Platelet mitochondrial membrane depolarization reflects disease severity in patients with sepsis and correlates with clinical outcome

Abstract

Introduction: Sepsis is still a leading cause of morbidity and mortality, even in modern times, and thrombocytopenia has been closely associated with unfavorable disease outcome. Decreases in mitochondrial membrane potential (depolarization) were found in different tissues during sepsis. Previous work suggests that mitochondrial dysfunction of platelets correlates with clinical disease activity in sepsis. However, platelet mitochondrial membrane potential (Mmp) has not been investigated in a clinical follow-up design and not with regard to disease outcome.

Methods: In this study, platelet mitochondrial membrane depolarization was assessed by means of a fluorescent Mmp-Index with flow cytometry in 26 patients with sepsis compared with control patients. Platelet Mmp-Index on admission was correlated with the clinical disease scores Acute Physiology and Chronic Health Evaluation Score II (APACHE II), Sequential Organ Failure Score (SOFA), and Simplified Acute Physiology Score II (SAPS II). Finally, platelet Mmp-Index on admission and follow-up were compared in the group of sepsis survivors and nonsurvivors. Expression of the prosurvival protein Bcl-xL in platelets was quantified by immunoblotting.

Results: Platelet mitochondrial membrane depolarization correlated significantly with the simultaneously assessed clinical disease severity by APACHE II (r = -0.867; P < 0.0001), SOFA (r = -0.857; P <0.0001), and SAPS II score (r = -0.839; P < 0.0001). Patients with severe sepsis showed a significant reduction in platelet Mmp-Index compared with sepsis without organ failure (0.18 (0.12 to 0.25) versus 0.79 (0.49 to 0.85), P < 0.0006) or with the control group (0.18 (0.12 to 0.25) versus 0.89 (0.68 to 1.00), P < 0.0001). Platelet Mmp-Index remained persistently low in sepsis nonsurvivors (0.269 (0.230 to 0.305)), whereas we observed recovery of platelet Mmp-Index in the survivor group (0.9 (0.713 to 1.017)). Furthermore, the level of prosurvival protein Bcl-xL decreased in platelets during severe sepsis.

Conclusion: In this study, we demonstrated that mitochondrial membrane depolarization in platelets correlates with clinical disease severity in patients with sepsis during the disease course and may be a valuable adjunct parameter to aid in the assessment of disease severity, risk stratification, and clinical outcome.

Figure 1

Correlation of platelet mitochondrial membrane potential with APACHE II, SOFA, and SAPS II scores. Dot-blot correlation of the individual mean platelet Mmp-Index of each patient calculated from triplicate JC-1 FL2 to FL1 fluorescence ratios by flow cytometry and the clinical disease-severity scores APACHE II (A), SOFA (B), and SAPS II score (C) in 26 patients with sepsis. Lower Mmp-Index values indicate a loss of mitochondrial membrane potential, whereas higher clinical disease scores indicate more-severe illness. r = statistical correlation coefficient; P ≤ 0.05 denotes statistically significant correlation of values.

Figure 2

Comparison of platelet mitochondrial membrane potential of patients with sepsis, severe sepsis, and control patients. Box-and-whisker plots illustrate platelet Mmp-Index of patients with sepsis (nonsevere without organ failure), severe sepsis including septic shock, and control patients without infection. Lower Mmp-Index values indicate a loss of mitochondrial membrane potential. Platelet Mmp-Index was significantly lower in patients with severe sepsis compared with sepsis without organ failure (P < 0.0006) and controls (P < 0.0001). No statistical difference was found between patients with sepsis and controls (P = 0.42). Box margins identify the upper and lower quartiles; the horizontal line marks the median; and whiskers indicate minimal and maximal values. Outliers are indicated with a circle. *Statistically significant difference, P ≤ 0.05.

Figure 3

Comparison of platelet mitochondrial membrane potential of survivors and nonsurvivors of the severe-sepsis group. (A) Box-and whisker plots illustrate platelet Mmp-Index of survivors (n = 10) and nonsurvivors (n = 7) of the severe-sepsis group on admission and during follow-up. Lower Mmp-Index values indicate a loss of mitochondrial membrane potential. Platelet Mmp-Index did not show significant differences between the groups on admission (P = 0.44). No relevant increase was noted in platelet Mmp-Index in nonsurvivors during follow-up (Mmp-Index <0.5), whereas Mmp-Index in survivors recovered to baseline levels of controls. Platelet Mmp-Index was significantly higher in survivors than in nonsurvivors on follow-up (P = 0.004). Box margins identify the upper and lower quartile; the horizontal line marks the median; and whiskers indicate minimal and maximal values. Outliers are indicated with a circle. *Statistically significant difference, P ≤ 0.05. (B) Individual mean Mmp-Index values from triplicate readings of patients in the group of sepsis survivors and nonsurvivors on admission and during follow-up.

Figure 4

Comparison of Bcl-xL expression in platelets of patients with sepsis and severe sepsis. Platelet Bcl-xL expression in patients with sepsis (lanes 1 to 3) and severe sepsis (lanes 4 to 7) on day 1 of admission was compared with immunoblotting. Bcl-xL expression was quantified with band-densitometry analysis with the help of a Bcl-xL-to-actin ratio. Mean Bcl-xL-to-actin ratio for the sepsis group was set as 1, and relative decrease in Bcl-xL expression in the severe-sepsis group was compared accordingly. Relative Bcl-xL expression between groups based on actin loading is shown.