Factor XIII (fibrin-stabilizing factor or fibrinoligase) is a plasma glycoprotein that plays an important role in the final stage of blood coagulation, in the regulation of fibrinolysis, and in tissue repair.
Thrombin generated during blood coagulation converts the proenzyme (factor XIII) to an active enzyme (factor XIIIa) in the presence of calcium ions. Factor XIIIa is a transglutaminase that catalyzes the crosslinking of fibrin monomers and the crosslinking of fibrin and α2-plasmin inhibitor through the formation of intermolecular γ-glutamyl-ϵ-lysine bonds. These reactions result in a fibrin clot with mechanical strength and increased resistance to proteolytic degradation by plasmin. The crosslinking of fibronectin to fibrin or to collagen is also catalyzed by factor XIIIa, and this reaction appears to be related to wound healing.
A deficiency of factor XIII results in a severe lifelong bleeding tendency and defective wound healing in affected individuals, and spontaneous miscarriage in affected females. Congenital factor XIII deficiency is caused by the absence of either the A or B subunit. This disorder is inherited as an autosomal recessive trait, since the genes coding for the A and B subunits exist on chromosomes 6 and 1, respectively. Disease-causing defects in these two genes have been identified in a number of patients with factor XIII deficiency.
Factor XIII circulates in blood as a tetramer, A2 B2 (~320 kDa) consisting of two A subunits (75 kDa each) and two B subunits (80 kDa each).135,136 The four polypeptides are held together by noncovalent bonds.136 The carbohydrate content has been reported to be 1.5 and 8.5 percent for the A and B subunits, respectively.137,138 The A subunit has more than six free sulfhydryl groups, whereas the B subunit has no free sulfhydryl groups.136,139 Factor XIII in plasma is complexed with fibrinogen.139,140 Both the native factor XIII (A2 B2) and that potentiated by thrombin, (activated) factor XIIIa′ (A′2 B2), bind similarly to calcium ions,7 although the native factor XIII remains a heterotetramer.136,142,143 Fibrinogen and factor XIII appear to bind through the B subunit. This observation is based on the fact that placental or platelet factor XIII (A2), which does not contain B subunits, does not bind fibrinogen.144 Thus plasma factor XIII (A2 B2) circulates in blood in a complex with both fibrinogen and calcium ions.
The three-dimensional structure of factor XIII was demonstrated by an electron microscopic study. The A and B subunits appear to be a globular particle and a filamentous strand, respectively.145 The A subunit of factor XIII has been purified and characterized,137,146–148 and crystallized.146 X-ray crystallography demonstrated that the A subunit is composed of five distinct domains: an activation peptide, β-sandwich, central core, barrel 1, and barrel 2 regions149,150 (Fig. 171-4). The C-terminal portion of the A subunit corresponding to the two β-barrels forms thermostable domains, whereas three thermolabile domains are formed by the N-terminal β-sandwich and core domains.151 The core domain is composed of two subdomains where three active-site residues are located as described below. It is of interest that the activation peptide, on cleavage by thrombin, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen152; moreover, the activation peptide blocks the entrance to the catalytic cavity in the core domain.149,152
Three-dimensional structure of the A subunit for factor XIII. The A subunit is composed of five distinct domains: from the N-terminus to the C-terminus, an activation peptide (residues 1–37), β-sandwich (38-183), central core (first half, 184–332; second half, 333–513), barrel 1 (514–628), and barrel 2 (629–731) domains. The central core domain contains the active site Cys314 and two additional residues (His373 and Asp396) important for enzymatic activity. Both N- and C-termini are identified by small circles. The activation peptide blocks the entrance to the catalytic cavity in the core domain (left). On cleavage by thrombin, the activation peptide remains in the same conformation and occupies the same position with respect to the rest of the molecule, although the catalytic cavity becomes wide open to substrates (right). (Modified with permission from Yee et al.149 and Yee et al.150 )
The A subunit of factor XIII consists of 731 amino acids.153,154 The molecular weight of the polypeptide portion of the molecule was calculated to be 83,150. The B subunit is composed of 641 amino acids155 with a calculated molecular weight of 73,183. The addition of 8.5 percent carbohydrate138 gives a molecular weight of about 79,700 for each of the B subunits of human factor XIII. These molecular weights are in agreement with those estimated by SDS-polyacrylamide gel electrophoresis.135,136
The N-terminus of the A subunit is acetylated,156 whereas that of the B subunit is a free Glu residue.155,156 The A subunit contains several functional regions, including an activation peptide (37 amino acids), an active site (Cys314), putative calcium-binding sites, and a thrombin inactivation site (Fig. 171-5). The amino acid sequence around the active site (Tyr-Gly-Gln-Cys-Trp, YGQCW in Fig. 171-5) is identical to those of other transglutaminases.157,158 Two additional amino acids, His373 and Asp396, seem to be important for the catalytic activity of factor XIII.149,159,160 The N-terminal subdomain of the central core domain contains the Cys314, whereas the His373 and Asp396 are located in the C-terminal subdomain.151 The proposed Cys-His-Asp catalytic triad, which is similar in arrangement to the active site of cysteine proteinases, suggests that the catalytic mechanism of transglutaminases may be similar to the reverse mechanism of the cysteine proteinases.161 Two nonproline cis peptide bonds were identified between Arg310 and Tyr311 close to the active-site Cys314 residue, whereas Gln425 and Phe426 were located at the dimerization interface, indicating their possible importance for factor XIII function.152
Primary structure of the A subunit and placement of introns. Arrows with capital letters indicate positions of introns. The N- and C-terminal residues are numbered, as are certain other key residues: Arg at an activation cleavage site by thrombin, Cys at an active site, Lys at the second cleavage site by thrombin, etc. The N-terminal Ser residue is acetylated. Wide arrows demonstrate the sites cleaved by thrombin. (Modified with permission from Ichinose.180 )
The A subunit of factor XIII is also present in other tissues and cells such as platelets, megakaryocytes, placenta, uterus, monocytes/macrophages, etc. (see “Synthesis of Factor XIII” below). The A subunits in plasma and in other tissues are identical; the amino acid sequences of the A subunit from plasma,153,156 platelets,156 and placenta153,154,162 are indistinguishable.
