In blood coagulation, thrombin cleaves fibrinogen (A?B??)2, sites for fibrin polymerization are revealed, and fibrin clot formation begins. Thrombin-activated Factor XIII (FXIII) is responsible for catalyzing the formation of ?-glutamyl-?-lysyl crosslinks between fibrin molecules and in fibrin-enzyme complexes. Abnormal fibrinogen levels, altered fibrin clot structure, and hindered FXIII-catalyzed crosslinking have each been associated with cardiovascular disease, arteriosclerosis, and bleeding disorders. The main objective of the research project is to examine how fibrin clot architecture can be regulated by fibrinogen (Fbg) and FXIII. Intermolecular cross- links involving the Fbg ?C region help secure lateral fibrin aggregation and lead to production of a more robust fibrin clot. Clot character can also be altered by the biochemical properties of FXIII and its mutants. A greater understanding of the molecular details associated with Fbg ?C and FXIII can be translated into new strategies to positively impact medical conditions. For the development of novel therapeutics to regulate blood clot structure, critical gaps in our knowledge must be resolved. More information is needed on 1) the kinetic and structural features of Fbg ?C, a key substrate for FXIII and 2) the role of individual FXIII A2 amino acids in controlling activation, conformation, and transglutaminase function. The proposed research for this application will thus address two hypotheses: 1) To be an effective substrate for FXIIIa, the Fbg ?C region (233-425) is hypothesized to contain distinct reactive glutamine environments and to employ a transglutaminase interaction site. Research will focus on the kinetics of the reactive glutamines and the structural features of ?C (233-425). Results will be correlated with pathophysiologic mutants within Fbg ?C. 2) FXIII A2 mutants within the activation peptide segment and the catalytic core are hypothesized to regulate transglutaminase activation, stability, and function. The FXIII A2 mutants will include the common polymorphism L34, a series of V34X mutants, and FXIII-A deficiency causative mutants associated with Y283 and R260. The biochemical methods required to address these aims include mass spectrometry, solution NMR, analytical ultracentrifugation, and measurements of fibrin clot formation and architecture. The knowledge gained from these studies will be used to improve the administration and utilization of these two key components involved in blood clot formation.
Abnormal fibrinogen levels, altered fibrin clot structure, and hindered FXIII catalyzed crosslinking have each been associated with cardiovascular disease and bleeding disorders. Using novel experimental approaches, the proposed research will provide critical information on how to control the activation and function of Factor XIII and how Fibrinogen regions are chosen for crosslinking sites. Stronger or weaker clots may be designed to handle particular medical conditions.
