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Aggregation of red blood cells (RBC) is one of important factors affecting hemorheology and microcirculation. During the last few decades it was studied intensively as a promising indicator of the hemorheological state. In the majority of cases, the mechanics of RBC interaction was studied in model solutions of neutral macromolecules, e.g. dextrans. In the plasma or plasma protein environments it was studied much less. Therefore, we focused our work on elucidating the mechanics of RBC interaction in various environments using the single cell force microscopy (laser tweezers) technique. We measured the interaction forces between pairs of RBCs in vitro in solutions of (1) fibrinogen, (2) fibrinogen and albumin, and (3) autologous plasma. The interaction force was determined as the minimum force required to fully disaggregate a pair of interacting RBCs (disaggregation force – DF). Our experimental procedure consisted of two steps. First, two single RBCs were captured by two independent optical traps and attached to each other with carefully controlled interaction area. After certain time one of the cells was slowly pulled from the other with the known (increasing step-by-step) pulling force until disaggregation. In this way, we measured the DF dependence on the interaction time and area. Measurements showed that the DF is independent of the interaction time but depends on the interaction area. In the cases of small (~20% of side surface) and large (~80% of side surface) ‘initial’ interaction areas the DF were 7±2 pN and 28±3 pN correspondingly. Surprisingly, the force required to separate two RBCs did not decrease along with the reduction of the interaction area but, on the contrary, it increased. We conclude that the mechanics of RBCs interaction in blood plasma and plasma proteins solutions are not in agreement with the depletion model but more likely are consistent with the moving cross-bridge model.