The A subunit of factor XIII is highly homologous to other human transglutaminases,157,163–168 including a novel transglutaminase identified in keratinocytes.169 The degree of identity between the A subunit and other transglutaminases ranges from 35 to 45 percent. The middle portion of these proteins contains most of the identical sequences, whereas their N- and C-terminal regions are more diverse. These structural features correspond to the domain organization of the A-subunit molecule; i.e., the middle portion forms a central core homologous with the transglutaminases, and the N- and C-terminal regions form independent domains,151 which may determine substrate specificity of each transglutaminase. Another transglutaminase has been isolated from horseshoe crab hemocytes, and its primary structure is found to be most homologous to factor XIII.170 This similarity provides strong evidence that these proteins are derived from a common ancestor. Human erythrocyte membrane band 4.2 protein is also similar to the transglutaminases171,172; however, it lacks enzymatic activity because of the substitution of the active site Cys by Ala.
The B subunit contains 10 tandem repeats (Fig 171-6). These repeats each consist of about 60 amino acids, two disulfide bonds, and highly conserved Pro, Gly, Tyr, and Trp residues.155 The 10 repeats are subclassified into four groups according to the degree of identity to each other. These repeats have been called Sushi domains because of their shape163 or GP-I structures 173 because the disulfide-bond pairing was first established in human β2-glycoprotein I to be between the first and third and the second and fourth Cys residues in each repeat.174 This pattern is also found in the bovine counterpart175 and in the a chain of human C4-binding protein.176 It is likely, therefore, that a similar pairing occurs with the disulfide bonds in the Sushi domains present in other homologous proteins. Each Sushi domain is independently folded177 and is predicted to consist of exclusively antiparallel β-sheets,178 which is consistent with the results obtained experimentally by an NMR study.179 At least 30 other proteins/genes are found to contain similar Sushi domains.163,180 Nearly half these 30 proteins are involved in the complement system, and the others are involved in diverse systems such as blood coagulation, lymphocyte regulation, etc. Sushi domains are found even in proteins in invertebrates and viruses, suggesting that the genes for these proteins may have evolved from a common ancestor.
Primary structure of the B subunit and identified mutations. Arrows with capital letters indicate positions of introns. The N- and C-terminal residues of the mature protein are numbered. Residues from (−)1 to (−)20 are enclosed as a signal peptide for secretion. An Arg-Gly-Asp sequence in the tenth Sushi domain is also enclosed. Solid and open diamonds indicate potential carbohydrate attachment sites of the Asn-X-Ser/Thr/Cys and Ser/Thr-X-X-Pro types, respectively. Three mutations identified in patients with the B-subunit deficiency (former type I factor XIII deficiency) are shown in boxes. (Reprinted with permission from Ichinose et al.303 )
Near the C-terminus of the B subunit of factor XIII within the tenth Sushi domain there is an Arg-Gly-Asp (RGD) sequence that is reported to be responsible for the cell attachment of various proteins.181 It remains to be determined, however, whether this RGD sequence in the B subunit is related to its function. The amino acid sequence of the mouse B subunit is 78 percent identical to the human counterpart, although the mouse subunit contained 7 amino acid residues at the C-terminus that were not found in the human.182
The A subunit of factor XIII contains the catalytic site, whereas the B subunit is thought to protect or stabilize the A subunit183–185 or regulate activation of the zymogen.136,186 The former role is supported by a study using a mammalian expression system demonstrating that recombinant A2 B2 tetramers are more stable than recombinant A2 dimers alone.187 The B subunit also mediates the binding of factor XIII to the C-terminus of the γ′ chain of fibrinogen.144
Factor XIIIa catalyzes a γ-glutamyl-ϵ-lysine crosslinking reaction between a number of proteins. Fibrin acts as both an amino donor and an acceptor,188 whereas α2-plasmin inhibitor and fibronectin preferentially serve as amino acceptors.189,190
Crosslinking of Fibrin Monomers.
