Polymorphisms on PAI-1 and ACE genes in association with fibrinolytic bleeding after on-pump cardiac surgery

Background Carriers of plasminogen activator inhibitor -1 (PAI-1) -675 genotype 5G/5G may be associated with lower preoperative PAI-1 plasma levels and higher blood loss after heart surgery using cardiopulmonary bypass (CPB). We speculate if polymorphisms of PAI-1 -844 A/G and angiotensin converting enzyme (ACE) intron 16 I/D also might promote fibrinolysis and increase postoperative bleeding. Methods We assessed PAI-1 -844 A/G, and ACE intron 16 I/D polymorphisms by polymerase chain reaction technique and direct sequencing of genomic DNA from 83 open heart surgery patients that we have presented earlier. As primary outcome, accumulated chest tube drainage (CTD) at 4 and 24 h were analyzed for association with genetic polymorphisms. As secondary outcome, differences in plasma levels of PAI-1, t-PA/PAI-1 complex and D-dimer were determined for each polymorphism. SPSS® was used for statistical evaluation. Results The lowest preoperative PAI-1 plasma levels were associated with PAI-1 -844 genotype G/G, and higher CTD, as compared with genotype A/A at 4 and 24 h after surgery. Correspondingly, 4 h after the surgery CTD was higher in carriers of ACE intron 16 genotype I/I, as compared with genotype D/D. PAI-1 plasma levels and t-PA/PAI-1 complex reached nadir in carriers of ACE intron 16 genotype I/I, in whom we also noticed the highest D-dimer levels immediately after surgery. Notably, carriers of PAI-1 -844 genotype G/G displayed higher D-dimer levels at 24 h after surgery as compared with those of genotype A/G. Conclusions Increased postoperative blood loss secondary to enhanced fibrinolysis was associated with carriers of PAI-1 -844 G/G and ACE Intron 16 I/I, suggesting that these genotypes might predict increased postoperative blood loss after cardiac surgery using CPB. Electronic supplementary material The online version of this article (doi:10.1186/s12871-015-0101-1) contains supplementary material, which is available to authorized users.

clinical characteristics of the patients are the same as presented in two previous studies where associations between fibrinolytic markers and blood loss were compared between bleeders and non-bleeders [30] as well as the influence of PAI-1 -675 (4G/5G) polymorphism to fibrinolytic activity was investigated [18].
Inclusion criteria: >18 years of age, first-time coronary artery bypass grafting (CABG) and/or valve replacement under CPB, EuroSCORE II [31] < 10%. To identify preoperatively the patients at similar risks of bleeding, those enrolled had baseline coagulation tests within normal ranges, including prothrombin time (PT) 70-120% or international normalized ratio (INR) 0.8-1.2 and fibrinogen plasma concentration 1.5 -3.5 g/L. Moreover, those included had platelet count (PLT) 150 -400 x 10 9 /L, hemoglobin (Hb) concentration > 135 g/L for men and > 120 g/L for women and no anticoagulant, -anti-aggregating or non-steroidal anti-inflammatory drugs for, at least, five days prior to surgery to exclude medicine-induced platelet dysfunction. The last dose of lowmolecular-weight heparin (LMWH) was administered the evening before the surgery.
Intraoperative factors such as duration of CPB, volumes of administered priming -and cardioplegia solutions, as well as bleeding tendencies where equally expressed in all polymorphism groups.
Immediately after the surgery, kaolin and heparinase-activated thrombo-elastography was performed to exclude residual heparin effects or coagulation abnormalities. Cell-saver blood was discarded for included patients.
Exclusion criteria: emergency -and redo operations, surgical bleeding approved during reexploration, preoperative hemostatic disorders with a history of hemorrhagic events or coagulopathy, severe renal and/or hepatic dysfunctions, and patients reluctant to allow their blood to be genetically analyzed.

Perioperative management
Anesthesia was induced with fentanyl (Fentanyl-Kalceks ® 0.05mg/ml, A/S Kalceks, Latvia) 0. Cryoprecipitate was transfused at a dose of 1 unit per 10 kg immediately after weaning off CPB if the baseline fibrinogen level was less than 2.5 g/L [1].

Demographic and laboratory data
The following variables were registered: age, gender, body mass index (BMI), ejection fraction (EF), type of surgery, time on CPB, aorta clamp -and reperfusion times (min), hemoglobin concentration (Hb, g/L) platelet count (PLT, x 10 9 /L) and fibrinogen (g/L). Fibrinogen plasma concentration was determined according to Clauss [32]. Prothrombin (PT) was analyzed with a complex assay (Lyophilized Dade ® and Innovin ® , Siemens Healthcare Diagnostics, USA). All the coagulation tests were determined using Sysmex ® CA-1500 (Siemens Healthcare Diagnostics, Germany). Hb and PLT were analyzed by means of a Beckman Coulter LH 750 Hematology Analyzer.
Genomic DNA was extracted from whole blood by using a standard fenol-chlorophorm extraction technique. DNA was then diluted in 1 ml of water and stored for future analysis. The region harboring the PAI-1 -844 A/G gene polymorphism was amplified using polymerase chain reaction (PCR). The primers had the following sequences: 5'-ATCCCTTTTCCCCTTGTGTC-3' and 5'-AACCTCCATCAAAACGTGGA-3'. The PCR products were then purified using Sap/Exo I (Thermo Scientific ® Fermentas, Lithuania) and sequenced on an ABI Prizm 3130xl genetic analyzer (Applied Biosystems ® , Life Technologies, USA).
For determination of ACE Intron16 I/D polymorphism the method described by Tomita et al [33] was used. Insertion and deletion alleles were identified by using PCR amplification of the respective fragments from Intron 16. Fragment size was determined by agarose gel electrophoresis.
The deletion allele was visualized at 191 base pairs (bp), and an insertion allele at 478 bp. For patients with /D genotype additional PCR was performed to verify the result of amplification due to the possible preferential amplification of deletion allele.

Postoperative bleeding
The postoperative bleeding volumes were recorded as chest tube drainage (CTD) in mL 4 h and 24 h after the surgery. The CTD system was connected to an aspiration pump providing a suction of 15 cm H2O. Indication for reoperation because of suspected surgical bleeding was based on a rate of blood loss > 100 mL/h or > 2 mL/kg in the first 4 h after surgery in combination with clinical 4 and hemodynamic changes. A surgical bleeding was ultimately diagnosed at the time of reexploration. Patients with diagnosed surgical bleeding during reoperation were excluded from further study.

Statistical analysis
Data were analyzed with SPSS (SPSS ® version 20.0, Chicago, IL). Continuous variables were presented as mean ± standard deviation (SD) and categorical variables as percentages (%). The data of the study groups were checked by an appropriate analytic test according to the data distribution. Comparisons between genotype groups were performed with Kruskal-Wallis H test for non-parametric variables, and with ANOVA for parametric variables. Chi-square test was used to analyze categorical data. Statistical significance was defined as P< 0.05.