Platelet-leukocyte aggregate is associated with adverse events after surgical intervention for rheumatic heart disease

Platelet-leukocyte aggregate (PLA) is implicated in the etiology of both vascular lesions and cardiovascular events. This prospective cohort study aimed to examine the prognostic value of PLA for major adverse cardiac and cerebrovascular events (MACCE) and perioperative adverse events (AEs) in patients with rheumatic heart disease undergoing surgical intervention by Cox proportional hazard regression and logistic regression. A total of 244 patients were included, of whom 7 were lost to follow-up. Among the analyzed 237 subjects who completed 3-year follow-up, 30 experienced MACCE and 38 experienced perioperative AEs. Preoperative PLA was higher in subjects who developed MACCE (13.32%) than in those who did not (8.69%, p = 0.040). In multivariate regression, elevated PLA was associated with increased MACCE (hazard ratio 1.51 for each quartile, 95% CI 1.07–2.13; p = 0.020), and perioperative AEs (odds ratio 1.61, 95% CI 1.14–2.26; p = 0.007). The optimal PLA cut-off for predicting MACCE was 6.8%. Subjects with PLA > 6.8% had a higher prevalence of MACCE (17.1% vs. 5.5%, p = 0.009) and perioperative AEs (19.9% vs. 8.6%, p = 0.018). Kaplan-Meier analysis showed shorter MACCE-free survival in patients with PLA > 6.8% (p = 0.007, log rank). Elevated preoperative PLA is associated with increased MACCE and perioperative AEs in patients with rheumatic valve disease undergoing surgical intervention.

incidence of MAcce and perioperative Aes. Primary outcome was major cardiac and cerebrovascular events (MACCE), a composite index consisting of stroke, heart failure, myocardial infarction, life-threatening arrhythmia, transient ischemic attack and MACCE-related death. A total of 13 (5.48%) patients died during the 3-year follow-up, and 8 deaths could be attributed to MACCE: 6 to cardiac failure, 1 to stroke and 1 to sudden death. The remaining 5 deaths were due to gastric cancer (n = 2), traffic accidents (n = 2) or lung infection (n = 1).

Discussion
In this prospective cohort study, we found that the incidence of MACCE was 4.2% per year in patients after valve surgery, indicating these patients may be at high risk of cardiovascular morbidity. Elevated PLA prior to surgery was associated with increased rate of perioperative AEs and MACCE during a 3-year period. Patients with preoperative PLA > 6.8% were more likely to suffer these outcomes. These results suggest that PLA could be used as a biomarker to identify patients with rheumatic valve disease at high risk of MACCE after surgical intervention. This is, to our knowledge, the first report suggesting an association of preoperative PLA with risk of perioperative AEs and MACCE.
Previous studies [23][24][25][26][27][28] have identified a variety of biomarkers that can predict morbidity and mortality after cardiac surgery. For example, postoperative B-type natriuretic peptide at >790 ng/L and troponin T at >0.08 μg/L have been associated with mid-and long-term mortality and major cardiac events after cardiac surgery. High preoperative C-reactive protein has also been associated with higher rate of mortality, cardiovascular events and myocardial damage after cardiac surgery. However, elevation of these biomarkers is the result, not cause, of tissue damage. Continued www.nature.com/scientificreports www.nature.com/scientificreports/ Both innate and adaptive immune cells are activated in inflammation-associated vascular lesions 5,29 . CD62L, an adhesion molecule expressed on lymphocytes, monocytes and neutrophils 5,15,30 , was used to identify leukocytes in the present study. CD62L has been shown to be up-regulated in stroke patients 15 , and to cluster along the leading edge of leukocytes during platelet-leukocyte interaction 30 . Lymphocytes from CD62L −/− animals have impaired ability to migrate to atherosclerotic aortas 5,31 . These results suggest that CD62L contributes directly to vascular lesions.
Based on double staining with CD62L and CD41a, the median PLA level in the current study was 9.6%, which was more than double the rate reported for a healthy population (3.5%) 32 . Increased PLA may induce and amplify inflammation via release of pro-coagulatory 33 and pro-inflammatory 34,35 factors. For this reason, increased PLA might partly explain the phenomenon that patients with rheumatic disease are at high risk of coronary artery disease (CAD) events 36 . Increased PLA can also be seen in patients with ischemic and non-ischemic cardiac and cerebrovascular events, such as angina 37 , acute coronary syndrome 12,13,16,38 , and cerebrovascular ischemia 15,17,39 . If increased PLA is a risk factor of CAD, it may also increase risk of MACCE by inducing similar cerebral vascular inflammation. Consistent with this, our results demonstrate an association of increased PLA with increased MACCE and perioperative AEs, and previous reports have linked increased PLA with stroke 15,32 and angina 13,16 .