The crosslinking reaction catalyzed by factor XIIIa leads to dimerization of the γ-chains of fibrin, followed by polymerization of the α-chains of fibrin.135 The crosslinking sites between two γ-chains are Gln398 or Gln399 and Lys406188 near the C-terminal ends of the polypeptide chain. Gln328 and Gln366191 and Lys508192 are involved in the α-chain polymerization. The γ-dimerization and α-polymerization reactions result in a fibrin clot with mechanical strength and elasticity.193,194 Thus these polymerization reactions aid in hemostasis.
The formation of a γ-trimer, γ-tetramer, and αγ2-triad has been demonstrated following the γ-dimerization of both fibrin and fibrinogen.195,196 These reactions lead to an increase in the resistance of fibrin to fibrinolysis.197
Crosslinking of α2-Plasmin Inhibitor.
The crosslinking of α2-plasmin inhibitor to the α-chain of fibrin198 or fibrinogen199 by factor XIIIa occurs at a faster rate than with other proteins. Accordingly, in plasma, α2-plasmin inhibitor and fibrin are considered the best substrates for factor XIIIa.200 The crosslinking site in each substrate has been identified as Gln2 in α2-plasmin inhibitor190,201 and Lys303 in the α-chain of fibrin(ogen).202
The crosslinking of α2-plasmin inhibitor to fibrin renders the fibrin clot resistant to digestion by plasmin.203 Consequently, the crosslinked α2-plasmin inhibitor protects the hemostatic fibrin clot from premature lysis by plasmin.203,204
Crosslinking of Fibronectin.
Factor XIIIa catalyzes the crosslinking of fibronectin to the α-chain of fibrin198,205 and to collagen.206 The crosslinking site of fibronectin is reported to be Gln3 at its N-terminus.189 The crosslinking of fibronectin to fibrin or collagen may result in anchoring of fibrin clots to cells or to the structural matrix in vessel walls at the site of vascular injury. In a culture system, fibronectin is crosslinked by factor XIIIa and accumulates in fibroblast cell layers. This reaction may be important for a particular assembly process to organize and stabilize the growing extracellular matrix.207,208 Factor XIII is also reported to enhance fibroblast proliferation.209 Cultured fibroblasts migrate optimally into gels prepared with fibrinogen at a concentration of about 3 mg/ml (i.e., normal plasma fibrinogen level), and migration is greatly enhanced by extensive crosslinking of the fibrin α-chains by factor XIIIa.210 In contrast, both the number of migrating macrophages and the distance of migration were reduced when the gel matrix included fibronectin and was crosslinked by factor XIIIa.211 These reactions appear to be related to wound healing.212–214
Other Protein Substrates.
The contractile proteins actin and myosin also have been shown to be crosslinked by factor XIIIa.215–217 Since these proteins exist in platelets, they may be involved in the crosslinking of structural proteins under certain conditions.216
Factor XIII is reported to generate a monocyte chemotactic factor by crosslinking of S19 ribosomal protein.218,219 This reaction may be related to inflammation. Most recently, it has been found that many A-subunit-positive microglia were associated with primitive senile plaques in the brains of patients with Alzheimer disease, whereas few or no microglia containing A subunits were found associated with classic plaques.220 Factor XIII may play a role(s) in the early phase of Alzheimer disease, since the tau protein is reported to be crosslinked by a transglutaminase.221
Several other plasma proteins such as von Willebrand factor,222,223 thrombospondin,224 factor V,225 plasminogen, and apolipoprotein(a)226 have been reported to be crosslinked to themselves or to other substrates by factor XIIIa. The precise functions of these reactions, however, have yet to be established.
Concentrations of the A and B subunits of factor XIII in normal plasma have been determined by an enzyme-linked immunosorbent assay (ELISA) to be 11 and 21 μg/ml, respectively.227 Since virtually all the A subunit in plasma is complexed with an equimolar amount of the B subunit, about half the B subunit (10 μg/ml) exists in the “free” form in plasma.184,228,229 The free B subunit may act as a reserve by binding and stabilizing the A subunit immediately on its release from cells into circulation.
Site of Synthesis for Factor XIII.
