A Modified Thromboelastometry Assay Enables Rapid, Real-Time Evaluation of Complement-Driven Immunothrombosis

Introduction Complement and coagulation are tightly interconnected systems that contribute to immunothrombosis and can drive inflammatory or thrombotic diseases. Leveraging this relationship and crosstalk we developed a method to functionally evaluate complement-induced coagulation activity using thromboelastometry (thermoelastometry of complement-driven immunothrombosis; TCDI). Methods To study the complement-dependent activation of coagulation, platelet-poor plasma (PPP) from patients was mixed with healthy blood in the presence or absence of the compstatin-based C3 inhibitor Cp40. PPP from healthy controls (n=10), or from patients with antiphospholipid syndrome (APS; n=6), severe COVID-19 (n=13), rheumatoid arthritis (RA; n=7), or synovial fluid (SF) from RA patients, were analyzed for their capacity to induce complement activation in healthy blood. Whole blood coagulation was analyzed by thromboelastometry and complement-driven immunothrombosis was quantified as clotting time (CT) prolongation following Cp40 treatment, expressed as fractional difference percentage (FD%). In parallel, C3a generation was measured by ELISA to monitor the C3 inhibitory activity of Cp40. Results Plasma from patients with APS and COVID-19 induced significant CT prolongation following C3 inhibition by Cp40 and increased FD% values compared with controls, indicating active complement-driven immunothrombosis. Higher TCDI levels were associated with mortality in severe COVID-19. In RA, TCDI positivity was detected in synovial fluid (SF) rather than peripheral plasma. Moreover, TCDI-positive samples treated with Cp40 exhibited significant inhibition of C3a generation, which strongly correlated with FD% values (r=0.67, p=0.0005). Conclusion The TCDI assay may provide a rapid, real-time evaluation of immunothrombotic activity in inflammatory and thrombotic disorders, which could inform timely medical prevention.