GG Polymorphism of Platelet ITGA2B Gene Increases the Magnitude of Interleukin-6 Release after Cardiopulmonary Bypass

1Marmara University School of Medicine Department of Cardiovascular Surgery, Turkey 2Health Sciences Institute Department of Biochemistry, Turkey 3Taksim Education and Research Hospital Pathology Division, Turkey 4Istanbul Süreyyapasa Thoracic Disease and Thoracic Surgery Education and Research Hospital, Turkey 5Department of Medical Genetics, Istanbul Medical Faculty, Istanbul University, Turkey 6Cyprus International University, Lefkosa, Cyprus


Introduction
Cardiac surgery with cardiopulmonary bypass (CPB) is associated with a systemic infl ammatory response syndrome (SIRS) that is known to be one of the main causes of postoperative organ dysfunction (1). Several factors such as contact of blood with foreign surfaces, surgical trauma, ischemia reperfusion to the organs, and release of endotoxin have been widely documented to cause SIRS after CPB. Recently it has been shown that individual genetic polymorphisms in the genes encoding infl ammatory cytokines, apolipoprotein E4 and lipoprotein lipase and the like have a major impact on the magnitude of SIRS in patients undergoing CPB (2)(3)(4).
ITGA2B is a receptor for fi bronectin, fi brinogen, plasminogen, prothrombin, thrombospondin and vitronectin and functions in platelet activation (5). Current dogma suggest that several genetic polymorphisms of ITGA2B have been associated with a wide variety of clinical events including myocardial infarction (MI) at a young age, stroke and resistance to antiplatelet agents (6)(7)(8). Furthermore, it has been stated that different mutations of this gene results in a loss in the ability of aggregate and the production of an immune response (9). Recently, a growing body of evidence suggests that platelets have the pivotal role in the pathogenesis of several poor prognostic mediacal conditions like infl ammation and cancer (10). We hypothesized that different polymorphism of the ITGA2B gene may infl uence the magnitute of infl ammatory Abstract Objective: Cardiopulmonary bypass (CPB) induces a systemic infl ammatory response which is thought to be a signifi cant cause of postoperative organ dysfunction and mortality. In this study we aimed to investigate the effect of ITGA2B (integrin alpha 2b, platelet glycoprotein IIb of IIb/IIIa complex) gene polymorphism on the magnitude of infl ammatory response after CPB Methods: Twenty patients undergoing coronary artery bypass grafting were included. Blood samples were taken at the three different times for analyses of Interleukin-6, Interleukin-10 and Nuclear Factor Kappa B by ELISA (t 1 : before operation, t 2 :10 minutes after removal of aortic cross clamping and t 3 : 24 hours after operation. ITGA2B gene polymorphisim, c.2621T>G, resulting missense alteration, p.Ile874Ser (rs5911) was studied in patients and 27 healthy volunteers by targeted polymerase chain reaction (PCR) and restriction enzyme digestion. Perioperative organ dysfunction was evaluated by cardiac surgery scorring (CASUS) system.

Results:
There was no perioperative mortality. The mean ages of the patients and the controls were 67.45±12.30 and 51,38±7,03 years, respectively (p=0.001). Thirty-fi ve percent (n=7) of the patients revealed TT, 45% (n=9) TG and 20% (n=4) GG polymorphism. The allele frequencies of the study group were similar to the controls (33,3%, n=9 revealed TT, 55,5 %, n=15 TG and 11,3%, n=3 GG). There was no signifi cant difference in the frequency of genetic polymorphism between the patients and the controls. In the study group, patients with GG allele had signifi cantly higher interleukin 6 levels 24 hours after operation than the others (GG (338,14±22.20pg/ml) versus TT (306,14±22, 10 pg/ml), p=0.025, and GG versus TG (308, 12±14,50pg/ml), p=0.039). response after coronary artery bypass grafting (CABG) since the critical role of ITGA2B receptor in infl ammation". We investigated the relationship between the ITGA2B polymorphism and the magnitude of perioperative infl ammatory cytokines and organ dysfunction in patients undergoing CABG with CPB.

Material and Methods
After obtaining approval to conduct the study from the local ethics committee and written informed consent from each patient, we enrolled 20 consecutive patients undergoing elective fi rst time coronary artery bypass grafting (CABG) procedure into the study. Exclusion criteria were previous CABG history, acute coronary syndrome, known infection, steroid or nonsteroidal anti-infl ammatory therapy within the last three months, an autoimmune condition, a known pathology of platelets, bone marrow disease, preoperative intra-aortic balloon pump insertion, emergency revascularization, chronic renal disease requiring dialysis, and hepatic failure. Twentyseven healthy control individuals (aged between 35 and 60 years) were used to investigate genotype distribution in comparison with patients. According to the results of medical history, physical examination, and laboratory data, they were judged healthy.
Medical history, demographic data, and the postoperative course for each patient were collected prospectively. Preoperative evaluation included routine blood biochemistry, complete blood count, pulmonary function test, transthoracic echocardiography, and coronary angiography. In the postoperative period, patients were followed up and treated according to the same institutional protocol.
Postoperative daily assessment of organ function in the intensive care unit (ICU) was performed by the cardiac surgery scoring (CASUS) system (11). In brief, CASUS contains 10 variables that were graded according to the severity for daily risk stratifi cation in the ICU. Operative day CASUS score was calculated when the patient was admitted to the ICU. Then, subsequent calculations were performed in the morning of each ICU day. Perioperative myocardial infarction was defi ned by new electrocardiographic changes and an increase of 3.5fold in creatine kinase (CK) isoenzyme MB level. Need for an inotropic agent was judged by systolic blood pressure lower than 90 mm Hg or more than 40 mm Hg below usual systolic pressure under appropriate fl uid management. Postoperative acute renal dysfunction was defi ned as a postoperative serum creatinine level greater than 200 μmol/L or need for dialysis therapy or hemofi ltration before hospital discharge. The surgical team and the clinicians who were responsible for the postoperative care of the patient were blinded to the study.

