Introduction
Cardiac surgical operations carried out in the cardio-pulmonary bypass (CBP) are invasive procedures with activation of various cell types and production of numerous biologically active molecules [1, 2]. Among other blood cells, platelets are involved in developing postoperative systematic inflammatory response syndrome (SIRS) [3]. It was shown previously that SIRS with clinical symptoms was noted in 30% of all individuals undergoing heart surgeries [4, 5]. Moreover, SIRS occurrence had an adverse effect on the postoperative course and led to higher postoperative morbidity, including organ failure [4, 6]. On the other hand, the postoperative activation, adherence and aggregation of platelets is mandatory to prevent blood loss at sites of vascular injury and to simultaneously initiate the healing process (a fibrin clot provides a scaffold for attracted inflammatory cells) [7, 8]. Contrary to hyperactivation of platelets, their impaired recruitment on damaged vessels is due to inherited or acquired defects that may promote excessive postoperative bleeding that is usually difficult to counteract by means of surgical maneuvers [9].
So far, many laboratory methods have been developed to test platelet function and metabolic activity that might have potentially applicability after surgical or intravascular procedures [10, 11]. Some of them, such as flow cytometry or light transmission platelet aggregation, are performed predominantly for basic research because they are time-consuming and require sophisticated analytic equipment [10]. Among rapid tests, a whole blood aggregometer (e.g., Multiplate Analyzer) based on impedance platelet aggregometry enables on-site evaluation of platelet function, but appropriate management with blood samples and analysis of its findings needs experienced laboratory personnel that usually are not available 24 hours a day. Moreover, interpretation of results when platelet count (PLT) is below 120 × 1012/L may be biased [12]. Of note, not uncommonly, PLT is relatively low after cardiac surgical interventions. Additionally, serum thromboxane B2, a stable metabolite of thromboxane A2 (TXA2) produced by activated platelets, has also been shown as a good marker of platelet’s reactivity. However, its measurement is laborious and time-consuming, also when using commercially available colorimetric assays. On the other hand, malondialdehyde (MDA), a broadly and easily assayed marker of lipid peroxidation and oxidative stress [13], is also known to be produced by the platelet’s thromboxane synthase in amounts that are equimolar to TXA2 [14]. Therefore, the aforementioned methods may not be considered as routine studies done in all patients in the early postoperative period. In particular, in view of a critical clinical setting that some of them can be related to the abnormal (hyper- or hypo-) activity of platelets, intensivists together with cardiac surgeons must undertake immediate actions to rescue patients. Thus, we need cost-effective and easy-to-perform tests/methods to define platelet metabolic status. It was previously showed that some information withdrawn from basic blood morphology analysis could be clinically relevant [15]. Mean platelet volume (MPV) and platelet distribution width (PDW) were observed to be increased during platelet activation [15]. The others, MPV-to-PLT ratio even better than MPV alone predicted long-term adverse outcomes in patients with ST-segment elevation myocardial infarction (STEMI) undergoing percutaneous coronary interventions (PCIs) [16].
The purpose of this study was to estimate which of the routinely examined basic morphological parameters or their derivatives correlate best with platelet MDA content, a marker of platelet metabolic activity and oxidative stress, after elective standard cardiac surgical procedures carried out from median sternotomy.
Methods
Patients
The study involved 22 consecutive patients with a mean age of 62.3 ± 10.3 years who underwent elective cardiac surgical procedures from full median sternotomy and in CPB during 1 month period in a single center. The patients who were operated out on a beating heart (off-pump coronary artery bypass grafting [OPCAB]), urgently or emergently, who had any previous cardiac surgical procedures or had to be treated surgically without cessation of antiplatelet (other than acetylsalicylic acid [ASA]) agents as well as on any anticoagulants (including low-molecular-weight heparin [LMWH]) irrespective of the indications, with proven inherited or acquired platelet dysfunction and/or other coagulopathy, PLT less than 120 × 1012/L on admission and those who had excessive postoperative bleeding followed by chest re-exploration and/or blood products transfusions (n = 2) were excluded from the study (Fig. 1). The selected demographic and preoperative clinical data are outlined in Table 1.