In patients with rheumatic disease, levels of the systemic inflammatory factor TNF-α play an important role in occurrence and development of disease 40 . However, we found in the present study that TNF-α did not correlate significantly with either MACCE or perioperative AEs. This may mean that TNF-α is not a reliable marker of vascular inflammation, despite its role in regulating immune cell activation 9 . This apparent paradox should not be surprising, given the complexity of the cytokine and chemokine networks involved.
Our results, together with others, suggest PLA may be more effective than TNF-α to predict MACCE in patients with rheumatic valve disease, which may be due to the role of PLA in cardiovascular lesions. Cardiac and cerebrovascular injury activate inflammatory and coagulation responses, as well as the corresponding antagonistic pathways. The interaction between platelets and leukocytes leads to leukocyte accumulation in the vascular wall, myocardium and many other organs. These PLA adhere to the endothelium at sites prone to plaque formation 11 . Leukocyte infiltration into tissue can promote collagen breakdown and structural remodeling processes, triggering oxidative stress 41 , leading in turn to ischemic heart failure 42 . Infiltrating leukocytes and PLA seem to promote structural and electrical remodeling in patients with atrial fibrillation 43 , which may also result in heart dysfunction and even failure.
To further elucidate the predictive value of PLA on MACCE, we used ROC analysis to identify an optimal PLA cut-off of 6.8% for stratifying our study population based on risk of MACCE. This level is similar to that previously reported in patients with cardiac and cerebrovascular ischemia, and lower than that reported in ischemic stroke patients (8.9%) 32 , and approximately twice the value reported in a healthy population (3.5%) 32 . These results suggest that the cellular biomarker PLA may predict higher risk of adverse cardiovascular events, perhaps because high PLA reflects a chronic proinflammatory state.  www.nature.com/scientificreports www.nature.com/scientificreports/ Some studies have reported that PLA correlates with platelet count and P-selectin, but not with white cell count or neutrophil activation (L-selectin) 44 . We found that elevated PLA correlated positively with platelet count before surgery but not with leukocyte count. No other significant associations were identified between increased PLA and pre-or perioperative characteristics.    www.nature.com/scientificreports www.nature.com/scientificreports/ The current study was conducted in a single center with a relatively small sample, 84% of them with poor cardiac function (NYHA III-IV), which may create bias, although the NYHA class was adjusted in logistic analysis. Second, although we composited small events (from 1 to 18) to MACCE and AEs, according to the similar studies [45][46][47][48][49][50] , there still a chance to create bias in analysis the PLA and the outcomes. At last, we did not attempt to identify which subtype(s) of leukocytes in PLA may explain our results. Monocytes, as macrophage precursors, migrate early to the endothelium 8 , while T helper 1 lymphocytes secrete cytokines to activate macrophages 51 , and infiltrating neutrophils help enlarge and destabilize atherosclerotic lesions by creating oxidative stress 8 . Future work should examine the contributions of these different subtypes to MACCE. clinical implications. Our findings suggest that PLA can serve as a useful cellular biomarker of MACCE risk in patients undergoing rheumatic valve replacement. It may be possible to monitor PLA in order to stratify patients according to MACCE risk, and it may be useful as a treatment target to reduce such risk. Indeed, animal studies 18,52 have proven that blocking PLA formation can effectively attenuate vascular lesions, although how this occurs requires further study. It is possible that PLA is equally useful for patients undergoing other types of cardiac surgery, such as CABG, which should be explored in future work. Our results suggest that future clinical and laboratory studies should focus on PLA as a cellular biomarker of MACCE risk and on the role of PLA in formation and progression of vascular lesions. We highlight the need for further research to identify preoperative variables that affect PLA levels. It is possible that inhibiting PLA formation via the preoperative variables might improve the patient prognosis, and this should be explored in larger samples.    www.nature.com/scientificreports www.nature.com/scientificreports/ IV status (n = 25), severe peripheral vascular disease (n = 3) or active infective endocarditis (n = 21). Another 60 patients were excluded because of preoperative complications in the respiratory system: PaO2 lower than 60 mmHg with room air (n = 11), asthma (n = 13), chronic obstructive pulmonary disease (n = 34) or pulmonary infection (n = 2). Another 21 were excluded because of a history of suspected liver injury (n = 10) or renal failure (n = 11). A further 70 patients were excluded for the following reasons: repeated surgery (n = 13), enrollment in other clinical trials (n = 34), or refusal to join the trial (n = 23). In the end, analysis of perioperative AEs included 244 subjects, while 7 patients (2.87%) were lost to follow-up and so only the remaining 237 patients were included in analysis of MACCE (Fig. 3). All 237 patients completed 3-year follow-up.
