Abstract
Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L−1 ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.
Highlights
Platelets are subcellular elements of blood and substantially contribute to hemostasis by clumping and initiating the formation of blood clots [1]
(27interquartile males and 2 range females) wereBar included the study columns were analyzed with the Wilcoxon matched-pairs signed rank test and bar graphs with an average age of 38.5 ± 7.3 years, body mass 92 ± 43 kg, and height 184.0 ± 7.2 cm
There was no significant dependence of PLT respiration on age, body mass, or body mass index (BMI) (Figure S1), nor on night shifts and cigarette smoking (Figure S2)
Summary
Platelets (thrombocytes, PLT) are subcellular elements of blood and substantially contribute to hemostasis by clumping and initiating the formation of blood clots [1]. Biomedicines 2021, 9, 1859 are fragments of larger cells located in the bone marrow, called megakaryocytes, which are released into the peripheral blood. Despite being absent of nucleus, PLT contain other organelles, such as mitochondria and endoplasmic reticulum. Mitochondria are key cellular organelles responsible for the production of adenosine triphosphate (ATP), redox homeostasis, regulation of reactive oxygen species and intracellular calcium concentration, activation of apoptosis, and many other functions. Fundamental mitochondrial functions can be studied by assessment of the rate of oxygen consumption related to substrate oxidation and coupling control [2,3]. Mitochondrial dysfunction of PLT was observed in several human physiological and pathological conditions, including type II diabetes [12,13], aging [14,15], asthma [16], sepsis [8,17,18,19,20], schizophrenia, Huntington’s, Parkinson’s, and Alzheimer’s diseases [13,21]
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