Identification of Factor XIII ß-Sandwich Residues Mediating Glutamine Substrate Binding and Activation Peptide Cleavage.

BACKGROUND: Factor XIII (FXIII) forms covalent crosslinks across plasma and cellular substrates and has roles in hemostasis, wound healing, and bone metabolism. FXIII activity is implicated in venous thromboembolism (VTE) and is a target for developing pharmaceuticals, which requires understanding FXIII - substrate interactions. Previous studies proposed the ß-sandwich domain of the FXIII A subunit (FXIII-A) exhibits substrate recognition sites. MATERIAL AND METHODS: Recombinant FXIII-A proteins (WT, K156E, F157L, R158Q/E, R171Q, and R174E) were generated to identify FXIII-A residues mediating substrate recognition. Proteolytic (FXIII-A*) and non-proteolytic (FXIII-A°) forms were analyzed for activation and crosslinking activities toward physiological substrates using SDS-PAGE and MALDI-TOF MS. RESULTS: All FXIII-A* variants displayed reduced crosslinking abilities compared to WT for Fbg αC (233 - 425), fibrin, and actin. FXIII-A* WT activity was greater than A°, suggesting the binding site is more exposed in FXIII-A*. With Fbg αC (233 - 425), FXIII-A* variants R158Q/E, R171Q, and R174E exhibited decreased activities approaching those of FXIII-A°. However, with a peptide substrate, FXIII-A* WT and variants showed similar crosslinking suggesting the recognition site is distant from the catalytic site. Surprisingly, FXIII-A R158E and R171Q displayed slower thrombin activation than WT, potentially due to loss of crucial H-bonding with neighboring activation peptide (AP) residues. CONCLUSION: In conclusion, FXIII-A residues K156, F157, R158, R171, and R174 are part of a binding site for physiological substrates [fibrin (α and γ) and actin]. Moreover, R158 and R171 control AP cleavage during thrombin activation. These investigations provide new molecular details on FXIII - substrate interactions that control crosslinking abilities.

Fbg αC 389-402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions.

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328-425 were studied by mutations to recombinant Fbg αC 233-425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233-402), 389 Stop (Fbg αC 233-388), and 328 Stop (Fbg αC 233-327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A* activities were observed for double mutants (D390A, E396A) and (W391A, E396A), relative to D390A and W391A, respectively. In contrast, cross-linking was reduced in (F394A, E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as key contributors in enhancing αC cross-linking.

Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates.

Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+. Cellular FXIII is activated nonproteolytically (FXIII-A°) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A° that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A° toward Q residues within Fbg αC (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α2-antiplasmin (α2AP), fibronectin, and Fbg αC (233-388, missing FXIII-binding site αC 389-402). Furthermore, the N-terminal α2AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233-425 WT, αC E396A, and truncated αC (233-388) further documented that FXIII-A* exerts greater benefit from the αC 389-402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII-binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are achieved.

Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A.

Factor XIIIA (FXIIIA) is a transglutaminase that cross-links intra- and extracellular protein substrates. FXIIIA is expressed as an inactive zymogen, and during blood coagulation, it is activated by removal of an activation peptide by the protease thrombin. No such proteolytic FXIIIA activation is known to occur in other tissues or the intracellular form of FXIIIA. For those locations, FXIIIA is assumed instead to undergo activation by Ca2+ ions. Previously, we demonstrated a monomeric state for active FXIIIA. Current analytical ultracentrifugation and kinetic experiments revealed that thrombin-activated FXIIIA has a higher conformational flexibility and a stronger affinity toward glutamine substrate than does nonproteolytically activated FXIIIA. The proteolytic activation of FXIIIA was further investigated in a context of fibrin clotting. In a series of fibrin cross-linking assays and scanning electron microscopy studies of plasma clots, the activation rates of FXIIIA V34X variants were correlated with the extent of fibrin cross-linking and incorporation of nonfibrous protein into the clot. Overall, the results suggest conformational and functional differences between active FXIIIA forms, thus expanding the understanding of FXIIIA function. Those differences may serve as a basis for developing therapeutic strategies to target FXIIIA in different physiological environments. ENZYMES: Factor XIIIA ( EC 2.3.2.13).

Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366).

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389-402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233-425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233-425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233-425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.

Thrombin Exosite Maturation and Ligand Binding at ABE II Help Stabilize PAR-Binding Competent Conformation at ABE I.

Thrombin, derived from zymogen prothrombin (ProT), is a serine protease involved in procoagulation, anticoagulation, and platelet activation. Thrombin's actions are regulated through anion-binding exosites I and II (ABE I and ABE II) that undergo maturation during activation. Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. However, the conformational basis behind such long-range communication and the resultant ligand binding affinities are not well understood. Protease activated receptors (PARs), involved in platelet activation and aggregation, are known to target thrombin ABE I. Unexpectedly, PAR3 (44-56) can already bind to pro-ABE I of ProT. Nuclear magnetic resonance (NMR) ligand-enzyme titrations were used to characterize how individual PAR1 (49-62) residues interact with pro-ABE I and mature ABE I. 1D proton line broadening studies demonstrated that binding affinities for native PAR1P (49-62, P54) and for the weak binding variant PAR1G (49-62, P54G) increased as ProT was converted to mature thrombin. 1H,15N-HSQC titrations revealed that PAR1G residues K51, E53, F55, D58, and E60 exhibited less affinity to pro-ABE I than comparable residues in PAR3G (44-56, P51G). Individual PAR1G residues then displayed tighter binding upon exosite maturation. Long-range communication between thrombin exosites was examined by saturating ABE II with phosphorylated GpIbα (269-282, 3Yp) and monitoring the binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual-ligand environment supporting the presence of interexosite allostery. Exosite maturation and beneficial long-range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.

Proline 36 of the Factor XIII Activation Peptide Plays a Crucial Role in Substrate Recognition and Zymogen Activation.

The activation peptide of blood coagulation factor XIII (AP-FXIII) has important functions in stabilizing the FXIII-A2 dimer and regulating FXIII activation. Contributions of many of its 37 amino acids to these functions have been described. However, the role of proline 36, which is adjacent to the thrombin cleavage site at Arg37, has not yet been studied in detail. We approached this question when we came across a patient with congenital FXIII deficiency in whom we detected a novel Pro36Ser mutation. We expressed the mutant FXIII-A Pro36Ser protein in Chinese hamster ovary cells and found that this mutation does not influence FXIII-A expression but significantly inhibits proteolytic activation by thrombin. The enzymatic transglutaminase activity is not affected as it can be induced in the presence of high Ca2+ concentrations. We performed nuclear magnetic resonance analysis to investigate AP-FXIII-thrombin interactions, which showed that the mutant Ser36 peptide binds less well to the thrombin surface than the native Pro36 peptide. The Arg37 at the P1 position still makes strong interactions with the active site cleft but the P4-P2 residues (34VVS36) appear to be less well positioned to contact the neighbouring thrombin active site region. In conclusion, we have characterized a novel mutation in AP-FXIII representing only the fourth case of the rare FXIII-A type II deficiency. This case served as a perfect in vivo model to shed light on the crucial role of Pro36 in the proteolytic activation of FXIII-A. Our results contribute to the understanding of structure-function relationship in FXIII.

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I.

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 of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (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 more weakly 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 the 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.

Activation of factor XIII is accompanied by a change in oligomerization state.

Factor XIII A (FXIIIA) is a member of the transglutaminase enzyme family that cross-links both intra- and extracellular protein substrates. To prevent undesired cross-linking, FXIIIA is expressed as an inactive zymogen and exists intracellularly as an A2 homodimer. In plasma, FXIII A2 is complexed with two protective factor XIII B subunits (A2 B2 ) that dissociate upon activation of the zymogen. Based on limited experimental data, activated FXIII was considered a dimer of two catalytically active A subunits. However, accumulating but indirect evidence has suggested activation may lead to a monomeric state instead. In the present study, we employed analytical ultracentrifugation (AUC) to directly explore the oligomerization state of zymogen as well as active FXIIIA in solution. We first confirmed that the zymogen was a FXIIIA2 dimer. When we activated FXIIIA nonproteolytically (by high mm Ca2+ ), the protein dissociated to monomers. More importantly, FXIIIA incubation with its physiological partner, the protease thrombin, led to a monomeric state as well. AUC studies of partially cleaved FXIIIA further suggested that thrombin cleavage of a single activation peptide in a zymogen dimer is sufficient to weaken intersubunit interactions, initiating the transition to monomer. The enzymatic activity of the thrombin-cleaved species was higher than nonproteolytically activated enzyme, suggesting that displacement of the activation peptide renders the FXIIIA more accessible to substrates. Thus, results provide evidence that FXIII undergoes a change in oligomerization state as part of the activation process, and emphasize the role of the activation peptide in preventing FXIIIA catalytic activity. ENZYMES: Factor XIIIA (EC2.3.2.13).

Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture.

In blood coagulation, thrombin converts fibrinogen into fibrin monomers that polymerize into a clot network. Thrombin also activates Factor XIII by cleaving the R37-G38 peptide bond of the Activation Peptide (AP) segment. The resultant transglutaminase introduces covalent crosslinks into the fibrin clot. A strategy to modify clot architecture would be to design FXIII AP sequences that are easier or more difficult to be thrombin-cleaved thus controlling initiation of crosslinking. To aid in this design process, FXIII V34X (28-41) Activation Peptides were kinetically ranked for cleavage by wild-type thrombin and several anticoagulant mutants. Thrombin-catalyzed hydrolysis of aromatic FXIII F34, W34, and Y34 APs was compared with V34 and L34. Cardioprotective FXIII L34 remained the variant most readily cleaved by wild-type thrombin. The potent anticoagulant thrombins W215A and W215A/E217A (missing a key substrate platform for binding fibrinogen) were best able to hydrolyze FXIII F34 and W34 APs. Thrombin I174A and L99A could effectively accommodate FXIII W34 and Y34 APs yielding kinetic parameters comparable to FXIII AP L34 with wild-type thrombin. None of the aromatic FXIII V34X APs could be hydrolyzed by thrombin Y60aA. FXIII F34 and W34 are promising candidates for FXIII - anticoagulant thrombin systems that could permit FXIII-catalyzed crosslinking in the presence of reduced fibrin formation. By contrast, FXIII Y34 with thrombin (Y60aA or W215A/E217A) could help assure that both fibrin clot formation and protein crosslinking are hindered. Regulating the activation of FXIII is predicted to be a strategy for helping to control fibrin clot architecture and its neighboring environments.

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII.

Factor XIIIa (FXIIIa) introduces covalent γ-glutamyl-ε-lysyl crosslinks into the blood clot network. These crosslinks involve both the γ and α chains of fibrin. The C-terminal portion of the fibrin α chain extends into the αC region (210-610). Crosslinks within this region help generate a stiffer clot, which is more resistant to fibrinolysis. Fibrinogen αC (233-425) contains a binding site for FXIIIa and three glutamines Q237, Q328, and Q366 that each participate in physiological crosslinking reactions. Although these glutamines were previously identified, their reactivities toward FXIIIa have not been ranked. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) methods were thus used to directly characterize these three glutamines and probe for sources of FXIIIa substrate specificity. Glycine ethyl ester (GEE) and ammonium chloride served as replacements for lysine. Mass spectrometry and 2D heteronuclear single quantum coherence NMR revealed that Q237 is rapidly crosslinked first by FXIIIa followed by Q366 and Q328. Both (15)NH4Cl and (15)N-GEE could be crosslinked to the three glutamines in αC (233-425) with a similar order of reactivity as observed with the MALDI-TOF mass spectrometry assay. NMR studies using the single αC mutants Q237N, Q328N, and Q366N demonstrated that no glutamine is dependent on another to react first in the series. Moreover, the remaining two glutamines of each mutant were both still reactive. Further characterization of Q237, Q328, and Q366 is important because they are located in a fibrinogen region susceptible to physiological truncations and mutation. The current results suggest that these glutamines play distinct roles in fibrin crosslinking and clot architecture.

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay.

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character, and the P-2 and P-3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Ligand binding to anion-binding exosites regulates conformational properties of thrombin.

Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exosite-exosite interactions. PAR3(44-56) and PAR1(49-62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54-65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. D-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54-65) was bound to ABE I, it was still possible to bind GpIbα(269-286) or fibrinogen γ'(410-427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligand-specific local and long range conformational events is proposed to help regulate this multifunctional enzyme.

Liberating R169 promotes anticoagulant protein C.

In this issue of Blood, Pozzi et al demonstrate that removing an anionic cage promotes exposure of R169 thereby generating a protein C (PC) that is far more readily activated.

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants.

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28-41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its K(m) upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.

Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS.

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A(2) can be activated proteolytically with thrombin and low mM Ca(2+) or nonproteolytically with high monovalent/divalent cations along with low mM Ca(2+). Physiologically, FXIII A(2) is poised to respond to transient influxes of Ca(2+) in a Na(+) containing environment. A successful strategy to monitor FXIII conformational events is hydrogen-deuterium exchange (HDX) coupled with mass spectrometry. FXIII A(2) was examined in the presence of different cations (Ca(2+), Mg(2+), Ba(2+), Cu(2+), Na(+), TMAC(+), and EDA(2+)) ranging from 1 to 2mM, physiological Ca(2+) concentration, to 50-500mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1mM Ca(2+) for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1mM Ca(2+) is insufficient for FXIII activation. The metals 1mM Mg(2+), 1mM Ba(2+), and 1mM Cu(2+) each led to conformational changes, many in the same FXIII regions as Ca(2+). FXIII could also be activated nonproteolytically with 500mM tetramethylammonium chloride (TMAC(+)) and 500mM ethylenediamine (EDA(2+)), both with 2mM Ca(2+). These different HDX studies help reveal the first FXIII segments that respond to physiological Ca(2+) levels.

Effects of introducing fibrinogen Aalpha character into the factor XIII activation peptide segment.

The formation of a blood clot involves the interplay of thrombin, fibrinogen, and Factor XIII. Thrombin cleaves fibrinopeptides A and B from the N-termini of the fibrinogen Aalpha and Bbeta chains. Fibrin monomers are generated that then polymerize into a noncovalently associated network. By hydrolyzing the Factor XIII activation peptide segment at the R37-G38 peptide bond, thrombin assists in activating the transglutaminase FXIIIa that incorporates cross-links into the fibrin clot. In this work, the kinetic effects of introducing fibrinogen Aalpha character into the FXIII AP segment were examined. Approximately 25% of fibrinogen Aalpha is phosphorylated at Ser3, producing a segment with improved binding to thrombin. FXIII AP ((22)AEDDL(26)) has sequence properties in common with Fbg Aalpha ((1)ADSpGE(5)). Kinetic benefits to FXIII AP cleavage were explored by extending FXIII AP (28-41) to FXIII AP (22-41) and examining peptides with D24, D24S, D24Sp, and D24Sp P27G. These modifications did not provide the same kinetic advantages that were observed with Fbg Aalpha (1-20) S3p. Such results further emphasize that FXIII AP derives most of its substrate specificity from the P(9)-P(1) segment. To enhance the kinetic properties of FXIII AP (28-41), we introduced substitutions at the P(9), P(4), and P(3) positions. Studies reveal that FXIII AP (28-41) V29F, V34G, V35G exhibits kinetic improvements that are comparable to those of FXIII AP V29F, V34L and approach those of Fbg Aalpha (7-20). Selective changes to the FXIII AP segment sequence may be used to design FXIII species that can be activated more or less readily.

Biophysical investigation of GpIbalpha binding to thrombin anion binding exosite II.

Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbalpha/beta-GpIX-GpV membrane-bound receptor complex. IIa's ABE-II is proposed to interact with an anionic portion of GpIbalpha which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa's ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)'s gamma' peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434-7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbalpha residues D274-E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbalpha (269-286) and gamma' (410-427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54-65), the data imply that GpIbalpha (269-286), GpIbalpha (1-290), and gamma' (410-427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbalpha and gamma' target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features.

Perturbations in factor XIII resulting from activation and inhibition examined by solution based methods and detected by MALDI-TOF MS.

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.