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Ulinastatin reduces postoperative bleeding and red blood cell transfusion in patients undergoing cardiac surgery

Supplemental Digital Content is available in the text


Introduction
The result of the blood conservation using antifibrinolytics in a randomized trial led to the suspension of aprotinin use in cardiac surgery by Food and Drug Administration in the USA in 2007 over concerns of increased mortality. [1] Subsequently, aprotinin was withdrawn from the Chinese market in December 2007. [2] Ulinastatin or urinary trypsin inhibitor, is a type of glycoprotein and a nonspecific wide-spectrum protease inhibitor. [3,4] Currently, Ulinastatin is used in China, Korean, Japan, and India. A large body of convincing evidence has indicated that, Ulinastatin can not only reduce the release of pro-inflammatory cytokines, but also provide vital organ protection in patients undergoing cardiac surgery for coronary artery diseases, heart valve diseases, congenital heart diseases. [5][6][7] A previous study found that Ulinastatin normalized coagulation function and prevented changes in thromboelastography (TEG) during liver surgery. [8] Another study by Ji et al demonstrated that, Ulinastatin shortened activated partial thromboplastin time (APTT) and activated coagulation time (ACT) after systemic heparinization in patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). [9] Whether Ulinastatin has similar beneficial effects on blood conservation in cardiac surgical patients as aprotinin remains undetermined. [5][6][7] Therefore, we performed this meta-analysis to evaluate the effects of Ulinastatin on bleeding and transfusion in patients undergoing cardiac surgery.

Ethical approval
This study was a meta-analysis of previously published literatures, ethical approval was not necessary under the ethical committee of Fuwai Hospital.

Search strategy
We conducted a systemic review according to the preferred reporting items for systemic reviews and meta-analysis quality of reporting of meta-analysis Guidelines (Supplement Table 1, http://links.lww.com/MD/D776). [10] The protocol of current meta-analysis was published in PROSPERO with the registration number of CRD42018115698. Relevant trials were identified by computerized searches of MEDLINE, Cochrane Library and EMBASE till January 6th, 2019, using different combination of search words as follows: (cardiopulmonary bypass OR heart OR cardiac surgery OR coronary artery bypass surgery) AND (Ulinastatin OR urinary trypsin inhibitor OR Miraclid OR Ulinase OR Bikunin OR Urinastatin) AND (bleeding OR blood loss OR transfusion) AND (randomized controlled trial OR controlled clinical trial OR randomized OR placebo OR randomly OR trial) (Appendix, http://links.lww.com/MD/ D771). No language restriction was used. We also searched the Chinese BioMedical Literature & Retrieval System (from 1978 to January 6th, 2019). Additionally, we used the bibliography of retrieved articles to further identify relevant studies.

Inclusion and exclusion criteria
We included all clinical trials comparing Ulinastatin with placebo or blank with respect to bleeding and transfusion in patients undergoing cardiac surgery. In studies which also included other comparator drugs, only data of Ulinastatin and placebo/ blank groups were abstracted. Primary outcomes of interest included intra-and postoperative blood loss, blood transfusion (red blood cells, plasma, platelet concentrates, cryoprecipitate), postoperative re-exploration for bleeding. Secondary outcomes of interest include perioperative hemoglobin level, platelet counts and functions, coagulation tests, inflammatory cytokines level, and so on.
The 2 authors (NXF and DHL) independently reviewed the titles and abstracts of all identified studies for eligibility, excluding obviously ineligible ones. The eligibility of those remaining studies for final inclusion was further determined by reading the full text.