All subjects signed written informed consent. The study protocol was approved by the Ethics Committee of Sichuan University and conducted in accordance with the Declaration of Helsinki. The study was registered in the Chinese Clinical Trial Registry (ChiCTR-OCH-12001922). patient management and procedure. Patients managements, including anesthesia, cardiopulmonary bypass (CPB), were performed according to standard hospital protocols ( Supplementary Information 3) 53 . www.nature.com/scientificreports www.nature.com/scientificreports/ All patients received postoperative warfarin, but not anti-platelet agents, as anticoagulation strategy. The target international normalized ratio (INR) in the present study was 1.5-2.5 for mechanical mitral valve replacement, 1.3-1.8 for mechanical aortic valve replacement; and 2.0-3.0 for tricuspid valve replacement, which is lower than the INR recommended in a Western study 54 . The lower INR is routinely used at our hospital and many medical centers in China because it appears to be more appropriate for Asian populations based on several studies 55-57 . Assessment of pLA and tnf-α. Blood samples for PLA and TNF-α were collected after anesthesia induction. Samples were incubated with CD62L-PE (BD, Basel, Switzerland), CD41a-FITC (BD) and DRAQ5 (Biotium, Hayward, CA, USA). An IgG1-FITC/PE antibody (BD) served as an isotype control. PLA was identified using CD62L/CD41a dot plots after confirming cell nuclei using DRAQ5. Putative PLA was verified by sorting stained samples using FACSAria and observation under a confocal fluorescence microscope (Supplementary Figure). Details of PLA determination are provided in Supplementary Information 4. For analysis of plasma TNF-α, arterial blood was centrifuged at 4 °C for 15 min at 1000 g. TNF-α concentration was determined using a commercial enzyme-linked immunosorbent assay kit (High Sensitivity Kit, R&D Systems, Minneapolis, MN, USA) with a detection limit of 0.038 pg/ml. Data collection. Baseline variables included demographics, smoking history, cardiac disease, NYHA class, history of medication, preoperative comorbidities and preoperative blood cell counts. Intraoperative variables included type of valve replacement, CPB time, cross-clamp time and blood transfusion.
Follow-up was carried out every 6 months for three years, either when patients came into hospital for treatment or by telephone. Patients or their family members were asked to report whether and when the patients had been hospitalized in any other hospitals. If the patients were hospitalized in our hospital, study events were determined during their hospitalization. For the other re-admission patients, events were determined based on medical records, and, when necessary, discussion with the consulting physician. Researchers conducting follow-up were blinded to patients' PLA results.
outcomes. Primary outcome was MACCE, including stroke, heart failure, myocardial infarction, life-threatening arrhythmia, transient ischemic attack and MACCE-related death. Re-hospitalizations for a combination of heart failure and arrhythmia were counted only once, and the reason was recorded as heart failure if heart failure persisted after treatment of arrhythmia; otherwise, the reason was recorded as arrhythmia.
Secondary outcome was AEs. Detailed definitions of the AEs are listed in Supplementary definition of outcomes.
Statistical analysis. The comparison of baseline characteristics was used to select potential risk and confounding factors. All variables, including demographics, medical history, preoperative medication, blood cell counts and intraoperative variables, were included into univariate and multivariate backward Cox proportional hazard regression to assess their association with 3-year outcomes. Logistic regression was used to access the association between variables and perioperative outcomes. To identify the effect of PLA on outcomes, hazard ratio (HR) was adjusted by age, gender, body mass index, smoking history, NYHA functional class, diabetes, stroke history, atrial fibrillation, hypertension, digoxin, type of valve replacement, concomitant tricuspid repair and valve material. Odds ratio (OR) was adjusted by all these factors as well as CPB time.
We performed receiver operating characteristic (ROC) curves, based on MACCE and PLA, from the logistic regression model, and then Youden's index were used to identify the optimal PLA cut-off value for predicting MACCE in 3 years. This threshold was used to classify patients as having low or high PLA, and MACCE-free survival was compared between the two groups using Kaplan-Meier analysis and the log rank test. Our pilot results with 88 patients showed that 2 of 34 patients with PLA ≤ 6.8% suffered MACCE, compared to 9 of 54 patients with PLA > 6.8%. The sample size was calculated by Cox model based on 2-Sided Equality, according to the Time-To-Event data. Power analysis indicated the need for at least 193 patients, based on 1-β = 0.8, α = 10%. Based on the number of cardiac surgery patients at our hospital in 2010 and the exclusion criteria in our study, we decided to screen all valve replacement patients within one year for eligibility.
All statistical analyses were performed using SPSS 19.0 (IBM, Chicago, IL, USA) and RStudio 1.1.453 (R-Tools Technology, Canada). In all analyses, p < 0.05 was considered significant. Detailed methods of the statistical analysis are shown in Supplementary statistical analysis.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.