**4. Discussion**

Erythrocyte damage caused by high shear stress generated by the rotation of the inner impeller is the main problem of VADs that cannot be ignored, and has been demonstrated to induce damage of the erythrocyte membrane, increase cellular fragility, reduce deformability, enhance aggregation and, finally, increase the risk of intervascular hemolysis and thrombosis [18]. The variation of PFH and LDH in the rheometer tests showed that high shear stress was the main factor causing red blood cell membrane injury. This result is consistent with our previous observations, and an effective interpretation of the influence of shear stress force on hemolysis [19]. Meanwhile, with the increase of exposure time in a fixed shear stress environment, the release of PFH and LDH rose gradually. This is consistent with the phenomenon reported by Shimono, that a lot of erythrocytes were suddenly ruptured in the long-term in vitro hemolysis tests [20]. The result indicated the cumulative damage of erythrocyte membrane increased with the extension of exposure time and, finally, aggravated the occurrence of hemolysis.

Increased blood viscosity, effects on the vessel wall, platelet activation, and thrombin generation, and the effects of fibrinogen and fibrin are established risk factors for thrombosis [21]. Blood consists of a large number of blood cells suspended in plasma. The total of white cells and platelets occupy less than 1% of blood volume, while RBCs are the dominant component of the blood, accounting for 40%–45% of blood volume [18]. Growing evidence suggests erythrocytes contribute to blood thrombosis because erythrocytes mediate blood rheology, interact with fibrinogen and fibrin, interact with other cells, and also support thrombin generation [22]. The rheological properties of blood are primarily governed by the concentration of RBCs, size, sharpness, deformability, intrinsic viscoelastic properties and fibrinogen-binding ability [23–25]. Therefore, the investigation of blood flow rheology and thrombus generation process directly in arterioles and its relationship with blood cell trauma under high shear stress environments is the key mechanism for research of thrombus formation. However, it is difficult to identify the mechanisms of thrombosis in clinical and epidemiologic studies, due to inaccurate identification of causation. Moreover, previous in vivo studies based on animal models, it was also difficult to interpret the effects of erythrocyte on thrombosis, due to the preparation and evaluation of the injured erythrocyte and the limitations of current observation techniques. Thus, microfluidic devices are applied to observe the dynamic movement patterns of erythrocyte. Previous microfluidic device experiments have confirmed that thrombosis can be induced by blood contact with exogenous materials and local flow conditions [26–28]. These studies depended on the parallel plate channel structure on microfluidic devices to provide similar arteriolar flow conditions in live vasculature. It is impossible to control the effect of specific shear stress on RBCs in the microchannel. Previous studies have shown that exposure to shear stress environments would induce erythrocyte membrane injury, resulting in swelling, irregular deformation, and reduce deformability. This phenomenon can also be observed directly in this research. All of these, especially when the stenosis of the blood vessel affects the blood fluid rheology, will aggravate the erythrocyte aggregation and eventually accelerate thrombosis. In this project, the erythrocyte damage in a fixed shear stress environment has been evaluated. The variations of flow patterns in bionic microchannels reflected accurately the effect of erythrocyte injury on aggregation ability and the flow state in small vessels. The captured images of bionic microfluidic device tests show that there was no significant difference for the migration rate at 0 and 25 Pa (*p* = 0.52 > 0.05). However, significant differences were found between other groups (*p* < 0.01). The results indicate that with an increase of shear stress suffered by the erythrocyte, the migration rate of damaged erythrocyte in bionic microchannels significantly decreased. Meanwhile the aggregation of erythrocyte was clearly observed in 75 and 100 Pa groups. The results indicate that mechanical shear stress caused by erythrocyte injury, which enhanced aggregation ability of erythrocytes and increased blood viscosity, resulted in decreased blood rheological performance, eventually leading to thrombus formulation and adhesion in arterioles.
