Congenital Factor XIII Deficiency With the Presence of Inhibitor: A Case Study.

Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor with additional roles in wound healing and interactions between the decidua and fetus. Congenital FXIII deficiency is rare bleeding disorder. Inhibitor development against FXIII in inherited FXIII deficency is also uncommon, but may cause severe, life-threatening bleeding. FXIII is the last step in the coagulation cascade with normal coagulation paramaters (PT, aPTT), the detection of inhibitor to FXIII is quite difficult. The treatment of inhibitor-positive congenital FXIII deficiency is challenging due to the lack of a role of by-pass agents such as FVII. The best known ways of treatment in these cases are the use of high-dose FXIII concentrates and immunosuppression. Herein, we report the management of postoperative bleeding diathesis in a patient with FXIII deficiency who developed inhibitors, and to follow the clinical course of the disease with FXIII concentrate and immunosuppression.

Structure-Based Design of FXIIIa-Blockers: Addressing a Transient Hydrophobic Pocket in the Active Site of FXIIIa.

Blood coagulation factor XIII (FXIII, F13) is considered to be a promising target for anticoagulants with reduced bleeding risk because of its unique position in the coagulation cascade downstream of thrombin. However, until now, no potent drug addressing FXIII has been available, indeed no compound has even entered clinical trials yet. In 2013, we published the co-crystal structure of FXIII in the active state (FXIIIa°), thereby providing a detailed map of the active site for the rational design of potent FXIIIa blockers. Here we report, for the first time, a structure-based approach to improving the affinity of FXIIIa inhibitors. FXIII was crystallized in complex with a methyl thiazole moiety to address a novel transient hydrophobic pocket close to the catalytic center. By subsequent structure-based design to rationalize the introduction of an ethyl ester, the potency of the inhibitor was improved significantly compared to that of the parent lead compound. The occupancy of the hydrophobic pocket described here might turn out to be a key step in the development of a potent reversible and orally available FXIIIa blocker.

Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation.

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model. SUMMARY: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Successful Management of a Patient with Autoimmune Hemorrhaphilia due to Anti-Factor XIII/13 Antibodies Complicated by Pulmonary Thromboembolism.

Autoimmune hemophilia-like disease (hemorrhaphilia) due to anti-factor XIII (FXIII) antibodies (AH13) is a very rare, life-threatening bleeding disorder. A 77-year-old woman developed macrohematuria and a right renal pelvic hematoma. The coagulation times were not prolonged, but FXIII activity and antigen levels were severely and moderately reduced to 9 and 29% of normal values, respectively. Accordingly, the FXIII-specific activity turned out to be low. FXIII inhibitor and anti-FXIII-A subunit autoantibodies were detected by a 1:1 crossmixing test and immunoblot and immunochromatographic assays. She was therefore diagnosed with "definite AH13" and treated with plasma-derived FXIII concentrates to arrest the hemorrhage. In addition to a highly compressed inferior vena cava by a huge renal pelvic hematoma, deep vein thrombosis (DVT) and pulmonary thromboembolism (PE) were identified by systemic computed tomography. The patient was immediately started on anticoagulation therapy with low-dose heparin. Emboli disappeared quickly, probably because under-crosslinked thrombi caused by severe FXIII deficiency are vulnerable to fibrinolysis. After about 1.5 years, anti-FXIII-A subunit autoantibodies still remained despite the use of rituximab, steroid pulse therapy, oral prednisolone, and oral cyclophosphamide treatments. In conclusion, an extremely rare AH13 case complicated by DVT and PE was successfully managed by balancing anticoagulation therapy with hemostatic therapy.

Autoimmune acquired factor XIII deficiency due to anti-factor XIII/13 antibodies: A summary of 93 patients.

Autoimmune acquired factor XIII (F13) deficiency or autoimmune hemophilia-like disease (hemorrhaphilia) resulted from the generation of anti-F13 antibodies (AH13) is a severe bleeding disorder that occurs mainly in the elderly. Although rare, the number of patients diagnosed with AH13 has recently increased. To improve understanding of this disease, the author summarized 93 ever reported/diagnosed AH13 cases. About 50% of cases were idiopathic. In the remaining half of the patients, autoimmune diseases and malignancies were the most common underlying diseases. Intramuscular and subcutaneous bleeding were the most frequently reported symptoms. Hemorrhage was the cause of death in 13 patients. In 4 patients, the diagnosis was established after hemorrhagic death. Therefore, physicians/hematologists must raise the awareness of AH13 as a life-threatening disease. Most patients were treated with F13 concentrates to arrest bleeding and with prednisolone and cyclophosphamide to eradicate anti-F13 autoantibodies. AH13 cases tend to become chronic and intractable and require close follow-up over an extended period.