|
Parameters* |
N = 22 |
|
Gender (male/female) |
16/6 |
|
Height [m] |
1.68 ± 0.10 |
|
Weight [kg] |
82.9 ± 14.5 |
|
BMI [kg/m2] |
29.0 ± 4.1 |
|
Obesity [BMI > 30 kg/m2] |
9 (40.9) |
|
Hyperlipidemia |
16 (72.7) |
|
Diabetes mellitus |
6 (27.3) |
|
Arterial systemic hypertension |
17 (77.3) |
|
Peripheral artery disease |
5 (22.7) |
|
CVA/TIA |
2 (9.1) |
|
CKD** 3+ |
2 (9.1) |
|
COPD |
4 (18.2) |
|
ACS in history |
7 (31.8) |
|
Previous PCI |
6 (27.3) |
All patients expressed written informed consent to participate in this trial. The study protocol was approved by the Local Bioethical Committee of the Poznan University of Medical Sciences (No. 918/15).
Surgical procedures
All study participants were operated out from a full median sternotomy. Every patient, before any surgical manipulation on the heart and big vessels received an intravenous injection of heparin in a dose of 300 IU/kg. CPB was conducted through cannulation of the ascending aorta and right atrium. Cold cardioplegic arrest and systemic moderate hypothermia (26 to 28oC) were applied as myocardial protective methods. At the end of the surgery, protamine sulfate was injected in relation 1:1 to heparin to reverse the latter one action. The anticoagulation effect’s accuracy was controlled by activated clotting time measured on-site and in the operating room. The target value of activated clotting time was greater than 400 s.
Perioperative antiplatelet and anticoagulant management
All but one antiplatelet agent (ASA) was stopped at least 5 days prior to surgery. After operation, the first subcutaneous injections of LMWH in the prophylactic doses were done 6 to 8 hours after surgery (if no excessive bleeding was observed), then twice a day. While postoperative ASA (75 mg) was given in the morning of the next day and then once a day.
Perioperative laboratory examinations
Sampling points. The blood samples were drawn from a peripheral vein before surgery (on the admission day, usually one date before operation), the second one after patient’s transfer from the operative room to the postoperative intensive care unit (post-ICU) (usually within 1 h after surgery) and then 24 and 48 hours later, irrespective of the patient stay (post-ICU or normal cardiac surgical ward).
Morphological platelets parameters. Besides typical laboratory examinations obligatory for safe monitoring of basic life parameters, including blood morphology and biochemistry, gases (exceptionally taken from artery), samples of 5 mL of peripheral venous blood were taken for further analysis of platelet metabolic status. Standard morphological parameters of platelets such as PLT, MPV and PDW were entered into an Excel sheet that served as a study database. Eventually, the MPV/PLT ratio was calculated.
Platelets malondialdehyde content
Platelet MDA content, one of the major products of lipid peroxidation, was to assess the effect of surgery on the platelet metabolic status. The platelets were collected in the following manner: the platelet-rich plasma was obtained by centrifugation (200 g, 12 min), transferred to acid citrate dextrose solution (1/10 vol.) and centrifuged again (900 g, 15 min).
After aspiration of plasma, platelets were suspended in distilled water (200 µL). The platelet MDA content was estimated by a commercial TBARS assay kit (Cayman Chemicals, USA) according to the manufacturer protocol and the previous studies [17]. The content was given as µM of MDA. Eventually, MDA content in a single platelet (MDA/PLT) was estimated since invasive surgical procedures can also change PLT significantly.
Data presentation and statistical analysis
First, continuous variables were tested for normality by means of the Shapiro-Wilk test. If they met the Gaussian distribution assumption, they were presented as the means ± standard deviation (SD), otherwise as medians and interquartile range (IQR). Time-related changes of all platelet indices were evaluated either by the analysis of variance (ANOVA) with repeated measurements followed, if applicable, by the post hoc Tukey honest significance difference test (for normally distributed continuous variables) or with the use of the Friedman repeated measures ANOVA supplemented by the Dunn multiple rank comparisons (for the other continuous data). Categorical variables were expressed as numbers (n) and percentages (%). The strength of association between platelet morphological (PLT, MPV, PDW, MPV/PLT) and functional parameters (MDA, MDA/PLT) was tested using the Spearman’s correlation coefficient. The latter one was interpreted using the scale provided by Chung and Salkin, where an r between 0.8 and 1.0 (or −0.8 and −1.0) was defined as very strong, between 0.6 and 0.8 — as strong, between 0.4 and 0.6 — as moderate, between 0.2 and 0.4 — as weak relationship [18]. A p value < 0.05 was considered statistically significant. All statistical analyzes were carried out using Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA).
