The effect of aspirin and lipopolysaccharide binding protein on hypercoagulability induced by lipopolysaccharide

Abstract

Inflammation is a well-known underlying cause in many diseases. Chronic inflammation has a role in a host of common and often deadly diseases, including Alzheimer’s disease, rheumatoid arthritis, cancer, type 2 diabetes, heart disease, and even conditions like depression. A highly potent inflammagen might be one of the drivers of systemic inflammation, when present in circulation and it is the bacterial component called Lipopolysaccharide. It is known that lipopolysaccharide is located on the outer membrane of gram-negative bacteria. Recently, research has shown that this bacterial (and other) membrane components, when in circulation, might have a direct effect on blood hypercoagulability, which is a prominent hallmark of chronic, systemic inflammation. In this study it is shown that Lipopolysaccharide caused marked changes in the nature of the fibrin network formed. Furthermore, irregular red blood cells’ membrane, platelets activation and formation of microparticles were seen when examining with scanning electron microscope and confocal microscope. Addition of Lipopolysaccharide caused significant hypercoagulation and changes to viscoelasticity of whole blood, as noted using Thromboelastograph. Airyscan confocal microscopy was used to study abnormal fibrin(ogen) protein folding in the presence of Lipopolysaccharide, using a well-known amyloid protein marker of anomalous blood protein clotting, called Thioflavin T. Previously, it was shown that this anomalous clotting of fibrin(ogen) fibres is amyloid in nature. Mopping agents are needed to reverse the hypercoagulation induced by Lipopolysaccharide. Biochemical agents (mopping agents) that will prevent anomalous blood clotting are therefore needed to reverse the hypercoagulation induced by Lipopolysaccharide. Such agents could, in future be used to prevent anomalous clotting in inflammatory diseases, where increased Lipopolysaccharide levels are known to be present. The first Lipopolysaccharide mopping agent of choice is Lipopolysaccharide binding protein. The Lipopolysaccharide binding protein is a serum molecule that arbitrates cellular activation in reaction to endotoxin by ensuring the delivery of Lipopolysaccharide to either soluble or membrane bound forms of CD14 also known as mCD14. The second Lipopolysaccharide mopping agent in this study, is aspirin. Aspirin is an anticoagulant and commonly used anti-inflammatory agent. In literature aspirin has been found to increase fibrin clot porosity and susceptibility to lysis. Lipopolysaccharide binding protein, and the combination with aspirin, and the effects on anomalous blood clotting, is novel, and has not previously been studied. The research questions that this thesis thus aims to address is, at what minimal concentration can the mopping agent Lipopolysaccharide binding protein be used to effectively “mop up” Lipopolysaccharide, and can Lipopolysaccharide binding protein be used in combination with Aspirin, an anticoagulant agent, to reduce the signs of hypercoagulation produced by Lipopolysaccharide ex vivo? Which techniques can be used to visually see the difference in structure and elastic function of fibrin(ogen) as well as cells in the coagulation system, namely red blood cells and platelets? The results shown here reveal that Lipopolysaccharide binding protein can reduce the effect of Lipopolysaccharides on fibrin(ogen) and cells of the coagulation system at an exposure concentration of 2ng.L-1. The coefficient of variation from Airyscan confocal between control and treatments indicated a decrease in the intensity of samples treated with Lipopolysaccharide binding protein mixed with Lipopolysaccharide and a decrease in intensity from whole blood treated with aspirin compared to control (p=0.0106). The coefficient of variation calculated from Scanning electron microscopy, showed a distinct alteration in the clots from samples treated with Lipopolysaccharide binding protein and aspirin alone and mixed together gave (more variation of light coming from fibres, neatly branched forming three layers) as significant difference, when aspirin was mixed with Lipopolysaccharide showed less variation light from fibres fussed into one layer with the contact surface and the same with aspirin mixed with Lipopolysaccharide (less light variation) compared to the control (untreated PPP) (p<0.0001). From the Thromboelastography® results there was a statistically significant differences in clot strength between whole blood and whole blood mixed with Lipopolysaccharide binding protein and Lipopolysaccharide, (p=0.0412). This shows a decrease in platelet and/or fibrin(ogen) interaction resulting in a less dense clot that is less rigid in fibrinogen or platelets. The time from clot initiation to maximum clot formation decreased when whole blood was mixed with aspirin together with Lipopolysaccharide binding protein and Lipopolysaccharide as compared with naïve whole blood, (p=0.0071). This usually occurs in hypercoagulation seen by decreased time from clot initiation to maximum clot formation. The amount of thrombus total resistance decreased significantly when whole blood was mixed with aspirin and Lipopolysaccharide verses whole blood alone, (p=0.0078). This shows that the addition of aspirin to Lipopolysaccharide can lead up to hypocoagulable state were the clot strength decreases. When measuring fibre thickness it was found that Lipopolysaccharide binding protein alone and in combination with aspirin effectively reduced the large fibres produced by Lipopolysaccharide, almost back to normal size which is +-110nm. The difference was statistically significant (p<0.05). Lipopolysaccharide binding protein and aspirin showed impeccable results reducing the signs of Lipopolysaccharide-induced hypercoagulation, both in plasma and whole blood. Many non-communicable diseases have been shown to have a bacterial component. The amyloid protein created with Lipopolysaccharide, can be reversed in the blood model with the study’s novel combination of Lipopolysaccharide binding protein/aspirin could be implemented in applications for treatment of conditions where there is presence of Lipopolysaccharide particularly sepsis.