(±) cis-Bisamido epoxides: A novel series of potent FXIII-A inhibitors.

A novel class of potent FXIII-A inhibitors containing a (±) cis-bisamido epoxide pharmacophore is described. The compounds display highly potent inhibition of FXIII-A (IC50 = 5-500 nM) in an in vitro assay. In contrast to other types of previously described covalent transglutaminase inhibitors, the bis-amido epoxides exhibited no measurable reactivity with glutathione, therefore possibly rendering this class of compounds suitable for future in vivo investigations. Additionally, the compounds show selective inhibition for FXIII-A against the cysteine protease, cathepsin S although they proved to have similar potency with a closely related transglutaminase, TGII, to that observed for FXIII-A.

Airway Compromise and Perioperative Management of a Patient with Acquired Factor XIII Inhibitor.

Perioral hematomas can lead to acute airway compromise and can present significant challenges in both direct and indirect approaches to airway instrumentation. In patients with normal cell counts and routine coagulation tests, spontaneous hematomas are rare, but when encountered, they elicit a limited differential diagnosis that includes von Willebrand factor deficiency, platelet dysfunction, and the acquired factor XIII (FXIII) deficiency. Although spontaneous hematoma formation resulting from FXIII inhibition has been reported, we describe what may be the first reported case of FXIII inhibitor-related hematoma leading to acute airway compromise. Successful management of this patient required multidisciplinary cooperation among anesthesiologists, intensivists, otolaryngologists, and hematologists.

Inhibitors of Factor XIII/13 in older patients.

Factor XIII/13 (FXIII or F13) is a plasma protransglutaminase, which stabilizes fibrin clots, and thus plays an important role in hemostasis. Autoimmune hemo(rrha)philia due to anti-F13 autoantibodies (AH13) has been on the rise in Japan, which has become the leading superaging society in the 21st century. The mean age of Japanese AH13 cases has risen to 70.4 years. A total of 83 AH13 cases have been diagnosed in the world as of July 2014. To raise the awareness of AH13, the author and members of the Japanese Collaborative Research Group first proposed "Criterion and Algorithm of Laboratory Tests for anti F13" in February 2012. AH13 is not just an acquired isolated defect of F13 molecule itself but a disturbance caused by autoantibodies. Accordingly, AH13 cases are diagnosed in patients with otherwise unexplained hemorrhages by a combination of a severe deficiency of F13 activity and the presence of anti-F13 autoantibodies. As patients with this disease manifest life-threatening bleeding symptoms, prompt diagnosis and proper treatment are essential. Because AH13 tends to become chronic and intractable, affected patients must be closely followed for a prolonged period.

Investigation of a link between raised levels of pepsinogen in blood as a mediator of in-vitro clot lysis in acid and a cause of abnormal factor XIII screening tests.

The in-vitro lysis of plasma clots in acetic acid generally indicates a Factor XIII deficiency that is confirmed by quantitative assay. However, there are two rare, poorly understood circumstances whereby clot lysis in acid occurs when factor XIII activity levels are normal: the presence of either an atypical antifactor XIII antibody, or an unknown acid-activated protease. Our centre has identified four patients with in-vitro clot lysis in acetic acid and normal FXIII levels by activity assay. Our aim was to determine whether the cause of this unusual result was an inhibitory antibody or an aspartic acid protease. In each case, we found an inhibitor that was not an IgG but showed characteristics of an acid protease, including that it was neutralized by pepstatin. The four patients had median pepsinogen I levels five-fold to 10-fold higher than the normal median of 89 µg/l. Pepsinogen II was increased by three-fold to six-fold, but from a lower baseline median of 6.5 µg/l. Cathepsin D levels were normal. Clot lysis in the acid test was observed when recombinant human pepsinogen I was added to normal plasma at similarly high concentrations as in patient samples, consistent with a role of an acid protease. Clot lysis also occurred with addition of pepsinogen II, but required four-fold to seven-fold more than in patient samples. Laboratories should be aware that a positive acid clot lysis test can be misleading if pepsinogen levels are raised and should not use this alone to diagnose FXIII deficiency.

