Platelet mitochondrial respiration and coenzyme Q10 could be used as new diagnostic strategy for mitochondrial dysfunction in rheumatoid diseases

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimunne disorder affecting both small and large synovial joints, leading to their destruction. Platelet biomarkers are involved in inflammation in RA patients. Increased circulating platelet counts in RA patients may contribute to platelet hyperactivity and thrombosis. In this pilot study we evaluated platelet mitochondrial bioenergy function, CoQ10 levels and oxidative stress in RA patients.

Methods: Twenty-one RA patients and 19 healthy volunteers participated in the study. High resolution respirometry (HRR) was used for analysis of platelet mitochondrial bioenergetics. CoQ10 was determined by HPLC method; TBARS were detected spectrophotometrically.

Results: Slight dysfunction in platelet mitochondrial respiration and reduced platelet CoQ10 levels were observed in RA patients compared with normal controls.

Conclusions: The observed decrease in platelet CoQ10 levels may lead to platelet mitochondrial dysfunction in RA diseases. Determination of platelet mitochondrial function and platelet CoQ10 levels could be used as new diagnostic strategies for mitochondrial bioenergetics in rheumatoid diseases.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Respirometric analysis of mitochondrial function in human platelets.

The trace from the measurement of platelet (PLT) respiration at 37°C in a respiration medium MiR05 and 20 mM creatine. The blue line represents oxygen concentration (μM), and the red trace represents oxygen consumption as flow per cells (pmol O2·s−1·10−6 cells). The modified substrate-uncoupler-inhibitor-titration (SUIT) reference protocol 1 (RP1) [38]. includes following steps: ce1: Oxygen consumption rate of intact PLT (routine respiration); 1Dig: Respiration rate of mitochondria in PLT permeabilized with digitonin; 1PM: Complex I-linked LEAK (State 4) respiration reflects the rate of mitochondrial respiration with exogenous substrates (pyruvate and malate); 2D: Complex I-linked OXPHOS (State 3) after ADP addition reflects CI-linked ATP production; 2c: Cytochrome c addition—a test for the integrity of outer mitochondrial membrane; 3U: The rate after addition of an uncoupler CCCP represents maximal CI-linked oxidative capacity with substrates pyruvate and malate (uncoupled from OXPHOS). 4G: Noncoupled Complex I-linked oxygen consumption after the addition of substrate glutamate; 5S: Noncoupled Complex I- and Complex II-linked oxygen consumption after the addition of CII substrate succinate; 6Rot: Noncoupled Complex II-linked oxygen consumption after the addition of rotenone—an inhibitor of Complex I; 7Ama: Residual oxygen consumption (ROX) after the addition of antimycin A—an inhibitor of CIII represents respiration that is not associated with electron transfer pathways. This respiration is subtracted from all values for the determination of mitochondrial electron transfer pathways-related oxygen consumption.

Fig 2. Platelet mitochondrial function in control subjects and groups of RA patients.

The parameters of mitochondrial respiration in human platelets expressed as oxygen (O2) flux per mitochondrial marker–the activity of citrate synthase (CS). The bars show mean±sem. The names on x-axis represent steps in the SUIT protocol RP1 (see the legend for Fig 1). Control = control subjects; RA_ALL = all patients with rheumatoid arthritis; RA_CRP = rheumatic patients with high c-reactive protein concentration; RA_CVD = rheumatic patients with cardiovascular diseases; +p<0.1.

Fig 3. Platelet count and platelets coenzyme Q10-TOTAL in control subjects and in groups of RA patients.

CoQ10-TOTAL (ubiquinol + ubiquinone); RA_ALL = all patients with rheumatoid arthritis; RA_CRP = rheumatic patients with high c-reactive protein concentration; RA_CVD = rheumatic patients with cardiovascular diseases; +p<0.1*p<0.05; **p<0.01, ****p<0.0001.

Fig 4

4A. Correlation between CoQ10-TOTAL in Platelets and Respiration of Intact Platelets in Control Subjects and RA_ALL Patients. ce1 = the rate of oxygen consumption in intact platelet; RA_ALL = all patients with RA; p = 0.069; CoQ10-TOTAL= ubiquinol + ubiquinone; PLT = platelet. 4B: Correlation between CoQ10-TOTAL in Platelets and Mitochondrial Respiration in Permeabilized Platelets in Control Subjects and RA_ALL Patients. 1PM = the rate of oxygen consumption in mitochondrial of permeabilized platelets (State 4); RA_ALL = all patients with RA; p = 0.010, p = 0.003 = statistically significant; CoQ10-TOTAL= ubiquinol + ubiquinone; PLT = platelet. 4C: Correlation between CoQ10-TOTAL in Platelets and 3U Platelets Mitochondrial Respiration in Control Subjects and RA_ALL Patients. 3U = the rate of oxygen consumption in platelet mitochondrial noncoupled respiration at CI; RA_ALL = all patients with RA; p = 0.039, in control group: p = 0.018; CoQ10-TOTAL= ubiquinol + ubiquinone; PLT = platelet. 4D: Correlation between CoQ10-TOTAL in Platelets and 4G Platelets Mitochondrial Respiration in Control Subjects and RA_ALL Patients. 4G = the rate of oxygen consumption in platelets mitochondrial noncoupled CI after addition of substrate glutamate CI; RA_ALL = all patients with rheumatoid arthritis; p = 0.060; CoQ10-TOTAL= ubiquinol + ubiquinone; PLT = platelet.

Fig 5. The OXPHOS-coupling efficiency in healthy control volunteers and groups of RA patients.

RA_ALL = all patients with rheumatoic arthritis; RA_CRP = rheumatic patients with high c-reactive protein concentration; RA_CVD = rheumatic patients with cardiovascular diseases; +p<0.5; *p<0.05 statistical significance vs control group.