In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a 2 dimer by cleaving the Factor XIII activation peptide segment at the Arg 37 -Gly 38 peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K m , k cat , and k cat /K m were determined for thrombin hydrolysis of fibrinogen A␣-(7-20), Factor XIII activation peptide-(28 -41), and Factor XIII activation peptide-(28 -41) with a Val 34 to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28 -41) exhibits a 10-fold lower k cat /K m value than fibrinogen A␣-(7-20). With the Factor XIII V34L mutation, decreases in K m and increases in k cat produce a 6-fold increase in k cat /K m relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.Fibrinogen is composed of three chains A␣, B␤, and ␥ arranged into the dimer (A␣B␤␥) 2 . In blood coagulation, the serine protease thrombin cleaves the N-terminal portions of the A␣ and B␤ chains. For the A␣ chain, cleavage occurs at the Arg 16 -Gly 17 peptide bond and fibrinopeptide A (FpA) 1 is released; whereas, for the B␤ chain, cleavage occurs at the Arg 14 -Gly 15 peptide bond and fibrinopeptide B (FpB) is released. Removal of the fibrinopeptides leads to exposure of fibrin polymerization sites that react to form an insoluble blood clot (reviewed in Ref. 1).Activated Factor XIII helps stabilize this clot structure by catalyzing the formation of intermolecular ␥-glutamyl-⑀-lysine cross-links in the fibrin network and in fibrin-enzyme complexes. Factor XIII is a member of a family of enzymes known as transglutaminases that have a catalytic triad, similar to cysteine proteases, composed of amino acids Cys 314 , His 373, and Asp 396 . In plasma, Factor XIII is expressed as a zymogen of the form a 2 b 2 . In the presence of thrombin and calcium, the a 2 unit is released and activated. By contrast, platelet Factor XIII is expressed as the zymogen a 2 unit (reviewed in Ref. 2).The Factor XIII a 2 dimer contains in the N-terminal portion of each monomer a sequence known as the activation peptide (3, 4). Each activation peptide segment crosses the dimer interface and extends over the catalytic site of the opposing Factor XIII a subunit. Cleavage of the activation peptide segments by thrombin at the Arg 37 -Gly 38 peptide bond aids in exposure of the Fact...

Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen gamma chain exists as the highly anionic gamma' variant (408VRPEHPAETEY(S)DSLY(S)PEDDL427). This segment has been demonstrated to target thrombin ABE-II and can accommodate sites of phosphorylation in place of sulfonation without sacrificing binding affinity. The present work employed 1D and 2D solution NMR to characterize the structural features of the bound gamma' peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the gamma' residues 414-427 make significant contact with the enzyme, a beta-turn exists between residues 422-425 in the presence of thrombin, and there is a large cluster of through-space interactions involving residues 418-422. Effective contact with ABE-II requires the presence of at least one phosphotyrosine residue with Y(P)422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the gamma' peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the gamma' peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the gamma' peptide also protects other regions of thrombin from deuterium exchange. Affected regions include segments of ABE-I, the autolysis loop, the edge of the active site region, and the A-chain. Finally, thrombin forms a ternary complex with the gamma' peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.