The effect of the neurotoxin lipopolysaccharide on the coagulability and red blood cell ultrastructure of blood from healthy individuals

Abstract

Lipopolysaccharide (LPS) is an endotoxin, which is found on the outer membrane of gram negative bacteria and is shed off during infections. This membrane contains a rich network of endotoxins that may circulate in the blood. Chronic inflammatory diseases are accompanied by hypercoagulable phenotypes. It is known that inflammation is typical initiated by pro-inflamatory cytokines but the origin of inflammation is still unclear. The study?s hypothesises is the LPS molecules from dormant microbes are resuscitated and bring about inflammatory changes seen in chronic inflammatory diseases, by changing red blood cells (RBCs) ultrastructure affecting coagulations. The following objectives direct the study: to investigate the effects of LPS on the ultrastructure plasma (fibrin networks) and RBCs, using scanning electron microscope (SEM) a high resolution CrioCrossbeam 540 from ZEISS; to investigate the effect of LPS on RBCs shape and morphology using light microscope (LM); to investigate possible damages to RBCs? elasticity using the atomic force microscope (AFM). For this investigation 30 healthy individuals were used, each person served as their own control. A miniature concentration of LPS (0.2 ng.L-1) was added. To study coagulation parameters blood was drawn into citrate tubes and aliquoted into two separate tubes one tube LPS was added to whole blood (WB) and PPP. To study the clotting properties of blood, Thromboelestograph (TEG) was conducted with and without added LPS. From TEG results it was observed that the initiation time of a clot in minutes and the kinetics of the clot decreased with the addition of LPS in both whole blood and platelets poor plasma. It was observed that when LPS added to blood an increase in clots strength was evident in treated blood compared to untreated blood. There was an increase in the angle of the clot given with variable strength size of the clot, making it difficult for the clot to be dissolved. Clot hypercoagulability and increased clot strength are common characteristics found in inflammation. From this investigation it was found that LPS changed the fibrin fibres forming matted mass deposits affecting coagulation. When viewing with SEM it was seen that LPS causes agglutination of plasma proteins on RBCs? surface changing the ultrastructure. The elasticity of RBCs decreased according to AFM results justified the shape changes of RBCs that have been exposed to LPS. These are also principal characteristics of hypercoagulation. Based on these results it was concluded that LPS could in part be the cause of physiological changes seen in inflammatory conditions where an underlying bacterial component is present.