Study quality assessment
Two authors (NXF and DHL) independently assessed the risk of bias, using the tool described in the Cochrane Handbook for Systematic Reviews of Interventions. [11] The Jadad score was used independently by 2 authors (NXF and DHL) to evaluate the methodological quality of each included trial. The Jadad scoring system (ranging from 0 to 5) includes: (1) randomization process; (2) blindedness assessment; and (3) reporting of withdrawals/dropouts.
Higher scores indicate excellent methodologic qualities, and lower scores suggest poor qualities. [12] 2.5. Data abstraction The following data were abstracted from the included studies to a data collection form by 2 authors (YTY and NXF) independently: (1) author, year of publication, and journal of included studies; (2) total number of patients, number of patients in Ulinastatin and control groups, gender, age; (3) type of surgical procedure, CPB time, and aortic crossclamping time; (4) data regarding outcomes of interest in both groups.
Disagreements were resolved by discussion among all authors during the process of data abstraction. The authors of the included studies were contacted if necessary. For trials in which continuous outcomes were reported as median and range, mean and standard deviation were estimated by utilizing the O'Rourke method. [13] When the results of the trial were reported as median and quartile, the Stela Pudar-Hozo method was used to estimate the mean and standard deviation. [14] 2.6. Statistical analysis All data were analyzed by utilizing RevMan 5.3 (Cochrane Collaboration, Oxford, UK). Pooled odds ratio (OR) and 95%  99:7 Medicine confidence interval (CI) were estimated for dichotomous data, and weighted mean difference (WMD) and 95% CI for continuous data, respectively. Each outcome was tested for heterogeneity, and randomized-effects or fixed-effects model was used in the presence or absence of significant heterogeneity (I 2 > 50%). Sensitivity analyses were done by examining the influence of statistical model on estimated treatment effects, and analyses which adopted the fixed-effects model were repeated again by using randomizedeffects model and vice versa. In addition to that, sensitivity analysis was also performed to evaluate the influence of individual study on the overall effects. The possible effects of patient age, gender, country, surgery type, and CPB on postoperative bleeding and transfusion were evaluated by meta-regression. Publication bias was explored through visual inspection of funnel plots of the outcomes, and evaluated by Begg test with STATA 14.0 (Stata Corp, College station, TX). All P-values were 2-sided and statistical significance was defined as P < .05.

First author
Year Journal Country Language Ulinastatin administration Control Chen TT [22] 2013 Heart Surg Forum China English 12,000 U/kg iv. after AI 12,000 U/kg added to CPB prime Saline Ji BY [21] 2007 J Cardiovasc Surg China English 300,000 U iv. before CPB 300,000 U iv. before AXC release 400,000 U iv. after protamine Saline Ji HW [9] 2009 Zhonghua Yi Xue Za Zhi China Chinese 400,000 U iv. after AI 400,000 U added to CPB prime 200,000 U ivgtt. (40,000-60,000/h)

Saline
Jin XG [32] 2005 Chin Pharm J China Chinese 300,000 U iv. after AI 300,000 U added to CPB prime 300,000 U during CPB Saline Kim NY [18] 2013 Korean J Anesthesiol Korea English 300,000 U iv. after AI Saline Le HB [30] 2005 Zhejiang Med China Chinese 300,000 U iv. after AI 300,000 U added to CPB prime 300,000 U during CPB Saline Nakanishi K [16] 2006 Crit Care Med Japan English 5,000 U/kg iv. before CPB Saline Pang XY [15] 2016 Am J Ther China English 5,000 U/kg iv. after AI 5000 U/kg iv. after heparinizatioin 5000 U/kg iv. after protamine Saline Park JB [19] 2013 Korean J Thorac Cardiovasc Surg Korea English 5000 U/kg iv. before ACC Saline Qiu Y [23] 2015 Chin Med J China English 5000-10,000 U/kg iv. before incision 5000-10,000 U/kg added to CPB prime

