Supplementary Materials Fig. with DMARDs, platelet activation and platelet aggregation reached values comparable to those of HD. Our preliminary data suggest that platelets L-NIO dihydrochloride might play an active role in the atherosclerosis occurring in RA patients. 0.01) higher percentage of platelets (76%) with hyperpolarized mitochondria membrane (HMM) as compared to healthy donors (30%). Interestingly, in treated patients the percentage of platelets with HMM decreased significantly ( 0.05) to values comparable to those measured L-NIO dihydrochloride in HD (44% vs 30%). Common flow cytometric measure of HMM in platelets from representative HD (left panel), RA\T0 (middle) and RA\F.U. (right) are in Fig. ?Fig.1B.1B. During endothelial dysfunction or under pathological conditions, reactive oxygen species (ROS) production increases and the platelets respond with specific biochemical and morphological changes. In this context, mitochondria play a key role, being able to both generate ROS driving redox\sensitive events, and respond to ROS\ mediated changes of the cellular redox state 16. Given that HMM involves an increased production of ROS, the levels of hydrogen peroxide (H2O2) in platelets were measured by using flow cytometry. As shown in Fig. ?Fig.1C,1C, the levels of H2O2 measured in platelets from RA\T0 were significantly ( 0.05) higher with respect to those measured in HD. At the follow\up, H2O2 levels decreased significantly ( 0.05) and reached values even below those measured in HD. Common flow cytometric measure of H2O2 levels in platelets from representative HD and RA patients (RA\T0 and RA\F.U.) are shown in Fig. ?Fig.1D.1D. In order to assess whether H2O2 production could lead to redox alterations within the platelets, total thiol levels were measured. As shown in Fig. ?Fig.1E1E the total thiol content decreased significantly ( 0.01) in platelets from RA\T0 patients with respect to those from HD. Interestingly, in RA\F.U. the total thiol content increased significantly ( 0.05) with respect to those measured in platelets from RA\T0 patients and reached values higher than those measured in platelets from HD. Common flow cytometric measure of total thiol content in platelets from a representative HD and a representative RA patient (RA\T0 and RA\F.U.) are in Fig. ?Fig.11F. Open in a separate windows Physique 1 Oxidative stress and mitochondria in platelets from RA patients. Flow cytometry evaluation of: (A) Percentage of platelets with hyperpolarized mitochondria membrane (HMM); (C) Levels of hydrogen peroxide (H2O2); and (E) Levels of total thiol content. The numbers refers to mean SD of 45 women with RA, baseline (RA\T0) and six months after DMARDs treatment (RA\F.U.), and 25 age\ and gender\matched healthy donors (HD). In B, common flow cytometric measure of HMM in platelets from HD (left panel), RA\T0 (middle) and RA\F.U typical subjects. In D, common flow cytometric measure of H2O2 levels in platelets from HD and RA (RA\T0 and RA\F.U.) representative patients. In F, common flow cytometric measure of total thiol content in platelets from a representative HD and a representative RA patient (RA\T0 and RA\F.U.). For each patient, flow cytometry analysis was conducted in triplicate. Bar graph MSH4 showing the results obtained in three impartial experiments and reported as mean SD of the median fluorescence intensity. The Student’s test was performed. In order to determine platelet activation by mitochondrial hyperpolarization and H2O2 generation, phosphatidylserine (PS) externalization, surface P\selectin and activation of integrin GP IIb3 were measured by flow cytometry. PS is one of the four major phospholipids distributed symmetrically in the bilayer of cell plasma membrane and is normally confined to the membranes inner leaflet. When uncovered on the outer membrane surface of activated platelets, PS mediates platelet pro\coagulant function and regulates platelet life span 17. PS causes coagulation and thrombosis by providing a suitable surface L-NIO dihydrochloride for assembly of the pro\thrombinase complex,.
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