Anesthesia and operative technique
Midazolam was used for premedication, and the anesthetic agent consisted of a combination of fentanyl, midazolam, and pancuronium. Anesthesia was maintained with midazolam and vecuronium infusion and with inhaled sevofl urane. Median sternotomy was carried out in all patients. Standard aortocaval cannulation was done to establish CPB. CPB was performed with a roller pump system (Jostra AG, Hirrlingen, Germany) and a hollow-fi ber membrane oxygenator. Also, a 40-μm arterial blood fi lter was used in all patients. Mild-to-moderate Aprotinin and steroids were not used. All patients were followed up in a standard fashion during postoperative period.  Fragments were separated by electrophoresis on a 2.75 % horizontal agarose gels.

Statistical analysis
Statistical analysis was performed with the SPSS for Windows 16.0 version (SPSS, Inc, Chicago, Ill). The  2 test was used to analyze relationships between categorical data. Nonparametric Mann-Whitney U test was used to compare IL-6, IL-10 and NFB levels and different outcomes among groups. Nonparametric testing was chosen because the study population was relatively small in size and the data did not follow a normal distribution. Association between two continuous variables was determined by Spearman rank correlation. Continuous variables were given as mean ± standard error of the mean. A  2 test was used to compare the observed numbers of each genotype with those expected for a population to establish whether they were in the Hardy-Weinberg equilibrium. Regression analyses were used to adjust for confounding factors. All tests were 2-sided. All measurements between and within the groups were checked for different time points by repeated-measures analysis of variance with Bonferroni corrections for multiple testing.

Results
The resuluts of genetic analyses related to the study group and the healthy controls were summarized in Table 1. Of the 20 patients enrolled in the study, 9 patients (45%) revealed polymorphic heterozygote pattern (T/G) of the ITGA2B gene which was found to be statistically nonsignifi cant to the control population (55.5%). The frequencies of the normal (G/G) and polymorphic homozygote (T/T) patterns did not differ between the study and the control group (for GG; 20% (n=4) versus 11,3% (n=3) and for TT, 35% (n=7) versus 33,3% (n=9), respectively). The mean age was 67.45±12.30 years in the study group and 51,38±7,03 years in the controls (p=0.001). The 70% of the patients (n=14) and 62.9% of the controls (n=17) were male (p=0.614). Comparison of the demographics and the perioperative profi le of the patients were given in the Table  2. All patients had signifi cant triple vessel CAD and revealed normal renal function preoperatively. Aortic cross clamp time, CPB time and the number of performed distal anastomosis did not differ amongh the diffrent polymorphisms. Moreover,   ionotrophic drug requirement, 24 hour blood loss, ICU and hospital stay did not create a difference.
There was no perioperative mortality in both groups. Postoperative daily assessment of organ dysfunction (CASUS scorring) in ICU revealed no difference in postoperative day 0 (operative day) and day 1 CASUS scores among three pomymorhisms (Table 3).
Among three alleles, we could not detect in any statistically signifi cant differences in the levels of IL-10 and NFB at three time points. However, IL-6 levels at t 3 were signifi cantly higher in patients with G/G polymorphism compared to those with T/G and T/T (G/G (338,14±22.20 pg/ml) versus T/T (306,14±22,10 pg/ml), p=0.025, and G/G versus T/G (308,12±14,50 pg/ml), p=0.039).

Discussion
In the present study, our results showed a similar frequency of the rs5911 SNP of the ITGA2B gene located on chromosome 17 in patients undergoing CABG compared to the healthy subjects. The study group was older than the healthy controls.      This binding triggers the tissue factor pathway and leads to leukocyte actication and production of infl ammatory mediators (23). Moreover r ecent evidence suggests that activated platelets also contribute to chronic infl ammatory milieu in the process of atherosclerosis (24). In our study, the magnitude of IL-6 level In conclusion, our results show that GG polymorphism of platelet ITGA2B gene attenuates the severity of SIRS by decreasing the levels of IL-6 24 hours after operation.
Preoperative genotyping for platelet ITGA2B gene might be benefi cial in detecting patients with a genetically determined risk for an exaggerated cytokine response.