The liver has long been thought to be the major site of synthesis for both the A and B subunits of plasma factor XIII.230,231 In addition, the biosynthesis and secretion of both subunits by the human hepatoma cell line HepG-2 have been reported.232 No cDNA clone for the A subunit, however, was obtained from liver libraries, despite extensive screening.153,233 Northern blot analyses of mRNA samples from normal liver, HepG-2 cells, and fibroblasts also showed little or no detectable hybridization signal, whereas a single mRNA species (about 4.0 kb) for the A subunit was obtained from mRNA samples from placenta,154 macrophages,234,235 and megakaryoblastoid cells (Kida and Ichinose, unpublished data). These results suggest that placenta, macrophages, and megakaryocytes do synthesize the A subunit, whereas liver does not.
Free A subunits are also present in a number of organs, tissues, and cells, such as placenta, uterus, prostate, platelets, megakaryocytes, and monocytes/macrophages.236–241 The A subunit in these cells, however, is localized in the cytoplasm by immunohistochemical and immunobiochemical methods. In particular, the amount of A subunit in platelets is equal to that in plasma,242,243 but the A subunit in platelets is present in the cytoplasm241,243 and is not secreted.242,243 An acetylated N-terminus and the absence of glycosylation and disulfide bonds are also consistent with the fact that the A subunit is a typical cytoplasmic protein. At present, the function of these intracellular forms of the A subunit is not known, although other cytosolic transglutaminases are thought to be related to apoptosis244,245 and the synthesis of a cornified envelope in the skin.246 Dermis-peculiar cells named dendrocytes were found to contain the A subunit of factor XIII.247 Since dendrocytes share several common surface epitopes with monocytes/macrophages, they are considered to be derived from bone marrow and become perivascular resident macrophages.
After bone marrow transplantation, the phenotype of the A subunit in the plasma of the recipient was replaced by that of the donor, whereas the phenotype of the B subunit remained unchanged.248 In addition, complete or partial conversion of the phenotype of the A subunit in monocytes and platelets as well as in plasma was detected in patients after bone marrow transplantation.249 Therefore, it is likely that the A subunit of plasma factor XIII is produced at least in part by hemopoietic cells.
The site of synthesis for the B subunit has been suggested to be the liver.230,231 HepG-2 and PLC/PRF/5 hepatoma cells secrete the B subunit,232 and cDNA clones coding for the B subunit have been obtained from a normal human liver library.155,233 In addition, the phenotype of a recipient’s B subunit changed to the donor’s phenotype after liver transplantation, whereas that of the A subunit remained unchanged.248 Therefore, it is clear that the liver is the major site of synthesis for the B subunit.
Mechanism of Release of the a Subunit of Factor XIII.
Like those of other transglutaminases, the 5′ end of the cDNA for the placental A subunit does not encode a typical hydrophobic leader sequence for secretion.153,154 A search for a possible preproleader sequence or internal signal has been unsuccessful, although the corresponding portions of the A-subunit gene have been examined extensively.233,250 Because the A subunit of factor XIII is known to remain in the cytoplasm of placenta,236 macrophages,239,240 megakaryocytes,238 platelets,241,243 and skin dendrocytes,247 it is very likely that the Met at position (−)1 functions as the initiator for biosynthesis. The removal of the Met by an aminopeptidase(s) would then be followed by acetylation of the N-terminal Ser residue.156
If any of the cells mentioned earlier is the major source of the A subunit of “plasma” factor XIII, there should be a unique mechanism(s) for its release into circulation, where the free B subunit can readily bind to and form an A2 B2 tetramer. Results of the expression of the A and/or B subunits in a mammalian cell system suggest that the A subunit is not secreted through the conventional secretory pathway but is released from cells after cell damage.187 There are more than 20 proteins that lack typical hydrophobic leader sequences and distinct internal hydrophobic signals but are present and function in extracellular spaces.251–255 In the case of IL-1b, two different mechanisms, a novel pathway and apoptosis, are proposed for its release from cells.256,257
The 5′ end of the cDNA and the corresponding exon in the gene for the B subunit code for a typical hydrophobic leader sequence that aids in its secretion from hepatocytes into the circulation.155,258 This is supported by the fact that the HepG-2 and PLC/PRF/5 hepatoma cells secrete the B subunit232 and by the results that the recombinant B subunit is secreted through the conventional secretory pathway in a mammalian cell system.187
Regulation of B-Subunit Levels.