Results
Standard platelet indices
Platelet count. PLT (223 ± 44 × 1012/L at baseline [BS]) decreased significantly markedly after surgery (p < 0.001). The post-hoc test revealed significant differences between all postoperative sampling points compared to the BS count. Of note, PLT after reaching the lowest value 24 hours after the operation (166 ± 57 × 1012/L) started to increase but 48 hours following surgery it was significantly lower (175 ± 60 × 1012/L) than it was before (Fig. 2A).
Mean platelet volume and platelet distribution width. Although MPV increased markedly after surgery (p = 0.041) but further analysis revealed a significant difference only between BS (8.4 ± 0.9 fL) and the last sampling point at 48th hour following operation (9.1 ± 1.2 fL; p = 0.021; Fig. 2B).
Mean PDW values ranges between 54.2 ± 5.0% at BS and 57.1 ± 6.5% on the 1st day after surgery (Fig. 2C). However, the present calculations failed to show any changes after operations (p = 0.364).
MPV-to-PLT ratio. MPV/PLT index increased significantly after procedures (p = 0.004) and in all postoperative study time points the value of this ratio was markedly higher (peak at the 24th hour following cardiac surgical operations) than it was in the preoperative period (Fig. 2D).
MDA and MDA/PLT
A significant increase in total MDA platelet content was noted after cardiac surgical interventions (p < 0.001). The highest level (median with IQR) was found soon after the operation (4.3 [2.9–5.2] µM; p < 0.001 vs. BS), then it systematically started to decrease (24 h: 2.6 [2.1–3.2] µM; p < 0.001 vs. BS), and even 48 h later (2.2 [1.5–2.9] µM; p = 0.027) it was still higher than before the procedures (1.0 [0.6–1.5] µM).
Since surgery impacted PLT, MDA content in a single thrombocyte (MDA/PLT) was also estimated. Detailed statistical analysis revealed higher medians of MDA/PLT at all sampling points after surgery in comparison to its BS one. The details are shown in Figure 3.
Correlations between platelet morphological and functional parameters
The strongest association between platelet morphological and markers of metabolic activity were observed between MPV/PLT and MDA/PLT (r = 0.56; p < 0.001), although more significant correlations were also found (Fig. 4).
Discussion
The most crucial finding of this study was the proven association, which was revealed for the first time according to available research, between metabolic activity of platelets and some basic morphological parameters such as MPV, PDW or MPV/PLT that are routinely monitored in the early period after cardiac operations. Therefore, such information about likely metabolic excitation may be easily made available within a short time. The other findings regarding postprocedural changes of morphological parameters, such as temporary decline in PLT as well as increase in MPV are not novel. Up till now, particularly the latter one has been studied extensively (usually together with PDW) in patients with coronary artery disease or after PCI, but not following cardiac operations. Of interest, the easily calculated MPV-to-PLT ratio, in the current study corresponds best with the metabolic activity of platelets, and has aroused scientific interest relatively recently.
