Identification and characterisation of novel plasma clot components

Plasma clot structure/function is a major determinant in cardiovascular disease risk and severity. Plasma proteins are incorporated into plasma clots via binding and factor XIII-dependent cross-linking, with complement C3 and factor H previously identified as plasma clot components using proteomics. The aim of this current project was to validate the role of C3 and factor H in fibrin structure and function and to establish a proteomics method for the identification of novel factor XIII substrates. C3 did not affect fibrin structure; however C3 induced a concentration-dependent prolongation of fibrinolysis. C3 was cross-linked to fibrin within purified and plasma clots and bound to plasma clot components. C3 was a substrate for plasmin, with cleavage occurring in the presence and absence of fibrin. C3 also influenced angiostatin production and t-PA and plasminogen interactions within fibrin clots to prevent plasminogen cleavage and plasmin generation. All of these interactions were found to influence fibrinolysis. Whereas factor H was confirmed to be a plasma clot component, was associated with inflammation and fibrin structure and function but was not associated with complement activation in individuals at risk of cardiovascular disease. Further in vitro analyses found that factor H did not affect fibrin structure or fibrinolysis. Factor H was not cross-linked to fibrin in purified and plasma clots, but did form homodimers in the presence and absence of fibrin and factor H was a substrate for thrombin and plasmin, with cleavage occurring within fibrin clots. The proteomic techniques were established for the identification of factor XIII substrates however no novel proteins were identified using these methods, suggesting the sensitivity of the technique may be insufficient to detect novel proteins. This study has added to the growing body of evidence which suggests complement and coagulation pathways interact for the purposes of preventing blood loss and pathogen invasion.