Patients with congenital factor XIII deficiency lack an immunologically detectable A subunit (<1 percent of normal) and have a reduced amount (about 50 percent of normal) of the B subunit.184,259 Heterozygotes have about 50 and 80 percent of the A and B subunits, respectively.260 Furthermore, administration of the A subunit obtained from placenta increased not only the A-subunit level in plasma but also the level of the B subunit, which reached a maximum after several days and was maintained at that level for a prolonged period.231,261 An ELISA assay revealed that the plasma concentration of the complexed form of the B subunit is decreased to almost 0 percent of normal in homozygotes and about 50 percent in heterozygotes of the A-subunit deficiency, whereas that of the free B subunit is essentially the same among normal individuals, homozygotes, and heterozygotes.227 Therefore, it is likely that the concentration of the complex form of the B subunit (A2 B2) depends on the amount of the A subunit in plasma and that the concentration of the free B subunit is regulated to be constant. The increase in the A subunit caused by the infusion of exogenous A subunit may induce the synthesis of the B subunit as a response to the increased A subunit or decreased free B subunit. This response was absent in a patient whose complete B-subunit deficiency was genetic in origin.262,263
Metabolism of Factor XIII
The half-life of the A subunit of factor XIII in circulation is about 10 days.214,231,264,265 It is of interest that the half-life of placental concentrates (A2) infused into a patient with complete B-subunit deficiency was shorter than those in patients with A-subunit deficiency,262 probably because the placental A2 dimer is stabilized by immediate formation of an A2 B2 tetramer with the free B2 dimer in the plasma of patients with A-subunit deficiency.
In rabbit, factor XIIIa (the activated form) is removed from circulation faster than factor XIII (the zymogen form), probably through the reticuloendothelial system in liver.230 The retention of factor XIIIa in fibrin clots266 could be an additional mechanism for its clearance from plasma.
Activation of the Zymogen.
During the final stage of blood coagulation, thrombin converts the proenzyme (factor XIII) to a potentiated form (factor XIIIa′) by releasing an activation peptide (4 kDa) from the N-terminus of each of the A subunits135,156 (Fig 171-5). The site of cleavage by thrombin is between Arg37 and Gly38. This reaction is stimulated by fibrin monomers.267,268 The cofactor activity of fibrin I (polymerized des-A fibrinogen) has been attributed to formation of a fibrin I-factor XIII complex (Kd = 65 nM), which is potentiated by α-thrombin 80-fold more efficiently than free, uncomplexed factor XIII.269 In contrast, fibrin crosslinked by factor XIIIa in the presence of calcium ions loses its cofactor activity as γ-dimers appear.268 Thus crosslinked fibrin may function as a negative feedback to prevent further generation of factor XIIIa.
In the presence of calcium ions, the potentiated A′2 dimer dissociates from the B2 dimer136,183,186 and binds to fibrin more tightly,270 whereas the B2 dimer remains in liquid phase.266 Calcium ions bind to the A′ subunit and unmask the active site.141,183,186,271 Fibrin(ogen) lowers the calcium concentration required both for the dissociation of the A′2 and B2 dimers and for the exposure of the active site to the physiologic level (1.5 mM).270,272
Several other enzymes, including trypsin, factor Xa, elastase, and cathepsin C, have been reported to activate (or potentiate) factor XIII.135,239,273–275 Factor XIII also can be activated by high concentrations of calcium and salt.276 This reaction may be specific for the intracellular form of factor XIII, since nonproteolytic activation of the A subunit in platelets is abolished by the addition of the B subunit in a stoichiometric amount.276 Thrombin-independent activation actually may play an important role(s) in the activation of intracellular factor XIII (A2 alone), since thrombin does not exist in the cytoplasm.
Degradation of the Enzyme.
The loss of biologic activity of factor XIIIa during prolonged incubation with thrombin occurs in parallel with the generation of fragments of 56 and 24 kDa from the A subunit, whereas the molecular weight of the B subunit remains unchanged.135,277 The cleavage site for thrombin is reported to be between Lys513 and Ser514162; however, Hornyak et al. have claimed that the loss of activity does not correlate with the appearance of the cleaved fragments.278 An expression study of mutant A subunits suggests that removal of the C-terminal portion makes the molecule unstable (Ichinose, unpublished data),279,280 which is consistent with the idea that the C-terminal domains are involved in the interaction of the two A subunits.151 Degradation of factor XIII or factor XIIIa also has been reported to occur by digestion with elastase and trypsin.135,151,274
Acquired deficiencies in factor XIII and its elevated plasma levels are seen in various disease states.214,281
Chromosomal Localization and Gene Structures.
The gene for the A subunit of factor XIII is located on chromosome 6p24-p25.282 The gene encoding the keratinocyte (epidermal) transglutaminase is localized to chromosome 14q11.2–q13,283–285 whereas the gene for tissue transglutaminase is located on chromosome 20q12167 and that for prostate transglutaminase (type IV, TGM4) on chromosome 3p21.33-p22.286 Thus gene loci for the various transglutaminases are not clustered but rather are dispersed throughout the genome. The gene for the B subunit of factor XIII is localized to chromosome 1q32-q32.1.287 In contrast, the genes for several proteins containing multiple Sushi domains, such as factor H, the a and b chains of C4b-binding protein, complement receptors type I and II, membrane cofactor protein, and decay accelerating factor, are clustered at the same locus.288,289 Because of the presence of a number of genes that are homologous to either the A or B subunit of factor XIII, their chromosomal localization was reexamined and confirmed by in vitro amplification of genomic DNAs from human-hamster hybrid cell lines employing gene-specific primers.233
The genes for both the A and B subunits of factor XIII have been characterized.250,258 The gene for the A subunit spans more than 160 kb (GenBank M21987, J03834). It consists of 15 exons interrupted by 14 introns, and each functional region is encoded by a separate exon. The genomic organizations of keratinocyte, tissue, and hair follicle transglutaminases and erythrocyte membrane band 4.2 protein are nearly identical to that of the A subunit.158,284,285,290–292
The gene for the B subunit is about 28 kb in length (GenBank M64554).258 It is composed of 12 exons interrupted by 11 introns, and each of the 10 Sushi domains is encoded by a single exon. This is also true of the genes for other Sushi domain-containing proteins, with few exceptions.
