In blood clotting, the enzyme thrombin cleaves fibrinogen, sites for fibrin polymerization are revealed, and fibrin clot formation begins. Activated Factor XIII is responsible for catalyzing the formation of covalent crosslinks between fibrin molecules and in fibrin-enzyme complexes. Factor XIII can be activated through cleavage of an activation peptide segment by thrombin or nonproteolytically in the presence of high calcium or high sodium/low calcium. The long-term objective of our research project is to examine in solution the structural features that govern the activation and substrate specificity of Factor XIII. Understanding these molecular details is critical considering the role of FXIII in increasing the risk of heart disease, stroke, and arteriosclerosis. The specific aims are an extension of ongoing studies and will address three hypotheses: 1) The reactive glutamines of certain FXIII substrates primarily target a distinctive surface within the transglutaminase active site region. Such studies are important since the substrate specificity of FXIII is not clearly defined, 2) FXIII undergoes subtle conformational changes upon activation. The degree and surface coverage of these changes will increase following introduction of a substrate or inhibitor, and 3) The FXIII activation peptide segment utilizes key positions to promote effective interactions with a thrombin surface whose properties are regulated by individual thrombin residues. A model system for such studies is FXIII V34L, a common polymorphism that has been correlated with protection against myocardial infarction and is more susceptible to thrombin cleavage than the native sequence. To test these hypotheses, a combination of kinetic studies, hydrogen/deuterium exchange, chemical modification methods, MALDI-TOF mass spectrometry, solution NMR, and surface plasmon resonance methods will be used. Information will be obtained on the selectivity of FXIII for glutamine-containing substrates, on the solvent accessibility changes that FXIII undergoes upon activation and ligand binding, and on the kinetic/structural features of the thrombin - FXIII Activation Peptide complex. Lay Summary: Factor XIII is a key enzyme in the formation of blood clots. While very important for wound healing, the clots can also increase risk for heart disease and stroke. Research will focus on understanding how Factor XIII is turned on and how it selects its targets. Studies may lead to novel medical strategies to control the actions of Factor XIII and the resultant clot architecture.