure, Q-wave myocardial infarction in the last six weeks, unstable angina, or poor left ventricular function, those with abnormal coagulation tests, including a history of coagulopathy and preoperative treatment with other anticoagulants.All the participants denied the intake of any antiagregation medication during the previous five days.All the patients were treated by the same surgical and anesthesiologist team.Eligible patients were assigned to one of the two study arms according to a computer-generated randomization list: the control (placebo) group (usual care), and the usual care plus PUFAs group.
The PUFAs infusion consisted of 100 mL of a lipid emulsion with a high content of omega-3 PUFAs (Omegaven ® 10%, Fresenius Kabi, Bad Homburg, Germany).The same batch of Omegaven ® was used throughout the study, and 100 mL of the lipid emulsion contained 1.25-2.82g of EPA and 1.44-3.09 of DHA.Infusion was given a day before surgery and repeated 4 h before starting CPB via the peripheral vein at single doses of 100 mL (25 mL/h).The patients in the control group received an equal volume of 0.9% saline.
Preoperative sedation with 5 mg of intramuscular midazolam was administered to patients on call to the operating room.All the patients received prophylactic preoperative antibiotics (cefazolin 2 g preincision, and 2 g post-CPB; or if allergic to penicillin, vancomycin 1 g preincision and 500 mg post-CPB).The same anesthesiologist administered standardized total intravenous anesthesia using sufentanil, midazolam, propofol and pancuronium.
Immediately before CPB, 300 IU/kg heparin was administered intravenously, followed by additional doses as necessary to maintain an activating clotting time exceeding 500 sec.Protamine was administered as 1 mg /100 IU of the heparin dose after complete separation from CPB.
All the patients had CABG with the use of CPB, which was conducted with a roller pump and a membrane oxygenator primed with a solution.During CPB, pump flow was set at 2.4 times the body surface area, and mean arterial pressure maintained between 50 mmHg and 60 mmHg.The temperature was allowed to drift with active rewarming at the end of CPB.Myocardial protection was afforded with cold potassium cardioplegia.A single-clamp technique was used, and cardioplegia was given in an anterograde fashion.In all the patients, the left internal mammary artery harvested and anastomosed to the left anterior descending artery.The rest of the grafts were constructed using the great saphenous vein.
After a total release of the aortic cross-clamp, epicardial atrial or ventricular pacing wires were placed.Aortic and venous cannulas were removed after an appropriate test dose of protamine, and the surgery proceeded with closure of the pericardium and sternum.
After the surgery, the patients were followed up in the Intensive Care Unit (ICU) and were weaned off mechanical ventilation when they fulfilled the following criteria: hemodynamic stability, peripheral temperature of more than 36 °C, cooperatively, and no major bleeding.
Blood for hemoglobin (Hb) concentration, hematocrit (Hct), platelet count and coagulation profile determination including international normalised ratio (INR) and activated partial thromboplastin time (aPTT), was taken from a radial arterial catheter before start CPB and 2 h after arrival in the ICU for all the patients in the two groups.Transfusion of blood products and management of postoperative bleeding intra-and postoperatively were determined by following institutional algorithm.Platelet function analysis was performed using the multiple electrode aggregometry (multiplate-analyzer) before started CPB and 2 h after arrival in the ICU for all the patients of both groups.The method has been described in detail elsewhere 14 .Platelet aggregation was initiated using arachidonic acid (ASPI test), adenosine diphosphate (ADP test), thrombin receptor-activating peptide (TRAP test) and collagen (COL test) using commercially available test reagents.Increased impedance caused by attachment of platelets to the test cell electrodes was continuously measured over 6 min.Platelet aggregation was quantified as the area under the aggregation curve [AUC(U)].Reference ranges for healthy subjects obtained from the manufacturer were 79-141 U for the ASPI test, 41-99 U for the ADP test, 92-151 U for the TRAP test, and 61-108 U for COL test.
The results were presented as mean values with standard deviation.The significance of differences betwen the study groups was analyzed using the t-test.Due to great variability of some data, the Wilcoxon matched pairs test and the Mann-Whitney U-test were also used.Comparison between more than two groups was done by using the Kruskal-Wallis test.
A p-value less than 0.05 was taken to be significant.The obtained data were processed through the Stat for Windows, R.4.5.Software package.