No Uli
Song JE [20] 2011 J Int Med Res Korea English 5000 U/kg iv. before ACC Saline Tan RD [29] 2011 Mod Hosp China Chinese 300,000 U iv. after AI 300,000 U added to CPB prime Saline Wang GY [24] 2010 Chin J ECC China Chinese 6000 U/kg iv. after AI 1000 U/kg/h ivgtt. till end of surgery Saline Xu CE [17] 2013 J Cardiothorac Vasc Anesth China English 20,000 U/kg total, 1/3 after AI, 1/3 before ACC and 1/3 before ACC release Saline Yang WH [26] 2010 Med Recap China Chinese 12,000 U/kg iv. after AI 12,000 U/kg added to CPB prime Saline Yu JG [33] 2003 Chin J Anesthesiol China Chinese 6000 U/kg iv. after AI 6000 U/kg added to CPB prime Saline Zhang BJ [31] 2004 J Cardiovasc Dis China Chinese 300,000 U iv. after AI 300,000 U added to CPB prime 300,000 U during CPB Saline Zhai YJ [25] 2004 Chin J ECC China Chinese 12,000 U/kg iv. after AI 12,000 U/kg added to CPB prime Saline ACC = aortic cross-clamping, AI = anesthesia induction, CPB = cardiopulmonary bypass, Uli = Ulinastatin.

Effects on TEG profiles
Park and colleagues [19] failed to detect any significant differences between Ulinastatin and saline with respect to postoperative TEG profiles including R (clotting time), K (clot formation time), and MA (maximum clot firmness) values in cardiac surgical patients. That is contrary to previous study by Okida et al reporting that Ulinastatin normalized the coagulation function and prevented changes in TEG during liver resection surgery. [8] 3.12. Effects on leukocyte and neutrophil counts As shown in Table 3, 4 trials [15,18,21,23] (348 patients) and 2 trials [18,23] (258 patients) reported leukocyte count and neutrophil

Sensitivity analyses and publication bias
Sensitivity analysis showed that treatment effects on all the outcomes were not affected by the choice of statistical model (Supplement Table 3, http://links.lww.com/MD/D778). Sensitivity tests were also performed by exclusion of some studies to analyze the influence of the overall treatment effect on high heterogeneity outcomes (Supplement Table 4, http://links.lww. com/MD/D779 but no contradictory results were found. No significant publication bias was detected by funnels plot examination for postoperative bleeding (Fig. 4) and RBC transfusion (Fig. 5)

