Essentials Roles of the two thrombin receptors in platelet signaling are poorly understood. Computational systems biology modeling was used together with continuous flow cytometry. Dual‐receptor system has wide‐range sensitivity to thrombin and optimal response dynamics. Procoagulant platelet formation is determined by donor‐specific activities of the two receptors. Summary Background Activation of human platelets with thrombin proceeds via two protease‐activated receptors (PARs), PAR1 and PAR4, that have identical main intracellular signaling responses. Although there is evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear. Methods We developed a multicompartmental stochastic computational systems biology model of dual‐receptor thrombin signaling in platelets to gain insight into the mechanisms and roles of PAR1 and PAR4 functioning. Experiments employing continuous flow cytometry of washed human platelets were used to validate the model and test its predictions. Activity of PAR receptors in donors was evaluated by mRNA measurement and by polymorphism sequencing. Results Although PAR1 activation produced rapid and short‐lived response, signaling via PAR4 developed slowly and propagated in time. Response of the dual‐receptor system was both rapid and prolonged in time. Inclusion of PAR1/PAR4 heterodimer formation promoted PAR4 signaling in the medium range of thrombin concentration (about 10 nm), with little contribution at high and low thrombin. Different dynamics and dose‐dependence of procoagulant platelet formation in healthy donors was associated with individual variations in PAR1 and PAR4 activities and particularly by the Ala120Thr polymorphism in the F2RL3 gene encoding PAR4. Conclusions The dual‐receptor combination is critical to produce a response combining three critical advantages: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease‐activated receptors do not allow combination of all three in a single receptor.

Aim: to investigate clinical properties of course and outcomes of infective endocarditis (IE) depending on source of infection, to find predictors of mortality in a Moscow general hospital. Materials and methods . We included in this study 176 patients with definite and possible infective endocarditis (the Duke criteria), admitted in our hospital in 2010–2017. Patients were divided in three groups according to source of infection. All patients underwent standard clinical and laboratory assessment, echocardiography, blood culture test combined with blood PCR with sequencing. Inhospital and 1­year outcome were evaluated. Results. Among 176 patients with IE 65.3 % were men (median age 57 [35–72] years), most patients (n=149, 84.7 %) had native valve IE. Etiological factor was identified in 127 (72.2 %) cases. Gram­positive infective agents prevailed (54 %). Surgery in active phase of the disease was performed in 30 (17 %) patients. Among patients with healthcare­associated IE (n=76, 43.9 %) prevailed those older than 60 years, with high Charlson comorbidity index, with culture­negative IE, and complicated clinical course (mainly progressing heart failure). Patients with intravenous drug use associated IE (n=50, 28.4 %) had low Charlson index, association with hepatitis C viral infection, involvement of tricuspid valve with big vegetations, high frequency of embolic complications, and low inhospital mortality. Group of patients with community acquired IE (n=50, 28.4 %) more often had uncommon causative microorganisms, and had better long­term outcome. In­hospital mortality was 30.1 % (n=53) mostly due to sepsis with multi­organ failure, and heart failure. Risk factors of inhospital death were history of cardiovascular diseases, old age, kidney damage, methicillin­resistant Staphylococcus aureus (MRSA) infection, uncontrolled infection, and embolic events. Risk factors of 1­year mortality were history of stroke, and heart failure as IE complication. Independent predictors of in­hospital death were MRSA infection (odds ratio [OR] 50.32, 95 % confidence interval [CI] 1.66–213.92; p=0.002), persistent infection (OR 18.6, 95 %CI 5.37–64.40; p=0.001), duration of fever >7 days after initiation of antibacterial therapy (OR 13.41, 95 %CI 3.51–51.24; p=0.001); and of death during first year – history of cerebral infarction (OR 4.39, 95 %CI 1.32–14.70; p=0.016)), and heart failure as IE complication (OR 8.1, 95 %CI 1.97–67.09; p=0.016). Among patients subjected to surgery there were no fatal outcomes during 1 year after hospital discharge, while among conservatively treated patients were 21 (14.4 %) deaths (p<0.009). Conclusion. Main clinical features of IE course in patients urgently admitted to a general hospital was dominance of healthcare­associated IE among patients, who were older than 60 years with severe comorbidities. These patients had more complications and worse outcome. Modeling of prognosis identified uncontrolled infection as key factor of unfavorable outcome. Surgery significantly reduced long­term mortality.

The aim of the study was to investigate the relationship between established clinical systemic biomarkers of ageing and the development of age-associated diseases and senescent cell biomarkers at tissue and cellular levels. Thirty-eight patients (mean age 70 ± 4.9 years) who were assessed for traditional risk factors for cardiovascular diseases were included. From all patients we obtained biomaterials (peripheral blood, skin, subcutaneous fatty tissue) and isolated different cell types (peripheral blood mononuclear cells (PBMC), fibroblasts (FB) and mesenchymal stem/stromal cells (MSC)). Isolated cells were analyzed using several senescent cells biomarkers such as telomere length and telomerase activity, proliferation rate, cell cycle inhibitor expression (p16 and p21), b-galactosidase activity, gH2AX expression. CD34+ cell content in peripheral blood was determined by flow cytometry. Systemic senescent cell-associated factors (insulin-like growth factor 1 (IGF-1), fibroblast growth factor 21 (FGF-21), osteoprogerin, ferritin, soluble vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule 1 (ICAM-1)) in peripheral blood as well as senescence-associated secretory phenotype (SASP) components in MSC and FB secretome were evaluated by ELISA. Skin and adipose tissue biopsy samples were analyzed histologically to assess senescent cell markers. A strong significant association of tissue p16 expression with age (r = 0.600, p < 0.001), pulse wave velocity (PWV) (r = 0.394, p = 0.015), vascular cell adhesion molecule (VCAM-1) content (r = 0.312, p = 0.006) in the systemic blood stream and p16 mRNA level in the blood mononuclear cells (MNCs) (r = 0.380, p = 0.046) were confirmed by correlation analysis. Statistically significant correlations were found with indicators of FBs and MSCs proliferation in culture and acquisition of SASP by the cells. Thus, p16 expression in tissues correlated significantly with interleukin-6 (IL-6) (r = 0.485, p < 0.05) and monocyte chemoattractant protein type 1 (MCP-1) (r = 0.372, p < 0.05) secretion by isolated cells. The results of regression analysis confirmed that, regardless of age, the expression of p16 was associated with the proliferation of isolated cells and IL-6 within SASP. Based on these findings, two models have been proposed to predict the level of p16 expression in tissues from the levels of other markers of senescent cell accumulation determined by non-invasive methods and available in clinical practice.