Gene Regulation of the a Subunit.
Recently, the cell type-specific transcriptional regulation of the A-subunit gene has been characterized.293 Although the A subunit and other transglutaminases share significant similarity in their gene organization, their 5′-flanking nucleotide sequences differ from each other, and the mechanisms for their gene regulation seem to be diverse as well.294–297
It has been reported that the conditioned medium from type II human T-cell leukemia virus-infected T cells induces the conversion of endothelial cells to a Kaposi sarcoma cell-like phenotype and that the endothelial cells cultured in the presence of hepatocyte growth factor acquired the ability to express the A subunit of factor XIII.298,299 These findings suggest that hepatocyte growth factor and factor XIII play a role in the initiation and maintenance of Kaposi sarcoma lesions. The expression of the A subunit may be induced by other cytokines and growth factors as well.
By use of agarose gel electrophoresis, several different allelic forms of the A subunit of factor XIII were identified in the normal population.300,301 Heterogeneity in these A subunits was confirmed by both amino acid and DNA sequencing.153,154,162,250,302 All amino acid substitutions can be explained by point mutations; these include Val34/Leu, Phe204/Tyr, Leu564/Pro, Val650/Ile, and Glu651/Gln.303 Although Arg77/Gly, Arg78/Lys, and Phe88/Leu polymorphisms have been described in the literature,154,162 genetic studies failed to identify the corresponding nucleotide changes. It has been reported that the Val34/Leu polymorphism is associated with myocardial infarction.304 A mechanism whereby Leu34 is protective against myocardial infarction remains to be determined. It also has been shown by restriction fragments length polymorphism (RFLP) that extensive DNA polymorphism exists in the A-subunit gene.250,307,308 Furthermore, a short tandem repeat polymorphism exists in the 5′-flanking region in the A-subunit gene.250,307,308
Microheterogeneity of the B subunit271 has been observed in several alleles.309 Differences in amino acid sequences or DNA sequences, however, were not detected.155,258 Several nucleotide substitutions were found in the noncoding and flanking regions of the B-subunit gene.263,310,311 A short tandem repeat polymorphism also was found in the 3′-flanking region of the B-subunit gene.258,312 Most recently, several nucleotide substitutions in the B-subunit gene corresponding to the heterogeneity described earlier have been identified (Umetsu and Ichinose, unpublished data).
Hereditary Disorders of Factor XIII
Congenital Deficiency and Molecular Abnormality.
The incidence of congenital factor XIII deficiency (<1 percent of the normal activity level) is about 1 in 5 million in the United Kingdom214 and in Japan. Most patients appear to be deficient in the A subunit in plasma. The mode of inheritance is autosomal recessive,214,260 which is consistent with the A-subunit gene being on chromosome 6.
In affected individuals, the first manifestation of bleeding is usually from the umbilical cord after birth, and this occurs in approximately 90 percent of the patients.212 Intracranial hemorrhage occurs in one-fourth of patients and is the leading cause of death. Superficial bruising and hematomas in subcutaneous tissue and muscle are common, and the bleeding at these sites may recur if not treated. Patients may bleed around the joint after trauma but have much less spontaneous hemarthrosis than hemophiliacs.
Deficiency of either factor XIII212,214 or α2-plasmin inhibitor313 results in “delayed bleeding” after trauma, whereas primary hemostasis in individuals with these traits is normal. The delayed bleeding is caused by premature lysis of hemostatic clots. Because of the absence of crosslinking between α2-plasmin inhibitor and fibrin, these clots have decreased resistance to proteolytic degradation by plasmin.203,204
In addition to a lifelong bleeding tendency, abnormal wound healing in affected individuals and habitual spontaneous abortion in affected females are not rare.212,214 It is noteworthy that recurrent miscarriage also has been described in patients with congenital hypofibrinogenemia or afibrinogenemia.314,315 Moreover, abnormal wound healing and repetitive spontaneous miscarriage are reported in patients with congenital dysfibrinogenemia (reviewed in ref.316). These symptoms underscore the importance of the function of factor XIII and fibrin in vivo.
Molecular and Cellular Bases.
