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.

Background: Thrombin utilizes active site and anion-binding exosites (ABE) to fulfill diverse roles. Results: ABE I ligands PAR3(44 -56), PAR1(49 -62), and Hirudin(54 -65) exert different effects on thrombin exosite-active site and exosite-exosite interactions. More consequences occur with added PPACK and ABE II ligands. Conclusion: Thrombin employs ligands to transmit unique events across the enzyme. Significance: Ligand binding may be tailored to therapeutically regulate multifunctional thrombin.

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge is lacking on changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I. Such changes are transient and failed to be captured by crystallography. Therefore, we employed NMR titrations to monitor development of ABE I using peptides based on Protease Activated Receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44–56) and weaker binding PAR3G (44–56) could already interact with pro-ABE I on prothrombin. 1H-15N Heteronuclear Single Quantum Coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.

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Factor XIII can be activated proteolytically by thrombin cleavage of the activation peptide or non-proteolytically by exposure to 50 mM Ca2+. The resultant transglutaminase cross-links Q and K residues within the noncovalently associated fibrin clot. Hydrogen deuterium exchange coupled with MALDI-TOF MS demonstrated that FXIII activation protects regions within the beta sandwich (98-104) and the beta barrel 1 (526-546) from deuterium, while exposing the potential Q substrate recognition site (220-230) to deuteration (Turner, B. T., Jr., and Maurer, M. C. (2002) Biochemistry 41, 7947-7954). Chemical modification indicated the availability of several residues upon activation including K73, K221, C314, and C409 (Turner, B. T., Jr., Sabo, T. M., Wilding, D., and Maurer, M. C. (2004) Biochemistry 43, 9755-9765). In the current work, activations of FXIII by IIa and by Ca2+ as well as FXIIIa inhibition by the K9 DON peptide (with the Q isostere 6-diazo-5-oxo-norleucine) and iodoacetamide were further examined. New findings unique for FXIIIaIIa included alkylation of C238 and C327, acetylation of K68, and increased proteolysis of 207-214. By contrast, FXIIIaCa led to increased proteolysis of 73-85 and 104-125 and to a loss of K129 acetylation. The FXIIIa inhibitors K9 DON and iodoacetamide both promoted even greater protection from deuteration for the beta sandwich (98-104) and beta barrel 1 (526-546). Interestingly, only K9 DON was able to block modification of catalytic core C409 near the dimer interface. The solution based approaches reveal that activation and inhibition lead to local and long range effects to FXIII(a) and that many are influenced by Ca2+ binding. Important glimpses are being provided on FXIIIa allostery and the presence of putative FXIIIa exosites.