Factor XIII Activation Peptide Research Articles

Factor XIII Activation Peptide Residues Play Important Roles in Stability, Activation, and Transglutaminase Activity.

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca2+ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca2+ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37-G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233-425, glutamine-substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A2 stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.

OC 28.5 Exploring Interdomain Interactions Mediated by the Factor XIII Activation Peptide

OC 28.5 Exploring Interdomain Interactions Mediated by the Factor XIII Activation Peptide

Factor XIII activation and substrate crosslinking can be influenced by amino acid substitutions from transglutaminase-2.

Factor XIII (FXIII) catalyzes formation of covalent crosslinks between reactive glutamines and lysines in plasma and cellular substrates. Related enzyme Transglutaminase 2 (TG2) also catalyzes crosslinking of cellular substrates, some common with FXIII. FXIII-A2 can be activated proteolytically to FXIII-A∗ by thrombin cleavage of the FXIII activation peptide (AP) and low mM CaCl2. FXIII-A2 can also be activated non-proteolytically by elevated Ca2+ levels (FXIII-A°) with up to three calcium binding sites Ca1, 2, and 3 filled. TG2 is allosterically regulated by Ca2+ and GTP. Kanchan et al (2013) reported that TG2 activity could be increased with a G224V substitution that improves Ca2+ binding. For the current project, two TG2 residues (V224 and N231) located near Ca3 were introduced into comparable positions of FXIII-A2. FXIII WT, G262V, K269N, and (G262V, K269N) were screened for their abilities to catalyze crosslinking of monodansylcadaverine (MDC, lysine mimic) into Fbg αC (233-425, glutamine substrate). For FXIII-A° generated with 100 mM CaCl2, MDC crosslinking ranked as FXIII G262V < FXIII WT ≪ FXIII K269N ≈ FXIII (G262V, K269N). FXIII V262 is proposed to impair a helical segment near Ca3 thus hindering the benefits of effective Ca2+ binding. FXIII K269N with its neutral N269 is hypothesized to enhance the lysine substrate binding surface near Ca3. Comparable crosslinkings occurred for FXIII-A∗ WT and FXIII-A∗ G262V likely due to presence of low mM Ca2+ that would primarily target Ca1 binding. The extent of MDC crosslinking ranked as FXIII-A∗ (WT ≈ G262V) ≪ (G262V, K269N) < K269N. For both FXIII-A∗ and A°, the K269N position in the vicinity of Ca3 and the lysine binding surface generated the greatest improvements in transglutaminase activity. Overall, TG2 substitutions were successfully used to tune properties of FXIII.

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.

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations.

Coagulation factor XIII activation peptide and subunit levels in patients with acute ischaemic stroke: A pilot study

IntroductionWe have recently shown that FXIII activation peptide (AP-FXIII) can be measured in plasma. The objective of this pilot study was to investigate for the first time if AP-FXIII can be detected in plasma from patients with acute ischaemic stroke. Materials and methodsWe included 66 patients with acute ischaemic stroke admitted between 1 and 7hours after the onset of clinical symptoms. We collected plasma samples upon admission and on the two following days and measured AP-FXIII and subunit levels by ELISA. Clinical stroke severity was assessed by NIHSS stroke score. ResultsAP-FXIII could be detected in 34 patients upon admission (range 0.2-26.3ng/ml), on day 1 in 15 patients (0.2-10.4ng/ml), and on day 2 in 11 patients (0.1-15.1ng/ml. AP-FXIII was higher in patients with severe stroke. Lower AP-FXIII levels upon admission were associated with clinical improvement. FXIII-A and FXIII-B subunit levels decreased significantly from day 0 to day 1. ConclusionsFor the first time, we detected AP-FXIII in patients upon an acute thrombotic event. The decrease in FXIII subunit levels during acute ischaemic stroke is evidence for ongoing coagulation activity and FXIII consumption. Our results suggest that FXIII activation and concomitant AP-FXIII release might be associated with an unfavourable short-term clinical outcome. Larger studies are needed to further investigate whether AP-FXIII might serve as a diagnostic and/or prognostic marker for acute thrombotic diseases.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

AbstractCoagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

Effects of Introducing Fibrinogen Aα 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.