Factor XIII deficiency has been classified previously into two categories: type I deficiency, characterized by the lack of both the A and B subunits, and type II deficiency, characterized by the lack of the A subunit alone.317 Based on genetic analyses, a new classification for factor XIII deficiency has been proposed: a deficiency of the A subunit (former type II), a deficiency of the B subunit (former type I), and a possible combined deficiency of both A and B subunits.303
Deficiency of the a Subunit.
Mutations in the gene for the A subunit have been detected by in vitro amplification of DNA samples obtained from patients with A-subunit deficiency.233 These include a variety of missense and nonsense mutations, small deletions and insertions with or without frameshift/premature termination, splicing abnormalities, and a large deletion280,318–333 (Fig 171-7). Effects of these mutations on A-subunit biosynthesis have been confirmed in several cases. For example, in one case a deletion of 4 bp was observed in exon XI, whereas in another case Gly562 was found to have been replaced by Arg.280 The deletion in the former case leads to a premature termination at codon 464. Reverse transcription (RT) polymerase chain reaction (PCR) analysis demonstrated that the level of mRNA was greatly reduced in the former case, whereas the level of mutant mRNA expressed in the latter case was normal. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the mutations were expressed in mammalian cells, showing that the mutants were synthesized normally but disappeared rapidly, whereas the wild type remained for a prolonged period of time.280 Molecular modeling also showed that the Arg562 substitution changed the conformation of the A subunit, causing misfolding and/or destabilization of the molecule (Fig. 171-8).
Structure of the A-subunit gene and identified mutations. (Above) Exons are indicated by wide vertical bars and Roman numerals. (Center) The 5′-untranslated region (5′-UT), region coding each exon (with Roman numeral), and the 3′-noncoding region (3′-NC) of the cDNA are shown by boxes. ACT, Ca1, C, H, D, Ca2, and INA stand for the activation cleavage site, a candidate for a calcium binding site, the active site Cys314, His373, and Asp396, another candidate for a calcium-binding site, and the inactivation cleavage site, respectively. (Below) The mutations were identified in various patients with the A-subunit deficiency (former type II deficiency). Short, medium, and long vertical arrows indicate mutations with premature termination, mutations resulting in exon skipping, and missense mutations, respectively. A large deletion is depicted by a long horizontal arrow at the bottom. The mutations discussed in the text are boxed. *, premature termination; #, exon skipping; −, deletion; +, insertion; =, substitution.
(Above) Close-up model view of the native (left, Gly562) or mutant (right, Arg562) A subunit. The tertiary structure of the normal A subunit is based on x-ray diffraction analysis. The main chain of the protein is drawn as a carbon trace; domains II and III are shown by light lines and domain IV by dark lines. Side-chain groups of labeled residues are shown as ball-and-stick structures; residues belonging to domains II and III are drawn in a light color and those in domain IV in a faint color. Although the mutant Arg562 residue can be accommodated by the chemical structure, it generates unfavorable short contacts with neighboring residues. Accordingly, the substitution of a small residue by a large charged amino acid is expected to yield an unstable, misfolded structure. (Below, left) Model view for the A-subunit dimer including the normal domain II (dark line), the first half of domain III (faint line), the second half of domain III (light line) in a monomer, and the same domains II and III in the counterpart monomer. Domains IV and V are not shown in this figure. A total of 268 residues consisting of the second half of domain III and domains IV and V are removed from the molecule in the deletion mutant. The C-terminal Leu463 residue of the truncated molecule is shown in a light color. Pro383 and Asp384 residues contact with the second part of domain III, which is absent in the mutant. (Below, right) Closer view of the environment around domains II and III. Since premature termination at position 464 would lead to the loss of a C-terminal part of the core domain and the entire domains IV and V (barrel 1 and 2), the protein is expected to misfold and/or be incapable of dimer formation. (Reprinted with permission from Takahashi et al.280 )
An amino acid substitution of Arg260 by Cys has been predicted by molecular modeling and mechanics to result in instability of the A-subunit molecule.334 Rapid degradation of this mutant has been confirmed by an expression study in yeast (Maeda and Ichinose, unpublished data). Rapid degradation of a novel Tyr283 to Cys mutant also has been ascribed to its instability by characterization in an expression system employing megakaryoblastoid MEG01 cells that endogeneously synthesize the A subunit (Souri and Ichinose, unpublished data). In contrast, a previously described Ala394 to Val mutation233 appears to be a rare polymorphism, since this mutation also was detected in a normal allele of the patient’s father. Moreover, molecular modeling and mechanics predicted no significant alteration in its conformation.334
A 20-bp deletion at the boundary of exon I/intron A and an insertion of T in the invariant GT dinucleotide at the splicing donor site of exon IV/intron D also were found in a patient.335 RT-PCR analysis demonstrated that only one kind of mRNA without exon IV was detected, although its level was reduced to less than 5 percent of normal. Transcript of the other defective allele of the A-subunit gene containing the 20-bp deletion was not detected. Thus both mutations impaired normal processing of mRNA for the A subunit.