Acquired factor XIII deficiency: a therapeutic challenge.

Less than 60 cases of acquired factor (F)XIII deficiencies have been reported, most having distinct clinical features. To illustrate the therapeutic challenges of acquired FXIII inhibitors, we report a case of a 65-year-old patient with no previous bleeding history who suddenly developed massive haemorrhages associated to a strong and isolated FXIII inhibitor. No underlying disorder has been detected till now after three years of follow-up. Despite aggressive treatment with prednisone, rituximab, cyclophosphamide, immunoglobulin, immunoadsorption and immune tolerance his inhibitor is still present, although at low titre and with a clinical benefit since the patient has no more bleed since more than one year. Moreover the patient had a venous thromboembolic complication. After a review of the management of acquired FXIII deficiency patients and based on the management of acquired haemophilia we discuss a possible strategy for such difficult cases.

Acquired FXIII inhibitors: a systematic review.

Coagulation factor XIII (FXIII) is a protein that promotes fibrin stabilization by forming multiple covalent cross-links between fibrin monomers. Beside congenital FXIII deficiency, due to FXIII gene mutations, severe acquired FXIII deficiency has been described in association with autoantibodies against coagulation FXIII. These inhibitors, which occurs very rarely but may cause life-threatening bleeding complications, may arise spontaneously or in association with autoimmune and lymphoproliferative disorders or medications. The management of patients with acquired FXIII inhibitors is very demanding and treatment regimens must be focused on eradication of the inhibitor and to increase the plasma FXIII levels. In this systematic review, we analyse all the published case-reports on anti-FXIII autoantibodies focusing on the clinical features and treatment modalities of this acquired hemorrhagic condition.

Aggressive fatal case of autoimmune hemorrhaphilia resulting from anti-Factor XIII antibodies.

Factor XIII (FXIII) is a fibrin-stabilizing factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). Congenital FXIII deficiency is a rare bleeding disorder. Acquired FXIII deficiency resulting from FXIII hypo-synthesis and/or hyperconsumption is a relatively common disorder in which patients seldom bleed. On the contrary, 'autoimmune/acquired hemorrhaphilia XIII/13 due to anti-FXIII antibodies (AH13)' is a rare but life-threatening bleeding disorder. Through a nationwide survey of AH13, we diagnosed aggressive AH13 in a 66-year-old woman. She consulted our department because of a spontaneous hematoma in her hand. After 1.5 months, she also developed an intramuscular hematoma but retained approximately half (52%) of the normal FXIII activities. The patient's bleeding symptoms were aggravated to catastrophic massive bleedings in the large abdominal muscles and intrapelvic and intraperitoneal spaces. Two months after the bleeding onset, she died despite undergoing plasma exchange, which was performed because we were deeply suspicious of the presence of an anti-FXIII inhibitor. Seven days after her death, extremely low FXIII activity (6%) and positive data on anti-FXIII inhibitor were reported by a commercial laboratory. Our dot blot assay detected anti-FXIII-A autoantibodies, afterwards. Thus, the diagnosis of aggressive AH13 as early as possible is necessary to save patients' lives.

Reduced difference of α2-plasmin inhibitor levels between plasma and serum in patients with severe factor XIII deficiency, including autoimmune hemorrhaphilia due to anti-factor XIII antibodies.

Coagulation factor XIII/13 (FXIII/13) stabilizes fibrin molecules by creating crosslinks with other fibrin molecules as well as with α2-plasmin inhibitor (α2-PI). "Hemorrhagic acquired FXIII/13 deficiency" was formerly considered rare, but has been increasing recently in Japan. During the 10 months of our nationwide campaign, we diagnosed five new patients with "acquired hemorrhaphilia due to anti-FXIII/13 autoantibodies," after examining 20 newly suspected cases of "hemorrhagic acquired FXIII/13 deficiency." When FXIII/13 activity was reduced to less than 50% of normal, it was proportional to the difference in α2-PI levels between plasma and serum (plasma-serum α2-PI), likely due to its cross-linking to fibrin by activated FXIII/13. Accordingly, decreased amounts of the plasma-serum α2-PI ex vivo may reflect reduced FXIII/13 activity in vivo. The plasma-serum α2-PI may thus also be a useful diagnostic marker for severe FXIII/13 deficiency.

