Drug Evaluation Inbal Recombinant factor XIII (rFXIII) 5 Drug Evaluation Recombinant factor XIII (rFXIII): a novel treatment of congenital factor XIII deficiency Clin. Invest. (Lond.) Factor XIII (FXIII) is composed of two catalytic A subunits and two carrier B subunits. Aida Inbal Following activation by thrombin FXIII becomes plasma transglutaminase, which cross- Thrombosis & Hemostasis Unit, Rabin links the γ-glutamyl-ɛ-lysine residues of fibrin chains stabilizing fibrin clot. Congenital Medical Center, Petach Tikva & Sackler Faculty of Medicine, Tel Aviv University, deficiency of factor XIII results in a severe life-long hemorrhagic disorder, abnormal Tel Aviv, Israel wound healing in about 30% of patients and recurrent abortions. Most of the FXIII Tel.: +972 3 937 7912 deficiency patients have mutations in the F13A gene. Only few mutations in F13B Fax: +972 3 920 1568 gene have been published. Plasma-derived concentrate of factor XIII used to be the [email protected] treatment of choice. Recently, recombinant FXIII concentrate has been developed and tested in multinational clinical studies. This new product appears to be safe and appropriate for life-long prophylactic treatment of patients with FXIII A deficiency. Keywords: congenital FXIII deficiency • recombinant FXIII concentrate Factor XIII molecular structure units is substantially higher than previously & function thought, confirming that 99% of plasma Factor XIII (FXIII) is part of the family of FXIII-A2 is in complex with FXIII-B2. protransglutaminases and plays a crucial role FXIII is activated by thrombin in the pres- in the final step of clot formation. It circu- ence of calcium in the final stage of the blood 10.4155/CLI.14.120 lates in plasma at an average concentration of coagulation cascade. Thrombin hydrolyzes 22 μg/ml and has a half-life of 9–14 days [1] . the R37-G38 bond in each A subunit, releas- Plasma FXIII molecule comprise an Mr ing a 37-amino acid activation peptide at the ∼340,000 heterotetramer composed of two N terminus (A’); calcium (Ca++) induces con- A and two B noncovalently linked subunits formational changes that lead to dissociation (FXIII-A B ). The A subunit (FXIII-A , Mr of the B subunits. Both steps are enhanced 2 2 2 2 ∼82,000) is synthesized in megakaryocytes by fibrin polymers [9]. As a result, the enzy- and packed into platelets during their for- matically active Cys314 residue on the trun- mation. An intracellular form (cFXIII-A2) cated A dimer (G38-FXIII-A2*) is exposed, has also been identified in monocytes, tissue allowing for cross-linking the peptide-bound 2015 macrophages/histiocytes, liver, chondrocytes glutamine residue (γ-glutamyl) and adjacent and osteoblasts [2–6]. The B subunit (FXIII- peptide-bound lysine residue (∈-lysine) of the B2, Mr ∼76,500) is synthesized in the liver fibrin α and γ chains. This creates an insol- and secreted as a dimer into plasma [7]. The uble fibrin network, preventing clot degrada- A subunit contains the catalytic domain, and tion [10] . FXIII also cross-links antiplasmin the B subunit serves as the carrier and regula- to fibrin to strengthen the clot [11] . tory protein. Their assembly probably occurs X-ray crystallography studies have pro- in the circulation and is functionally essen- vided further insight into the structure of tial, because in the absence of the B subunit, FXIII and its relationship to other cysteine the A subunit is spontaneously activated and proteinases. Researchers showed that the cleared. Recently, Katona et al. [8] determined A subunit may be further divided into four that the affinity between the A and B sub- sequential domains, namely, β-sandwich part of 10.4155/CLI.14.120 © 2015 Future Science Ltd Clin. Invest. (Lond.) (2015) 5(2), 137–143 ISSN 2041-6792 137 Drug Evaluation Inbal (Glu43 – Phe184), catalytic core (Asn185 – Arg515), causing FXIII-A deficiency, 50% are missense muta- barrel 1 (Ser516 – Thr628) and barrel 2 (Ile629 – tions and the rest are deletions/insertions, splice site Arg727). Cys314 is located in a catalytic triad with and nonsense mutations. His373 and Asp396, forming one hydrogen bond Clinical manifestations include umbilical stump with His373, while the other nitrogen atom forms a bleeding during the first few days of life, postoperative hydrogen bond with Asp396 [12] . bleeding and intracranial hemorrhage – more frequent The noncatalytic FXIII-B subunit regulates the in FXIII deficiency than in other inherited bleeding calcium-dependent activation of FXIII and stabilizes disorders – in addition to ecchymoses, hematomas and the A subunit [13] by protecting it from proteolysis, prolonged bleeding following trauma [28]. Bleeding into thereby prolonging its half-life [14] . It is composed of the joint spaces or muscles also occurs, but less often ten homologous consensus or ‘sushi’ repeats [15], each than in hemophilia. About 30% of FXIII-deficient approximately 60 amino acids long and containing patients have delayed wound healing [25]. Habitual four disulfide bonds, Cys1–4 (with Cys1 linked to pregnancy loss has been reported in affected women Cys3 and