Employing Mutants to Study Thrombin Residues Responsible for Factor XIII Activation Peptide Recognition: A Kinetic Study

In the last stages of coagulation, thrombin helps to activate Factor XIII. The resultant transglutaminase introduces covalent cross-links into fibrin thus promoting clot stability. To better understand the roles of individual thrombin residues in recognition and hydrolysis of the Factor XIII activation peptide, mutations within thrombin's aryl and apolar binding site were explored. The thrombin mutants W215A, E217A, W215A/E217A, L99A, and I174A were examined through HPLC kinetics against the substrates FXIII (28-41) V34 AP and FXIII (28-41) V34L AP. Several mutants responded differently to FXIII (28-41) V34 AP vs the cardioprotective V34L AP. W215 provides an important platform for binding and directing FXIII APs for proper hydrolysis. Loss of this platform leads to decreases in kinetics, particularly to the kcat of FXIII V34L AP. E217 also plays a supporting role, but the E217A mutation is not as detrimental as W215A. W215A/E217A is unfavorable for both activation peptides and its coupling effect has been characterized. This mutant can readily bind the peptides but cannot orient them for effective hydrolysis. Kinetic studies with I174A indicate that this thrombin residue is more crucial for interactions with the larger V34L AP segment. The L99A mutation causes deleterious effects to binding and hydrolysis of both APs. The V34L, however, is able to partially compensate for the loss perhaps by increasing contact within the aryl and apolar sites. Understanding how specific FXIII and thrombin residues participate in binding and control hydrolysis may lead to the design of coagulation enzymes whose degree of activation and optimal target site can be controlled.

Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.

Probing thrombin's ability to accommodate a V34F substitution within the factor XIII activation peptide segment (28-41).

In blood coagulation, thrombin helps to activate factor XIII (FXIII) by cleaving the activation peptide (AP) at the R37-G38 peptide bond. The common polymorphism V34L yields a FXIII that is more easily activated than the wild type enzyme. Peptides based on the FXIII (28-41) (28TVELQGVVPRGVNL41) sequence serve as an important model system to evaluate the substrate specificity of thrombin and thus how to regulate FXIII activation. Our previous kinetic and nuclear magnetic resonance (NMR) studies have suggested that the P4-P1 amino acids on this FXIII segment provide key anchors to the thrombin active site surface. Furthermore, the most effective amino acid to have at the P4 position is a leucine. In the current work, a peptide containing V34F was examined to probe the ability to accommodate an aromatic residue at this position. Kinetic parameters for thrombin-catalyzed hydrolysis of FXIII AP (28-41) V34F are comparable with that of the wild type V34. One-dimensional proton line-broadening studies reveal that the 34FVPR37 segment encompassing the P4-P1 positions makes the most contact with the thrombin surface. Two-dimensional transferred-nuclear overhauser effect spectroscopy (NOESY) studies indicate that when the peptide is bound to thrombin, the F34 aromatic ring is oriented to promote P4-P2 interactions with P36. This characteristic has been viewed as a hallmark for V34L. An ability to generate this interaction may promote the ability of FXIII AP (28-41) V34F to remain a viable substrate for thrombin.

V34I and V34A substitutions within the factor XIII activation peptide segment (28-41) affect interactions with the thrombin active site

In the blood coagulation cascade, thrombin helps activate Factor XIII by cleaving the Factor XIII Activation Peptide at the R37-G38 peptide bond. The common polymorphism V34L yields a Factor XIII that is more easily activated than the wildtype enzyme. Peptides based on the Factor XIII (28-41) ((28)TVELQGVVPRGVNL(41)) sequence serve as an important model system for evaluating how to regulate thrombin activation of Factor XIII and subsequently fibrin clot character. Kinetic and NMR (1D proton line broadening and 2D transferred NOESY) studies have revealed that the P(4)-P(1) region of the activation peptides is critical for binding to the thrombin surface. These results have led to an interest in exploring two new mutations at the P(4) position including V34I and V34A. The V34I peptide was found to have the lowest K(m) of the peptides studied. However, unlike the V34L peptide, there are no P(4) to P(2) interactions observed in the transferred NOESY spectrum. The Leu thus promotes a bound conformation that cannot be achieved with the similar amino acid Ile. The V34A peptide exhibited a decrease in K(m) but also a substantial decrease in k(cat). With this smaller amino acid, 1D proton line broadening NMR studies indicate that further contact of the Q32 residue with the thrombin surface is now possible. From these studies, valuable kinetic and structural information is being obtained to characterize interactions between thrombin and the Factor XIII activation peptide.

Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation.

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.

Interaction of the Factor XIII Activation Peptide with α-Thrombin: CRYSTAL STRUCTURE OF ITS ENZYME-SUBSTRATE ANALOG COMPLEX

The serine protease thrombin proteolytically activates blood coagulation factor XIII by cleavage at residue Arg(37); factor XIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot. The 2.0-A resolution x-ray crystal structure of human alpha-thrombin bound to the factor XIII-(28-37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the factor XIII activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. A comparison of this structure with the crystal structure of fibrinopeptide A complexed with thrombin highlights several important determinants of thrombin substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28-37) and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding site of thrombin determine the substrate conformation further away from the catalytic residues. In the case of factor XIII-(28-37), the aryl binding site is shared by hydrophobic residues P4 (Val(34)) and P9 (Val(29)). A bulkier residue in either of these sites may alter the substrate peptide conformation.

The Role of γA/γ′ Fibrinogen in Plasma Factor XIII Activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

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 Aalpha-(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 Aalpha-(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.