FXIII plays a critical role in supporting coagulation and fibrinolysis due to both the covalent crosslinking of fibrin polymers, rendering them resistant to plasmin lysis, and the crosslinking of fibrin to proteins of the fibrinolytic system. FXIII is a proenzyme; its active form (FXIIIa) is a transglutaminase (TG; protein-glutamine: amine γ-glutamyltransferase, EC 220.127.116.11) that forms ε(γ-glutamyl)lysyl crosslinks between two polypeptide chains [1
]. FXIIIa crosslinks fibrin γ- and α-chains into γ-chain dimers and α-chain polymers, respectively. The rapid γ-dimer formation is the result of a reciprocal intermolecular bond formation between the Lys406 of one γ-chain and the Gln398/399 residue of another aligning γ-chain. Cross-linking of the α-chains, a much slower process, occurs among multiple glutamine and lysine residues, resulting in α-oligomers and high molecular weight α-polymers. The cross-linking of α-and γ-chains confers final stability to the fibrin clot, providing strength, rigidity, and resistance for fibrinolysis [1
FXIII is a heterotetrameric proenzyme FXIII-A2B2 that consists of two single-stranded catalytic A subunits (FXIII-A2), each having a molecular weight of 83 kDa, and two identical single-stranded inhibitory/carrier B subunits (FXIII-B2), with a molecular weight of 80 kDa each [1
]. All four subunits are held together by non-covalent bonds [1
The catalytic FXIII-A subunit is a single polypeptide chain of 731 residues, including nine cysteine residues, none of which form disulfide bonds [1
]. The polypeptide chain forms five distinct structural domains: an N-terminal 37 amino acid long region on the FXIII-A subunit called the activation peptide (FXIII-AP), β-sandwich (Gly38-184Phe), the catalytic core domain (Asn185-515Arg) containing the active site (residues Cys314, His373, and Asp396) and β-barrel 1 (Ser516-628Thr), and β-barrel 2 (Ile629-731Met) [1
The non-catalytic carrier/regulatory FXIII-B subunit is spatially folded into ten sushi domains, each of which is formed by approximately 60 amino acid residues with no free cysteine groups [3
]. The structure of either sushi domain is stabilized with two disulfide bonds [1
The conversion of FXIII-A2B2 into the active form of the enzyme (EC 18.104.22.168) is a multistage process. The first stage involves thrombin-catalyzed proteolytic cleavage of the Arg37-Gly38 bond at the amino-terminus of the FXIII-A subunit leading to the release of the activation peptide [4
]. This cleavage leads to the conversion of the FXIII-A2B2 heterotetramer into the FXIII-A′2B2 heterotetramer. The second stage of activation is accompanied by the binding of calcium ions to FXIII-A′ resulting in conformational changes to trigger the dissociation of the FXIII-A′2B2 heterotetramer into the dimer, FXIII-A′2 and FXIII-B2 subunits. During the last stage, in the presence of calcium ions, FXIII-A′2 also undergoes large-scale conformational rearrangements that cause the opening up of the closed zymogenic dimeric form of FXIII-A′2 into an open form of the enzyme, FXIII-A2*, thus giving the substrate access to the catalytic site [4
]. Most recently, novel results have provided evidence that the dimeric molecule FXIII-A2* tends to split into two monomers of FXIII-A*, each one retaining transglutaminase activity [7
]. As shown by Singh et al. [8
], the transition of the zymogenic heterotetramer to active, open, monomeric FXIII-A* involves the formation of a transient FXIII-AB heterodimer in which the FXIII-A subunits are incompletely saturated with calcium and are still loosely bound to one of the FXIII-B monomers.