Previous reports that PLT usually drops soon after cardiac surgical procedures are supported herein, for both adults and in children [19, 20]. Many factors have been suggested which contribute to low PLT, particularly when CPB is applied this includes; mechanical damage, hemodilution, hypothermia and perioperative treatment with platelet-inhibiting agents [21]. Moreover, blood loss is inevitable during such invasive procedures, especially if more complex procedures with longer CPB time are carried out [22, 23]. Of note, this effect is usually temporary and not uncommon post 24 hours, at least a partial recovery is usually observed [24]. The latter finding was also confirmed by the present observations. The other basic platelet indices such as MPV and PDW were found to be elevated after procedures associated with iatrogenic injury [25, 26]. Higher values of MPV and PDW after surgical procedures may be a net result of two biological processes both driven by tissue damage. The trauma-induced mixture of cytokines and reactive oxygen species provokes bone marrow for production and release of platelets to the vascular system due to a differentiation of megakaryocytes [27]. Newly released platelets tend to be larger than mature forms and MPV is often considered a reflection of the average age of these cells. However, the aforementioned reaction of bone marrow in response to surgical trauma may not be harmful. Miceli et al. [26] found that aortic valve replacement in CPB resulted in thrombocytopenia, higher PDW and MPV in the early postoperative period but in the absence of adverse clinical events. On the other side, circulating platelets respond to vessel injury by changes in morphological shape, secretion of granule contents, and aggregation to prevent blood loss. It was previously proved that large size platelets manifested increased enzymatic and metabolic activity as well as prothrombotic potential [28]. Our study supports earlier reports regarding MPV but not PDW. We cannot exclude that recruitment of a larger volume of patients would have revealed more changes of statistical significance.
Monitoring of platelets activity as well as their metabolic status is of importance after medical procedures with extensive iatrogenic tissue damage and SIRS. To assess the metabolic activity of platelets, we have chosen the content analysis of MDA, a product of thromboxane synthase and the marker of lipid peroxidation, oxidative stress and redox imbalance [17]. Oxidative stress altering platelets redox state may lead to their activation and eventually thrombus formation. It was previously observed that increased intraplatelet MDA content was also shown to be related to higher platelet activity, including aggregation [14, 29]. This association was confirmed by the other studies that found suppression of platelet MDA levels resulted in inhibition of arachidonate- and collagen-induced aggregation [30]. Therefore, pronounced changes in platelet metabolic status may play a role in the functional pathologies responsible for either excessive bleeding or thrombo-embolic adverse events [4, 7].
Of note, some basic morphological platelet indices have been suggested to be of clinical prognostic value after both coronary artery bypass grafting (CABG) and acute coronary syndrome patients treated with PCI [31–34]. One of them, MPV was previously found to predict not only the development of saphenous vein graft disease but also mortality and morbidity (e.g., atrial fibrillation) following CABG) [31, 32] Wang et al. [33] showed dynamic changes of MPV during the acute phase of myocardial infarction and higher MPV in patients with high Killip class, suggesting its predictive value in ventricular dysfunction and adverse clinical outcome after angioplasty for acute coronary syndrome. In their study, antiplatelet treatment with pre-used clopidogrel resulted in significant MPV reduction on admission. Not only MPV, but also PDW was found to be associated with no-reflow phenomenon after primary PCI, and may be one of the mechanisms responsible for a poor prognosis if the platelet were hyperactive (reflected by rapid elevation of MPV and PDW) [34]. Herein, among platelet indices, the strongest correlation with platelet metabolic activity (MDA) was found for MPV/PLT ratio. The latter one was shown to predict long-term adverse outcomes in patients with STEMI undergoing PCI [16]. In another study, a high MPV/PLT ratio was associated with early vein-graft occlusion and poor postoperative outcomes, including both early and late reduced survival rates [35].
Limitations of the study
Some limitations of the present study should be stressed. Firstly, the number of patients participating in this clinical study was relatively small. However, even the small group size did enable revealing significant findings. Of course, one cannot exclude the that recruitment of a higher number of individuals would have increased the practical value of the examined parameters. On the other side, the primary stress is put on the findings in the laboratory studies. At least one of them is not routine and requires experienced staff and a well-equipped research laboratory. In the vast majority of studies, such a number is considered sufficient to make the conclusions that follow. There is an awareness that the present findings must be further supported by the clinical outcomes of patients undergoing cardiac surgical procedures. Therefore, it must be stressed that the present research results are treated as groundwork for further prospective clinical studies.
Conclusions
Heart surgeries as complex and invasive procedures have significant impact on both morphological parameters and indices of their metabolic status. Among basic morphological parameters and indices, the MPV-to-PLT ratio best reflects the metabolic status of platelets in cardiac surgical patients.
