Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Simone Douglas - Georgia Institute of Technology
Co-Author(s): Sarah E. Lamothe, Georgia Institute of Technology, GA; Tatiyanna S. Singleton, Georgia Institute of Technology, GA; Rodney Averett, University of Georgia, GA; Manu O. Platt Georgia Institute of Technology, GA
Fibrin-based constructs are favored due to proangiogenic properties, and controllable gelation time and network geometry, making fibrin a promising scaffold for tissue engineered systems in regenerative medicine. Proteolytic activity is needed for matrix remodeling during tissue engineered construct development, however, uncontrolled proteolysis could lead to their destabilization. Tissue destructive mechanisms due to fibrin degradation should be considered, even in the absence of plasmin, the canonical fibrinolytic enzyme. Cell types in these constructs could synthesize matrix depositing non-plasmin proteases leading to destabilization due to fibrin degradation. We hypothesize that cysteine cathepsins, powerful proteases first identified in lysosomes, but now known to be secreted by several cell types in tissue destructive disease progression, can hydrolyze fibrin(ogen) and destabilize fibrin-based constructs. Identification of cathepsin-mediated fibrin degradation, an undetermined mechanism in biology, will allow for improved inhibitory design strategies to sustain stability in fibrin-based tissue engineered constructs. Fibrin gels were polymerized by combining fibrinogen and thrombin, then incubated with increasing amounts of recombinant cathepsins (cat) K, L, or S, or plasmin as a control, for 24 hours. Soluble fraction, containing released fragments, and insoluble fraction, containing fibrin gel, were collected and prepared for reduced SDS-PAGE and multiplex cathepsin zymography to assess fibrin degradation, and amounts of active cathepsins, respectively. CatK, catL, and catS all degraded fibrin as indicated by loss of the α, β or γ fibrin chain in the PAGE as protease concentration increases compared to the no enzyme control. However, we determined catL is associated with fibrin degradation products as it degrades the fibrin gel, as indicated by active catL higher molecular weight bands (37-75 kDa) which correlate with degraded α, β or γ fibrin chains. We hypothesize that fibrin acts as a reservoir for catL, which may serve to stabilize and sustain its activity in the system, allowing it to further degrade fibrin fragments in the soluble fraction. Thus, in fibrin-based constructs catL can be bound to fibrin and fibrin fragments maintaining its fibrinolytic activity, which explains a mechanism for why constructs will continue to fail. CatS only yielded a zymogram active signal at the free catS size (25kDa) and catK yielded no zymogram active signal after the incubation period. These studies demonstrate that catK, L, and S are fibrinolytic, and specifically degrade fibrin polypeptide chains, in cleavage patterns that differ from plasmin. We are investigating if catS has the greatest fibrinolytic capabilities compared to catK or L by kinetic studies, and are modifying our zymography protocol to include fibrinogen as the in situ substrate, to demonstrate directly fibrinolysis by cathepsins. This will motivate the inclusion of cathepsin regulatory and inhibitory strategies in the design of fibrin-based tissue engineered constructs, while also generating new hypotheses for abnormal cathepsin-based fibrinolysis in the fields of thrombosis and hemostasis.
SDouglas_ERN Conference Abstract.docxFunder Acknowledgement(s): Emergent Behavior of Integrated Cellular Systems (EBICS)- NSF STC ; NSF Graduate Research Fellowships
Faculty Advisor: Manu O. Platt, manu.platt@bme.gatech.edu
Role: Experimental design; all experiments, assays, and data analysis