The potentially active FXIII subunit is also present in the cytoplasm of platelets, monocytes, monocyte-derived macrophages, dendritic cells, chondrocytes, osteoblasts, and osteocytes. In addition to its contribution to hemostasis, FXIII has multiple extra- and intracellular functions [1
Proteins are well known to be among the main targets of reactive oxygen species (ROS), which can alter a protein’s structure and functions [9
]. Currently, studies focusing on oxidative modifications of FXIII are rare in the literature [10
]. The ozone-induced oxidation of FXIII has previously been demonstrated to affect the transglutaminase activity of FXIIIa, which depends largely on the stage of the conversion of FXIII into FXIIIa, during which protein oxidation is carried out [10
]. High-resolution mass spectrometry showed that the numerous oxidation sites (including Met residues) on the catalytic subunit found upon the ozone-induced oxidation of native FXIII plasma, FXIII plasma activated solely with Ca2+
, and fully activated FXIII plasma (i.e., treated with thrombin and Ca2+
) differ significantly [11
]. These findings suggest that in the process of the proenzyme’s conversion into FXIIIa, a portion of the amino acid residues originally buried within the protein globule becomes exposed to the outside, thus becoming a vulnerable target for oxidizing agents.
Unlike ozone, another oxidizing agent, hypochlorite, which was chosen in the current study, is a physiological oxidant. Hypochlorite (produced in vivo through the reaction of hydrogen peroxide and Cl−
with aid from the enzyme myeloperoxidase released from neutrophils upon their activation) is considered to be an important pathophysiological factor in oxidative stress [13
]. Activated neutrophils can generate up to 425 μM HOCl/h in vitro [14
] and are able to damage a number of plasma proteins, such as alpha 1-protease inhibitor [15
], α1-antitrypsin [16
], human plasminogen [17
], proteinase inhibitor alpha-2-macroglobulin [18
], human serum albumin and ceruloplasmin [19
], and fibrinogen [20
However, to date, the hypochlorite-induced oxidation of coagulation factor XIII remains completely unexplored. For this purpose, by applying HPLC-MS/MS and biochemical methods, the effect of hypochlorite (HOCl/−OCI) on the chemical and spatial structure of FXIII and its enzymatic activity is studied in this paper for the first time.
The present study aims to provide evidence that (1) the FXIII proenzyme is the least vulnerable target for hypochlorite compared to its activated forms, with FXIII partly activated by Ca2+ or FXIII fully activated by Ca2+/thrombin; (2) fully activated FXIII is highly vulnerable to hypochlorite action; (3) a portion of the Met and Cys residues on the catalytic subunit is solvent-exposed, allowing them to serve as innate antioxidants; and (4) the regulatory subunits contribute to antioxidant defense of the catalytic domain; the FXIII proenzyme structure is adapted to an oxidative attack.
A comparison of the mass-spectrometry data obtained for the native and hypochlorite-oxidized samples of FXIII convincingly demonstrates that the catalytic subunit undergoes significant chemical alterations. Since, in proteins, Cys and Met are the primary targets for hypochlorite [36
], these residues in the catalytic subunit were the main residues subjected to modifications. The HPLC-MS/MS data indicate that of the 19 Met residues present in the primary structure of the protein, 14 were identified in the untreated sample of FXIII, while only five Met residues, Met136, Met247, Met380, Met679, and Met 731, were not covered. For the FXIII sample untreated with HOCl/−OCl, the 12 Met residues proved to be oxidatively damaged to varying degrees. These residues belong to the catalytic core domain (Met242, Met350, Met406, Met474, Met475, Met499, and Met512), the β-barrel 1 domain (Met520 and Met595), and the β-barrel 2 domain (Met646, Met676, and Met709). That the above methionines exhibit increased susceptibility to oxidation suggests that they are surface-exposed residues [38
]. Exposed methionine residues are oxidized with negligible effects on biological activity and serve as a pool of targets to scavenge ROS and protect functionally crucial residues [38
]. The other two non-oxidized Met residues, Met159 and Met265, are likely buried within the protein globule and become exposed to the surface during conformational rearrangements of the molecule due to its activation.