Cys2 linked to Cys4). and FXIIIA-deficient rats, perhaps due to intrauterine The gene for FXIII-A is located on chromosome bleeding [29]. The reported impaired formation of the 6p24-p25 [16,17]. It spans 177 kb and contains 15 cytotrophoblastic shell, perhaps due to deficient fibrin/ exons. Exon I comprises a 5′ noncoding region; exon II fibronectin cross-linking at the implantation site, may encodes the activation peptide (amino acids 1–37) [17] . lead to placental detachment [30]. The gene for FXIII-B has been localized to chromo- some 1q31-q32.1 [18]. It spans 28 kb and contains 12 Diagnosis of FXIII deficiency exons. Exon I encodes the leader sequence; exons II-XI In patients with suspected FXIII deficiency, the usual each encode a single ‘sush’ repeat [18]. coagulation tests (prothrombin time, activated par- There is an increasing body of evidence to sup- tial thromboplastin time, thrombin time) are normal, port the multiple functions of FXIII beyond its role so clinicians need to test directly for plasma FXIII-A in hemostasis. These functions include: angiogen- and FXIII-B levels. In patients deficient in FXIII-A, esis through downregulation of thrombospondin1 this subunit is absent or low in plasma and platelets. (TSP-1) [19]; bacterial immobilization through cross- In patients deficient in FXIII-B, the FXIII-A level linking of bacterial surface proteins to fibrin within a is decreased by 5–40% in plasma and normal in plasma clot [20]; wound healing through cross-linking platelets [31] . of extracellular matrix proteins and enhancement of Traditionally, screening for FXIII deficiency was monocyte and fibroblast proliferation, migration[21] performed by incubating the patient’s plasma with and phagocytosis [22]. Although at present, the main thrombin and calcium and testing the formed clot for indication for FXIII treatment is in FXIII deficiency, increased solubility in 5 M urea, diluted monochlo- its administration in a series of clinical conditions roacetic acid or acetic acid. However, this approach including bacterial infections and wound healing may is feasible only in cases of severe FXIII deficiency provide a potential clinical benefit. (<0.5–2% activity) and can be false positive in patients Furthermore, researchers have identified several with fibrinogen deficiency. Furthermore, solubil- other substrates for activated FXIII including factor V, ity tests are poorly standardized, and their sensitivity plasminogen activator inhibitor-2, collagen, thrombo- depends on the features and concentrations of the solu- spondin, von Willebrand factor, vinculin, vitronectin, bilizing agent as well as the concentration of fibrino- fibronectin, actin, myosin and lipoprotein(a) [23]. gen. They are not recommended for diagnosis of Fac- tor XIII deficiency. TEG is not standardized and is not Congenital FXIII deficiency recommended as well. Therefore, the solubility tests Congenital FXIII deficiency is a rare autosomal reces- are being largely replaced by quantitative photometric sive bleeding disorder. It was first described 54 years FXIII activity assays that measure the incorporation ago in a Swiss boy with bleeding diathesis and impaired of fluorescent or radioactive amines by proteins [32]. wound healing [24] and has since been reported in more Yet this method too has an important disadvantage, than 500 patients [25]. because the commercial assays are inaccurate at FXIII Most congenital FXIII deficiency is caused by activity levels between 0 and 10% of normal, poten- FXIII-A subunit deficiency; this occurs at a fre- tially leading to misdiagnosis of severe deficiency. To quency of approximately 1 in 2 million [26]. Congeni- counter this problem, researchers recommend to sub- tal deficiency of the FXIII-B subunit, which has been tract a plasma blank [33]. For this reason, more sensi- reported in only 11 patients, is a rare cause of clinically tive photometric assays with a detection limit of greater significant FXIII deficiency[27] . Among the mutations than 0.6% are being developed (Reanal-Ker, Buda- 138 Clin. Invest. (Lond.) (2015) 5(2) future science group Recombinant factor XIII (rFXIII) Drug Evaluation pest, Hungary), in addition to specific ELISA tests of and hepatitis A. It also does not destroy prions, and there FXIII-A, FXIII-B and FXIII-A2B2 antigen levels [34]. is currently no screening test for blood donors for the prion that causes Creutzfeldt-Jakob disease. Therefore, Treatment of FXIII deficiency given that affected patients require life-long treatment To prevent the severe bleeding associated with con- purified recombinant FXIII concentrate has a substan- genital FXIII deficiency, affected patients require tial advantage with regard to patients’ safety. The USA monthly replacement therapy. Prophylactic treatment price for NovoThirteen (called Tretten in the USA) is has yielded highly satisfactory results owing to the 13.3 US$/IU. The USA price for Corifact is 8.6$/IU [41] . long half-life of FXIII (9–14 days) and the need for A new recombinant FXIII (rFXIII) concentrate only small quantities (∼5%) for effective hemostasis.