Abstract 5483: The Inflammatory Protein Complement C3 is a Novel Substrate for Factor XIII and Impairs Fibrin Clot Lysis
Introduction: Inflammation is fundamentally important in the pathogenesis of cardiovascular disease (CVD) and increasing evidence supports a functional role for complement C3. Using a functional proteomics approach we have identified zymogen and activated C3 in perfused solubilized clots, suggesting C3 influences thrombus formation.
Hypothesis: In this study we assessed the hypothesis that complement C3 interacts with coagulation and fibrinolytic factors to influence thrombosis.
Methods and Results: Analysis of 123 paired plasma and serum samples revealed that 24.8% (SD 7.6) of plasma C3 was incorporated into the clot. To explore the mechanism of incorporation we investigated binding interactions between C3, fibrin and FXIII using ELISA based binding assays and surface plasmon resonance and found that C3 bound to immobilized fibrinogen and FXIII in a concentration dependent manner. Further analysis of the interactions between C3 and FXIII demonstrated that activated FXIII (FXIIIa) incorporated a known FXIIIa substrate, 5-(biotinamido)pentylamine, into immobilized C3 in a concentration dependent manner and FXIIIa also induced the formation of high-molecular weight C3 polymers, indicating that C3 is a FXIII substrate. Furthermore, FXIIIa incorporated plasma C3 into immobilized fibrin and Western blot analysis of clots formed from purified fibrinogen and C3 indicated high molecular weight bands that cross-reacted with specific antibodies against C3 and fibrinogen, suggesting that C3 is cross-linked to fibrin alpha chains. In turbidimetric assays using purified proteins C3 prolonged fibrin clot lysis times in a concentration dependent manner and Western blot analysis of lysed clots indicated that C3 was cleaved by plasmin, suggesting impaired fibrinolysis is due to substrate competition for plasmin. Increased plasma C3 concentration was significantly associated with increased ex vivo plasma clot density and prolonged fibrinolysis times, after adjusting for fibrinogen concentrations, to support our in vitro results. In conclusion, taken together these data indicate that complement C3 influences key processes involved in fibrin formation and fibrinolysis supporting a role for C3 in cardiovascular thrombosis.