Deficiency of the B Subunit.
A rare case of complete deficiency of the B subunit of factor XIII was found in Japan and has been characterized.262 The patient, who manifests a mild bleeding tendency, has no B subunit and a significantly reduced level of the A subunit in plasma. The half-life of an infused placental concentrate (A2) in the patient was shorter than that in the plasma of patients with A-subunit deficiency; therefore, the lack of the B subunit most likely causes instability of the A subunit. Nucleotide sequence analysis of the B-subunit gene of this patient revealed that the patient is a compound heterozygote for two separate defects in the B-subunit genes. The deletion of an A at the splicing acceptor junction of the intron A/exon II boundary and the amino acid substitution of Cys430 by Phe in exon VIII (see Fig 171-6) result in abnormal splicing of pre-mRNA and the breakup of a disulfide bond in the seventh Sushi domain, respectively.263 The Cys430 to Phe mutation does not prevent the de novo synthesis of the B subunit but alters the conformation of the mutant protein sufficiently to impair its intracellular transport, resulting in its deficiency in this patient.336
Additionally, two unrelated Italian families were found to be deficient in the B subunit.265,317,337 Surprisingly, these families share the same AAC insertion mutation in exon III and identical polymorphisms in the 3′-noncoding and 3′-flanking regions of the B-subunit gene.310,311 These results suggest that this mutation reflects a founder effect, at least among Italians.
Diagnosis for Factor XIII Deficiency.
The diagnosis of a homozygote with congenital deficiency of factor XIII is based on the pattern of inheritance, clinical symptoms, and laboratory tests. In addition to the typical umbilical cord bleeding after birth, the characteristic delayed bleeding after trauma strongly suggests this disorder. Deficiencies and molecular abnormalities of α2-plasmin inhibitor and fibrinogen should be ruled out before the final diagnosis, since these disorders show symptoms similar to those described earlier.
Tests for factor XIII deficiency are based on transglutaminase activity. These include thromboelastography and, more specifically, a solubility test of the recalcified plasma clot in 5 M urea or 1% monochloroacetic acid. Visualization of a γ-dimer or α-polymers of fibrin by SDS-polyacrylamide gel electrophoresis is useful to obtain a rough estimate of the functional level of factor XIII in plasma. The transglutaminase activity of factor XIII is quantitatively measured by amine incorporation assays.338,339 A standardized method for screening of factor XIII deficiency, however, needs to be established so that patients with this disease will not be misdiagnosed or completely overlooked.
Immunologic quantitation of the A and B subunits in plasma is essential in determining which subunit is primarily deficient, although the incidence of B-subunit deficiency appears to be rather rare. Although concentrations of the A and B subunits were measured routinely by the Laurell rocket electrophoresis method,184,259 these measurements can now be determined more precisely by an ELISA.227 Laboratory coagulation tests other than factor XIII are within the normal range.
Diagnosis of a heterozygote, who usually lacks symptoms of factor XIII deficiency, can be made only by specific quantitative measurements of both the A and B subunits. Genetic diagnosis at the DNA level will help both in prenatal detection of affected patients and in determination of carrier states. It would be difficult, however, to perform genetic diagnosis for a new subject unless the type of mutation is known in at least one member of the subject’s family. This is so because mutations causing factor XIII deficiency are highly heterogeneous, as discussed earlier.
Therapy for Factor XIII Deficiency.
Both congenital and acquired factor XIII deficiencies have been treated successfully with fresh frozen plasma, cryoprecipitate, and crude factor XIII concentrates from placenta.340,341 Maintaining the level of plasma factor XIII at 10 to 20 percent of the normal level is sufficient to alleviate symptoms, since bleeding occurs frequently in patients with less than 1 percent of the normal level, and levels of 1 and 10 percent of normal are adequate for the in vitro γ-dimerization and α-polymerization of fibrin, respectively. The long half-life of factor XIII in plasma and its minimal requirement for hemostasis are beneficial both for the treatment of acute bleeding and for prophylaxis. Prophylactic infusion of factor XIII concentrates would be desirable for severe deficiency patients, who otherwise may bleed frequently. Although development of inhibitors to factor XIII following multiple infusions is rare, it must be considered when bleeding is uncontrollable by a therapeutic dosage of factor XIII. In such a case, immunosuppressive therapy may be required as well.
The cloning of human factor XIII153,154 made it possible to prepare recombinant A subunit as a therapeutic material that is free of viral contamination. The recombinant A subunit is comparable with the native A-subunit protein with respect to structural and functional properties.148,187 Thus it is a safe substitute for the placental and plasma factor XIII concentrates currently used in transfusion therapy. It also has been introduced as an essential component in fibrin sealant, which has been used widely for many types of surgery and for the treatment of trauma.