Methionine oxidation in proteins is one of the most commonly occurring oxidative modifications in proteins due to the special susceptibility of methionine to oxidative conditions. Accordingly, this observation indicates that methionine residues act like intramolecular antioxidants and protect other amino acids from oxidation. In other words, methionine residues embedded in the primary structure of plasma proteins may serve as innate ROS interceptors [39
]. The ability of exposed methionines to scavenge different forms of ROS to protect crucial amino acid residues from being oxidized was previously demonstrated for some plasma proteins [40
]. Hence, that the FXIII-A subunit is exceedingly plentiful in Met residues, many of which are likely to be located on the protein surface and could contribute to primary antioxidant defenses against oxidant-induced injury.
The results of electrophoresis and colorimetry demonstrate that the transglutaminase activity of oxidation-modified FXIIIa decreased, strongly depending both on the oxidant amount chosen and the stage of FXIII conversion into FXIIIa in which oxidation was carried out (Figure 1
and Figure 2
). The proenzyme exhibited the least vulnerability to oxidation. The proenzyme treated with 150 μM HOCl/−OCl completely retained its enzymatic activity inherent to the unaffected protein, while the FXIIIa treated with 50 μM HOCl/−OCl demonstrated drastically reduced enzymatic activity. These results are completely consistent with those obtained earlier for the ozone-induced oxidation of FXIII (Figure S1
When comparing the results of mass spectrometry with the results obtained by electrophoresis and colorimetry, none of the oxidized amino acids residues found in the proenzyme treated with HOCl/−OCl are functionally significant for the enzyme, whose activity does not decrease in this group of samples regardless of the amount of hypochlorite used. However, the degree of damage to the residues correlates with the amount of oxidizing agent.
Another interesting observation is that an increase in the degree of damage to amino acid residues belonging to one group of samples occurs not only with growth in the amount of oxidizing agent but also for different groups of samples subjected to the same amount of hypochlorite. This is clearly a consequence of the process of the conversion of the proenzyme into the enzyme, during which the catalytic FXIII-A subunit becomes an increasingly more vulnerable target for oxidation.
As shown by the mass-spectrometry data, the crucial residues, Tyr560 and Trp279, remained in their native form in all of the samples. However, since FXIIIa dramatically reduced its activity when exposed to hypochlorite, the effect was driven by damage to the amino acid residues in this group of samples (Trp130 and Cys695), as well as Tyr311 and Trp315, located in the immediate vicinity of the catalytic center of the enzyme (Figure 7
). Due to a lack of knowledge regarding the functionality of each of these residues, it can be assumed that they are endowed with certain functions, which, to one degree or another, can affect the activity of the enzyme.
Interestingly, the human α2-macroglobulin treated with hypochlorite at a molar protein/ratio close to that used in the present study, completely lost its antiproteolytic activity [40
]. Although the quaternary structures of the proteins differ, the various effects of hypochlorite on the proteins suggest a high antioxidant capacity of the FXIII proenzyme structure.
In the oxidized samples of FXIII, FXIII + Ca2+
, and FXIII + Ca2+
/Thr, some of Cys residues are also targets for the oxidant. The Cys238 residues in FXIII-A2′B2 and FXIIIa are involved in the oxidative damage only with 150 μM of the oxidant. Cys238 is believed to be the surface-exposed residue in the fully activated molecule [1
]. Nevertheless, that the residue remains unaffected by moderate oxidation contradicts this finding. Most likely, when FXIII is activated, the residue initially localized within the protein core begins to migrate to the hydrophilic region, while still being partly spatially inaccessible. The Cys423 residue is not covered in the control samples of FXIII and FXIII + Ca2+
/Thr. Since the Cys423 residue is covered and non-oxidized for the control FXIII + Ca2+
sample, this residue possibly does not undergo oxidative alterations in the control FXIII sample. Upon both mildly and strongly induced oxidation, the Cys423 residue is oxidatively modified in all the oxidized samples. Furthermore, the oxidation degree of the Cys423 residue (like Met residues) mainly trends toward a more significant modification. In many cases, the unwanted oxidation of Cys may result in oxidative damage thereby modifying protein function [44
]. As mentioned above, none of the oxidized amino acid residues found in the oxidized proenzyme are functionally significant for the enzyme. Consequently, the Cys152, Cys188, and Cys423 located on the FXIII-A subunits may serve as antioxidant residues that, together with the Met residues, are capable of protecting the catalytic protein center against oxidation.