Discussion
To our knowledge, this is the first meta-analysis dedicated to evaluate whether Ulinastatin could reduce blood loss and transfusion requirements. The present meta-analysis suggested that, Ulinastatin administration could reduce postoperative bleeding and RBC transfusion requirement, preserve platelet function, inhibit hyperfibrinolysis, and attenuate systemic inflammation in cardiac surgical patients. Systemic heparinization, hemodilution, and hypothermia applied during cardiac surgery with CPB significantly influence coagulation and fibrinolysis systems. The present meta-analysis showed that Ulinastatin did not influence coagulation parameters such as PT and APTT values, fibrinogen levels, AT-III:A, and in vivo thrombin generation. CPB-induced fibrinolysis activation is associated with increased postoperative bleeding volume. [49] Ddimer is an index for measurement of fibrinolytic activity in cardiac surgery. The present meta-analysis confirmed the antifibrinolytic effect of Ulinastatin, as manifested by lowering postoperative D-dimer levels without influence on PA and a2-AP concentrations.
Platelets play an important role in hemastasis by means of adhesion, activation, and aggregation. CPB and hypothermia can result in significant changes in platelet function. [50] Upon platelet activation, CD62P (also known as P-selection) was rapidly released from a-granules to external surface. [36] Therefore, CPBinduced platelet activation could be evidenced by increases in CD62P expression. [36] The present meta-analysis suggested that, Ulinastatin did not influence platelet count, but PAGM% was better preserved post-operatively in Ulinastatin-treated patients. Inflammation contributes to thrombotic response and influence the initiation and propagation of blood coagulation. [51] Evidence has suggested that, Ulinastatin can reduce proinflammatory cytokines, elevate anti-inflammatory cytokines, and provide organ protection in patients undergoing cardiac surgery. [5][6][7] The conclusion is further confirmed by our metaanalysis which indicated that Ulinastatin not only reduced postoperative bleeding and transfusion requirement, but also inhibited the release of inflammatory mediators such as TNF-a, PMNE, IL-6, and IL-8. Open heart surgery triggers systemic inflammatory response syndrome via the action of leukocytes, especially polymorphonuclear neutrophils (PMNs). PMNs degrade or inhibit the activity of fibrin, fibrinogen, platelets and coagulation factors, and lead to increased blood loss and transfusion requirements. [19,52] The inhibitory effect of Ulinastatin on inflammatory cytokines may be related to the activity inhibition of the widely distributed serine proteases, inhibition of migration and activation of leukocytes, reduction in inflammatory cell infiltration and release of tissue toxic substances. [53,54] It has also been proved that Ulinastatin is effective in lowering allogeneic blood transfusion induced PMNE and cytokines release. [55][56][57] Interestingly, it has been indicated that Ulinastatin addition could attenuate in vitro storage lesion (eg, hemolysis, erythroptosis) of human red blood cells. [58,59] Notably, the administration strategy of Ulinastatin could significantly affect the outcomes of interests. The maximum recommended daily dosage of Ulinastatin in its product instructions is 30 Â 10 4 U (Guangdong Techpool Bio-pharma Co. Ltd., Guangdong, China). However, the doses required to treat severe acute diseases (eg, sepsis, acute pancreatitis) is much higher. In a recent randomized, double-blinded, placebo-controlled and singledose escalation study, Chen et al [60] demonstrated that, 2 hours of intravenous infusion of Ulinastatin ranging from 30 Â 10 4 U to 800 Â 10 4 U was well tolerated in healthy volunteers. The effects of Ulinastatin on blood loss reduction might be dose-dependent. In our meta-analysis, larger doses (eg, above 1.2 Â 10 4 U/kg or 300 Â 10 4 U) of Ulinastatin tended to reduce bleeding and blood transfusion requirement, while smaller doses (eg, 5000 U/kg) might not be sufficient to be effective. Ulinastatin administration only by intravenous bolus were unable to reduce bleeding in 5 trials, [16,17,[19][20][21] while 5 included trials [25,[27][28][29]33] demonstrated that adding Ulinastatin to CBP prime could significantly reduce blood loss. The half-life of Ulinastatin in healthy adults is only 40 minutes, [61,62] which is shorter than the duration of cardiac surgery or CPB. It is also possible that CPB-induced hemodilution could reduce the effectiveness of Ulinastatin. [20] Therefore, it is highly possible that, larger doses and longer period of Ulinastatin infusion may be necessary to be effective in reducing bleeding in cardiac surgical patients. It is also possible that, there are different effective Ulinastatin concentrations for different therapeutic purposes, such as anti-fibrinolysis, anti-inflammation, and so on. The optimal doses of Ulinastatin for different therapeutic purposes remain to be investigated. Notably, both Ulinastatin and aprotinin are trypsin inhibitors although derived from different sources. Whether ulinastatin have any effect on short-and long-term outcomes of cardiac surgical patients remains to be examined in adequately powered RCTs.
This study has some limitations. Meta-analysis can increase the power of analysis by pooling many small low-quality studies, but varied Ulinastatin dosages and surgical operation types, different clinical practices, quality and heterogeneity issues of included studies may limit the certainty of the findings of meta-analysis. To clarify the hemostatic effectiveness of Ulinastatin in cardiac surgical patients, a prospective randomized, placebo-controlled, triple-blinded trial is ongoing in our center (ClinicalTrials.gov Identifier: NCT01060189).
To conclude, Ulinastatin reduces postoperative bleeding and transfusion requirement in cardiac surgical patients, possibly by inhibiting hyperfibrinolysis, preserving platelet function, and alleviating inflammation. To confirm this, more well-designed and adequately-powered randomized trials are needed.