Results
The results of the study are presented in Tables 1 to 3 dealing with baseline and operative characteristics of the patients (Table 1), the effect of CPB procedure on hematologi- cal parameters (Table 2), the effect of CPB procedure on the activity of platelets in the control and the PUFAs group of patients (Table 3), and peri-and postoprative complications.
Table 1 shows that the baseline and operative characteristics of the patients included in the study did not differ between the control and the PUFAs group in any of the observed parameters.This equally relates intraoperative CPB (101.4 min vs 95.5 min; p = 0.29), aortic cross-clamp time (42.5 min vs 38.9 min; p = 0.66), CABG number (2.9 min vs 2.8; p = 0.65) and total heparin use (27.7 min vs 27.6 units 1,000; p = 0.86), and postoperative interventions: total protamine dose (279 mg vs 282 mg; p = 0.63).
Table 2 shows that hematological data regarding preoperative and on the arrival to ICU, as well as intraoperative and postoperative requirements for allogenic RBCs, FFP and platelet units were similar in both groups of patients, with no statistically significant difference.
Table 3 shows that the level of platelet aggregation reached the reference values in both groups of patients and in all four performed tests, indicating thus their normal values.In all instances, the observed values were above, but closer  to the lower levels of aggregators reference potencies given in the brackets.
The second part of the results concerns the intergroup differences in the platelet aggregation pre-, and postoperatively.In that respect, almost all the tests showed equal activity of plateletes before the surgical intervention in the PU-FAs group in relation to the control group of patients.Postoperatively, platelet aggregation was not significantly different between the PUFAs and the control group in the ADP test (39 ± 11 vs 42 ± 15; p = 0.701), ASPI test (64 ± 24 vs 70 ± 27; p = 0.525) and TRAP test (68 ± 25 vs 75 ± 26; p = 0.396), while their aggregation in COL test was statistically significantly lower in the PUFAs related to the control group (32 ± 15 vs 47 ± 20; p = 0.009).
Postoperative complications were similar in both groups of patients.In the control group, one patient died of cardiac failure on the second postoperative day, two patients had perioperative infarction, three patients needed inotropic support.In the PUFAs group, one patient underwent reexploration for bleeding, one had a respiratory failure and two patients needed inotropic support.Due to the low number of the observed complications, no statistical comparison was performed.

Discussion
We studied the relatoinship between omega-3 PUFAs, which may influence the activity of platelets, and the hematological parameters liable to imapirment in patients with CPB, and found that in spite of the marked decrease in the postoperative activity of platelets, more pronounced in the PUFAs group of patients in relation to placebo in the COL test, they did not affect any of the observed intra-and postoperative hematological parameters (blood loss, RBC, FFP and platelet requirements).
Due to the separate study types, discussion is given in two parts: the effect of CPB on hematological parameters, and the influence of omega-3 PUFAs in CPB procedure on the activity of platelets.

The effect of CPB on hematological parameters
Statistically significant differences were postoperatively found in both groups of the studied patients in some hematological parameters, like Hb and Htc levels and platelet counts, but not in the others as INR and aPTT.This is very important, since bleeding after cardiac surgery may ensue either from surgical (anastomoses, sternum, cannulation sites) or nonsurgical sites.If bleeding becomes excessive or causes hemodynamic disorders, reexploraton of mediastinal wound is necessary.In our study, one patient in the PUFAs group underwent reexploration for bleeding.Studies from the other authors have shown that reexploration can be associated with multiple negative outcomes such as renal failure, sepsis, atrial fibrilation, prolonged mechanical ventilation and hospital stay and, most notably, increased mortality and costs [1][2][3][4] .
There have also been studies in which patients with coronary bypass grafting [15][16][17] , endarterectomy 18,19 , and femoral artery catheterization [20][21][22] were given omega-3 PU-FAs.In these studies, identically with our results, the risk of clinically significant bleeding was virtually nonexistant.However, in this respect, one has to keep in mind the review of Bays 23 in which he concluded that although there is little evidence for increased risk of clinically significant bleeding with omega-3 PUFAs supplementation, clinicians should be aware of this as a theoretical possibility.