Rapid determination of blood coagulation factor XIII activity using protein arrays for serodiagnosis of human plasma.

We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca(2+) and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FXIII activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca(2+), thrombin, and 5-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.

In vitro inhibition of factor XIII retards clot formation, reduces clot firmness, and increases fibrinolytic effects in whole blood.

BACKGROUND: Thrombelastography has received renewed interest in the perioperative setting. The main determinants of thrombelastographic results are coagulation factor concentrations (various zymogens and fibrinogen) and platelet count; thus, platelet inhibition renders these assays mainly coagulation factor dependent. Assays with and without platelet inhibition are thus increasingly used to trigger and monitor replacement therapy with blood products. In this study, we evaluated the effect of factor XIII inhibition and additional glycoprotein (GP) IIb/IIIa blockade on (platelet-inhibited) whole blood thrombelastography and whether a modified routine assay (using factor XIII antibody) can be used to detect factor XIII deficiency. METHODS: Normal whole blood was incubated with increasing amounts of a nonspecific antibody, an anti-GPIIb/IIIa antibody, or a neutralizing anti-factor XIII antibody; samples were analyzed with a tissue factor-activated and platelet-inhibited whole blood thrombelastographic assay. Clotting time, clot formation time, maximum clot firmness, and clot lysis at 60 min were evaluated in triplicate. Also, 25 whole blood routine samples were evaluated for factor XIII deficiency using a new thrombelastographic assay incorporating a factor XIII antibody and using a standard factor XIII assay for comparison. RESULTS: Although GPIIb/IIIa inhibition did not alter the results of the platelet-inhibited whole blood thrombelastography, factor XIII inhibition significantly reduced maximum clot firmness (P = 0.020) and increased clot formation time (P = 0.025) and clot lysis (P = 0.007), leaving clotting time unchanged; a ceiling effect seemed to be present with increasing antibody concentrations in whole blood (but not plasma). The thrombelastographic assay for factor XIII deficiency (<70% activity) had a 90% sensitivity and negative predictive value (area under receiver operating characteristic curve 0.803, P = 0.0015); for a deficiency <60%, sensitivity and negative predictive value were 100% (area under receiver operating characteristic curve 0.84, P = 0.0037). CONCLUSION: Factor XIII has significant impact on platelet-inhibited activated whole blood thrombelastography. This phenomenon should be considered when interpreting thrombelastographic results in the bleeding patient, especially when the results trigger procoagulant therapy. Antibody-mediated factor XIII inhibition can be used to establish thrombelastography-based assays to detect factor XIII deficiency.

Coagulation factor XIII serves as protein disulfide isomerase.

Tissue transglutaminase was reported to act as protein disulfide isomerase (PDI). We studied whether plasma transglutaminase - coagulation factor XIII (FXIII) - has PDI activity as well. PDI activity was measured by determining the ability to renature reduced-denatured RNase (rdRNase). We found that FXIII can renature rdRNase, with efficiency comparable to commercial PDI. This PDI activity was inhibited by bacitracin. Like tissue transglutaminase, FXIII-mediated PDI activity is independent of its transglutaminase activity and is located on the A subunit. Surface-associated PDI has been previously shown to catalyse two distinct functions: transnitrosation with subsequent release of intracellular nitric oxide and disulfide bond rearrangement during platelet integrin ligation. Our results imply that FXIII-PDI activity may have a role in platelet function.

Possible role for cellular FXIII in monocyte-derived dendritic cell motility.

The A subunit of plasma factor XIII (FXIII-A) is thought to function as an intracellular transglutaminase (TG) in the monocyte/macrophage lineage to regulate certain intracellular processes involving cytoskeleton remodeling, but its precise role and the functional consequences of its absence remain poorly understood. In the present study, we show that cellular FXIII (cFXIII) expression is largely upregulated during in vitro differentiation of monocytes into dendritic cells (DCs). Monodansyl-cadaverine, a competitive substrate of TG activity, inhibited basal and CCL19-stimulated migration of mature DCs. In agreement, FXIII-A-deficient DCs showed a reduced chemotactic response to CCL19. Consistent with these findings, CHO cells stably expressing human FXIII-A showed enhanced motility in transwell and scratch-wound assays. These cells displayed increased formation of membrane blebs, dynamic cell protrusions implicated in cell movement that were also observed in DCs. The results provide evidence suggesting that upregulation of cFXIII in DCs has a role in regulating cell motility.