Likely, the FXIII-B subunits also contribute to the antioxidant defense of the catalytic subunit. In their native conformations the interactions between all four subunits of FXIII are well known to provide support for the most compact, globular structure of the multimeric protein complex. As a result, the entrance to the catalytic center is closed to any substances, including small molecules [4
], such as HOCl/−OCI.
The mass-spectrometry data indicate the significant contribution of sulfur-containing residues of methionines and cystines to the total amount of oxidation sites emerging in the XIII-B2 subunits due to hypochlorite-induced oxidation of FXIII. The abundant cystine residues present in FXIII-B [3
] have high reactivity to hypochlorite [44
]. It is important to note that, like the catalytic subunit, the vulnerability of the regulatory subunit to induced oxidation increases with FXIII activation in the following order: native FXIII < FXIII + Ca2+
< FXIII + Ca2+
/Thr (Figure 6
). Additionally, in the last stage of FXIII activation, the new oxidation sites (Tyr26, Cys39, and Met94) that arise in FXIII-B2 provide insight into the conformational changes in FXIII-B2 that occur upon FXIII dissociation.
A recent report by Protopopova et al. [7
] suggests partial wrapping of the B subunits around the central core of FXIII-A2, thus additionally protecting the catalytic center from being involved in oxidation. The protective functions of the FXIII-B subunits (as antioxidants) might explain why their deficiency leads to a dramatic reduction in the FXIII-A concentration in plasma—due to its instability [4
]. Finally, as recently shown, the cell-derived FXIII composed only of the two catalytic FXIII-subunits (cFXIII), subjected to oxidation under conditions similar to those of the blood coagulation factor XIII, exhibited profound changes in the spatial structure of their proteins, which resulted in a much greater loss of transglutaminase activity compared to that of oxidized FXIIIa [47
Thus, the tight packing of both the tetrameric FXIII structure and the exposed Met and Cys residues on the FXIII-A subunits, as well as the protective function of the FXIII-B subunits, could be the main factors underlying the high tolerance of the protein to oxidizing agents.
It is worth noting that the results of the current study were obtained using FXIII alone, while in the bloodstream, FXIII circulates together with other proteins that are present in greater abundance than FXIII. In one way or another, each of the blood plasma proteins is able to intercept the ROS, which is likely to limit FXIII oxidation under healthy conditions. Most recently, it was shown in plasma samples collected from healthy volunteers that 500 HOCl/−OCl μM does not alter the enzymatic activity of FXIII [48
]. In this regard, it should be emphasized that the authors added the oxidizing agent to blood plasma in which FXIII was present in an inactive form. As shown in the current study, the proenzyme has maximum resistance to the action of hypochlorite, and it seems reasonable to suppose that only these amino acid residues could undergo oxidation (for example, Met residues), which is not vital.
However, at the inflammation site, the local HOCl/−OCl level can reach millimolar concentrations [50
]. In the blood plasma, FXIII is known to be non-covalently associated with fibrinogen [51
], which, by binding to the integrin alphaMbeta2 (Mac−1) expressed on activated leukocytes, provides a key link between thrombosis and inflammation [53
]. Therefore, the effect of neutrophil oxidants on FXIII is likely local.
For this reason, future studies should aim to identify the sites of oxidative modifications employing FXIII extracted from the blood plasma of patients suffering thrombotic disorders associated with inflammation and the production of oxidants.