The influence of omega-3 PUFAs in CPB procedure on the activity of platelets
The results of our study show that preoperative activity of platelets in both groups of patients were in the range of reference values for all of the four used tests.Conversely, after the surgical intervention, their activity was statistically significantly reduced, with easily noticable lower values in PUFAs group in relation to placebo, particularly in COL test.
This finding is very relevant, because platelets play an important role in maintaining normal hemostatic function.Their disfunction is a major cause of excessive bleeding in the early postoperative period after CPB procedures 24,25 , not found in our patients.Transient impairment of platelet function is mediated by platelet activation during passage through the synthetic, nonendothelial surface of the extracorporeal circuit, used also in our patients with twofold decrease in their count, and involves the secondary release and partial depletion of -granules.Platelet dysfunction may also be related to other factors 24 .Hypothermia related CPB influences platelet function and coagulation 26 , the effect which can persist into the ICU 27 , and be more pronounced as the time on CPB increases 28,29 .
Multiple electrode platelet aggregometry (MEA) used in our study, allows the assessment of platelet function without centrifugation steps 30 , and has proven sensitive for platelet inhibition induced by aspirin and clopidogrel, as well as for the effects of CPB and of hypothermia on platelet aggregation [31][32][33][34] .The test has also been found to be able to detect impaired hemostasis after CPB surgery [35][36][37] and to identify patients before and after cardiac surgery with enhanced risk of bleeding and of blood transfusion 38,39 .
The mechanism of favorable antithrombotic effects of omega-3 PUFAs found in our study are complex.It has been shown that alteration of fatty acid composition by omega-3 PUFAs incorporation into platelet membranes can alter not only membrane permeability, but also modulate function and activity of membrane receptors and transporters 40,41 .The COL test reagent, being the most sensitive in our study, contains collagen, which activates the platelets by the collagen receptor.Following its binding to the receptors, AA is released, which is the substrate of platelet enzyme cyclooxygenase (COX).COX transforms AA into TXA 2 , a potent platelet activator.With a blockade of COX, the formation of TXA 2 is inhibited and therefore inhibited platelet activation is usually detected, as happened to be in our patients.
In most studies either no effect on platelet aggregation was found with omega-3 fatty acids or no difference in effect was seen between the treatments and the control.Kwon et al. 42 noted that with 2 mg of collagen, a significant decrease in Strana 401 platelet aggregation was found at three weeks on canola oil diet, which reverted to baseline by eight weeks.Freese et al. 43 reported that the decrease in collagen-induced aggregation in the fish oil supplement arm did not return to baseline during a 12 week follow-up period.
Overall, although there is heterogeneity among the studies, there is a trend toward a net reduction of coronary artery restenosis with fish oil supplementation, estimated by the meta-analysis to lower such a risk for 14% 21,44 .The optimal degree of platelet inhibition is unclear and must be confirmed in trials evaluating cardiovascular outcomes and sould be balanced with the excessive risk of bleeding 45 .In any case, the results of our study show that the postoperative inhibition of platelet aggregation by PUFAs, particularly pronounced in the COL test, did not affect the intra-and postoperative hematological parameters, with the risk of bleeding, as the most dangerous, being equal to the placebo group.This finding undoubtedly speaks in favour of omega-3 PUFAs use as cardioprotectors in patients with open heart surgery, found in our recent study 12 .

Conclusion
The results of our study show that acute omega-3 PUFAs pretreatment of patients subjected to CBP grafting did not affect INR, aPTT and bleeding volume, while the postoperative platelet count dropped twofold, equally in the PUFAs and placebo treated groups.The activity of platelets was statistically significantly lower after surgical intervention in both groups of patients, particularly markedly pronounced in the COL test in the PUFAs group, but with no negative effect on bleeding.

Table 2 Hematological data in the patients subjected to CPB
Data presented as mean value ± standard deviation.CPB -cardiopulmonary bypass; PUFA -polyunsaturated fatty acids; INR-international normalization ratio; aPTT -activated partial thromboplasin time; ICU -intensive care unit; RBC -red blood cells; FFP -fresh frozen plasma.