Characterizing the specificity of activated Factor XIII for glutamine-containing substrate peptides.

Activated Factor XIII (FXIIIa) is a transglutaminase that catalyzes the formation of gamma-glutamyl-epsilon-lysine crosslinks in the fibrin network. To better understand the source of FXIIIa substrate specificity, Q-containing substrates based on beta-casein, K9-peptide, and alpha(2)-antiplasmin were characterized. alpha(2)AP (1-15, Q2, Q4) and alpha(2)AP (1-15, Q2, Q4N, K12R) are highly promising peptide models since they exhibited k(cat)/K(m) values comparable to intact beta-casein. In the absence of a lysine-like donor, FXIIIa could promote deamidation of a reactive Q to an E and solution NMR served as an effective strategy for monitoring this reaction. A tendency toward deamidation allowed greater investigations of the alpha(2)-antiplasmin based peptides. FXIIIa preferentially selects the Q2 residue for carrying out crosslinking processes. The E3 and Q4 provide supporting roles in binding. When a crosslinking reaction occurs at Q2, the Q4 position is sterically blocked from reactivity. By contrast, deamidation of Q2 to E2 allows, for the first time, observation of reactivity at Q4. The K12 position provides an additional favorable site of interaction with the FXIIIa surface. The sensitivity of alpha(2)AP (1-15, Q2, Q4) to amino acid changes at Q2, Q4, and K12 suggests the importance of individual FXIIIa subsites that are controlled by chemical environment and sterics.

Establishing the inhibitory effects of bradykinin on thrombin.

Bradykinin, RPPGFSPFG, has been reported to be an inhibitor of thrombin's roles in blood clotting, platelet activation, and cellular permeability. The exact target, magnitude, and type of inhibition occurring are not well characterized. Based on the individual kinetic parameters calculated here, bradykinin is classified as a weak competitive inhibitor against hydrolysis of S-2238 and of a PAR4-like peptide. The K(m) values increased twofold in the presence of bradykinin, whereas the k(cat) values remained constant. The K(i) values ranged from 170 to 326 microM. Other biochemical studies indicated that bradykinin inhibits release of fibrinopeptide A from fibrinogen. Furthermore, bradykinin hindered the time required for fibrin clot formation. The weak inhibitions observed in vitro suggest that the direct effects of bradykinin on the thrombin active site become significant only at high concentrations, levels that may be difficult to achieve physiologically. Clearly, bradykinin can target thrombin but whether this direct interaction can be achieved in vivo and is sufficient to elicit a response without contributions from other cofactors requires further investigation.

Structural features associated with the binding of glutamine-containing peptides to Factor XIII.

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.

Protease-activated receptor 4-like peptides bind to thrombin through an optimized interaction with the enzyme active site surface.

Protease-activated receptor 4 (PAR4) is cleaved by thrombin at the R47-G48 peptide bond. Unlike PAR1, PAR4 does not contain a sequence readily predicted to interact with thrombin anion binding exosite-I. HPLC kinetic results on hydrolysis of PAR4 peptides (38-51 and 38-62) reveal that extending the sequence from the active site toward the exosite does not promote further binding interactions with thrombin. One-dimensional-proton line-broadening NMR indicates that the amino acids occupying the P(4)-P(1) positions of PAR4 (38-47), 44PAPR(47), come into direct contact with the thrombin surface. Less contact arises from the Leu43 at the P(5) position. Two-dimensional total correlation spectroscopy and two-dimensional transferred nuclear Overhauser effect spectroscropy studies on this complex reveal that Leu43 is flexible and can exhibit two conformational states. The binding mode observed for PAR4 peptides is similar to that of PAR1 peptides. PAR4 takes advantage of a distinctive sequence to optimize its interactions with the thrombin active site surface.