ИСТИНА

At the cellular level, the blood flow regulation is mostly conducted via red blood cell (RBC) spontaneous aggregation and shear forced disaggregation. The mechanisms of RBC aggregation are presumed to be connected with protein content in blood plasma. However so far they are not completely understood as, in the experiments, the roles of different proteins often seem controversial [1]. In this work, we aimed for assessing the roles of albumin and fibrinogen affecting RBC aggregation in plasma with optical techniques. Holographic multiple laser tweezer (LT) allows for effective measuring of the interaction forces between RBCs on the single cell level [2]. The aggregating force (FA), the force leading to overlapping of two adjacent RBCs, was measured by comparing it with the minimum optical trapping force required for holding the trapped cells from aggregate formation. The RBC interaction at disaggregation was estimated by measuring the probability of cells separation with LT – a ratio of successfully dispersed RBC doublets to the total number of separation attempts at defined trapping force. Force measurements were performed in diluted suspensions of RBCs (~0.05%) in plasma with varied concentrations of fibrinogen and albumin. Diffuse light scattering technique is based on measuring the intensity of light scattered from a whole blood sample. Further analysis of the obtained data allows for estimating the RBC interaction via the aggregometry parameters. In this work, the aggregation index (A.I., %), aggregation time (T1/2, s) and critical shear stress (CSS, mPa) were measured using the aggregometer RheoScan AnD-300. Blood samples were prepared by mixing the RBCs with platelet poor plasma at 40% hematocrit level with EDTA as anti-coagulant agent. FA measurements showed that increasing the albumin concentration from 40 to 80 mg/ml led to the significant growth of the aggregating force at 4 – 8 mg/ml fibrinogen concentration. For fibrinogen concentration of 2 - 6 mg/ml the same growth of albumin concentration led to a decrease in the separation probability. However at 8 mg/ml fibrinogen concentration the separation probability was increasing with addition of albumin. Aggregometry measurements showed A.I. and T1/2 highly depended on fibrinogen concentration and were less sensitive to a possible synergetic effect of albumin. CSS measurements showed that at 2 – 4 mg/ml of fibrinogen concentration adding albumin either didn’t affect or led to non-significant growth of the RBC interaction strength. But at high fibrinogen concentrations (>4 mg/ml) CSS was decreasing when albumin concentration changed from 40 to 80 mg/ml indicating that RBCs aggregates became less strong. We conclude that fibrinogen and albumin have a complex synergetic effect on RBC aggregation in plasma. CSS and disaggregation forces measurements showed correlation revealing a change in albumin role in RBC aggregate strength regulation from agonist to inhibitor. Possible underlying mechanisms of the observed synergetic effects could be due to a change in the RBC membrane potential or formation of albumin-fibrinogen complexes preventing strong RBC aggregation. These ideas require further studies. [1] Lee K., Kinnunen M., Danilina A.V., Ustinov V.D., Shin S., Meglinski I., Priezzhev A.V, Characterization at the individual cell level and in whole blood samples of shear stress preventing red blood cells aggregation, Journal of Biomechanics, vol. 49(7), pp. 1021-1026 (2016). [2] Lee K., Wagner C., Priezzhev A.V., Assessment of the “cross-bridge”-induced interaction of red