Human Factor XIII Research Articles

Discovery of a new lead molecule to develop a novel class of human factor XIIa inhibitors.

Factor XIIa (FXIIa) is a plasma serine protease within the contact activation pathway. Inhibiting FXIIa could offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks that accompany the use of existing anticoagulants. Therefore, we investigated the anticoagulant properties of an amidine-containing molecule (inhibitor 1) to identify a potential lead molecule for subsequent development of FXIIa inhibitors. Results indicated that inhibitor 1 primarily inhibits human FXIIa with an IC50 value of ~30 µM. The inhibitor demonstrated variable selectivity against thrombin, factor IXa, factor Xa, factor XIa, and activated protein C. Michaelis-Menten kinetics indicated that the molecule is an active site inhibitor of FXIIa. Molecular modeling studies revealed that the molecule recognizes residues His57, Asp189, and Ala190 in FXIIa's active site. The inhibitor selectively and concentration-dependently prolonged the clotting time of human plasma under activated partial thromboplastin time assay conditions. The inhibitor did not exhibit significant cytotoxicity in human HEK293 cells and the in silico pharmacokinetics and toxicology data were comparable to known anticoagulants. This study introduces inhibitor 1 as a lead platform for further development as an anticoagulant to provide a more effective and safer approach to preventing and treating thromboembolic diseases.

Discovery of Heparin Mimetic, Potent, and Selective Inhibitors of Human Clotting Factor XIIIa.

Factor XIIIa (FXIIIa) is a cysteine transglutaminase that catalyzes the last step in the coagulation process. An anion-binding site inhibition of FXIIIa is a paradigm-shifting strategy that may offer key advantages of controlled inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We previously reported a flavonoid trimer-based allosteric inhibitor of FXIIIa with moderate potency and selectivity. To further advance this approach, we evaluated a series of 27 variably sulfonated heparin mimetics against human FXIIIa. Only 13 molecules exhibited inhibitory activity at the highest concentration tested with IC50 values of 2-286 μM. Specifically, inhibitor 16 demonstrated an IC50 value of 2.4 ± 0.5 μM in a bisubstrate, fluorescence-based trans-glutamination assay. It also demonstrated a significant selectivity over other clotting factors including thrombin, factor Xa, and factor XIa as well as other cysteine enzymes including papain and tissue transglutaminase 2. Inhibitor 16 did not affect the viability of three human cell lines at a concentration that is 5-fold its FXIIIa-IC50. The molecule had a very weak effect on the activated partial thromboplastin time of human plasma at a concentration of >700 μM, further supporting its functional selectivity. Importantly, molecule 16 inhibited FXIIIa-mediated polymerization of fibrin(ogen) in a concentration-dependent manner as shown by the gel electrophoresis experiment. Michaelis-Menten kinetics revealed that the molecule competes with the Gln-donor protein substrate, i.e., dimethylcasein, but not with the Lys-donor small substrate, i.e., dansylcadaverine. Molecular modeling studies revealed that this type of molecule likely binds to an anion-binding site comprising the basic amino acids of Lys54, Lys61, Lys73, Lys156, and Arg244 among others. Overall, our work puts forward a new anion-binding site, selective, nontoxic, sulfonated heparin mimetic FXIIIa inhibitor 16 for further development as an effective and safer anticoagulant.

P146 A phase II, randomised, double-blind, placebo-controlled trial exploring pharmacokinetics, safety and efficacy of intravenous factor XIII treatment in adults with systemic sclerosis

Abstract Background/Aims Systemic sclerosis (SSc) is a multi-system autoimmune disease with significant morbidity and mortality, and an unmet therapeutic need. The pathogenesis of SSc involves dysfunctional immune signalling, fibroblast behaviour and vascular development. Factor XIII (FXIII) is an enzyme involved in coagulation, and in wound healing where it promotes angiogenesis via inhibition of thrombospondin-1 (TSP-1). Therapeutic administration of FXIII in SSc is proposed to increase angiogenesis via suppression of TSP-1, which is known to be dysregulated in SSc. Previous preclinical and clinical studies suggest that therapeutic administration of factor XIII could improve vascular function in SSc. Methods Two interlinked clinical trials were conducted at a single centre. First, a single-dose open-label study was performed to assess the safety and pharmacokinetics of FXIII treatment in SSc. Eight adult patients with limited or diffuse SSc received a single dose of intravenous purified human FXIII, followed by a 6-week monitoring period. Second, a phase II, double-blind, randomised, placebo-controlled study was performed to investigate the safety and efficacy of factor XIII treatment in SSc. 18 adult patients with limited or diffuse SSc were randomised 2:1 to receive study intervention (intravenous purified human FXIII) or placebo (intravenous 0.9% sodium chloride solution). Safety endpoints were assessment of the safety and tolerability of FXIII treatment via review of adverse events, physical examination and monitoring of physiological function. Efficacy was explored as change in skin severity by modified Rodnan skin score (mRSS) at week 24 compared to baseline, and change in severity of Raynaud’s phenomenon, assessed by review of the Raynaud’s Condition Score (RCS) at week 24, compared to baseline. The study is registered with ClinicalTrials.gov (number NCT02551042). Results The main objective of the clinical trial was met by demonstrating that FXIII is safe and well tolerated in SSc. Pharmacokinetic analysis showed a predictable rise and fall in FXIII level following therapeutic administration, with the mean time taken for the FXIII level to return to within 10 IU/dL of the endogenous level being 16.6 days (SD 8.7). Adverse event and safety monitoring did not reveal any concerning pattern. Study intervention-related adverse events were self-resolving and tolerable. There was no statistically significant change in mRSS or RCS during the trial, but there was a trend towards improvement in RCS in the FXIII group (mean change in RCS of -1.2 (95% CI -2.26 to -0.14) in the FXIII group versus -0.83 (95% CI -3.44 to 1.77) in the placebo group). Conclusion Our results suggest that therapeutic strategies to modulate links between coagulation and tissue repair using FXIII are safe and feasible. Further studies with different patient populations and FXIII doses could investigate the role of FXIII in SSc further. Disclosure A. Leslie: Other; A.L. is an employee of GSK. S. Vigneswaran: None. K. Khan: None. V. Ong: None. C.P. Denton: Consultancies; C. D. has received consulting fees from Roche, Janssen, GlaxoSmithKline, Sanofi, Galapagos, Boehringer Ingelheim, and CSL Behring, and Acceleron. Grants/research support; C. D. has received research funding from CSL Behring, Horizon, Abbvie, and GlaxoSmithKline.

Detection of human platelets antigen polymorphism (HPA-1 and HPA-3) and human factor XIII mutation in Sudanese women with recurrent pregnancy loss

BackgroundRecurrent pregnancy Loss (RPL) is common problem affecting many couples. A certain genetic variants link to increase the danger of this condition particularly HPA-1, HPA-3 and Human Factor XIII Val34Leu Mutation. The present study aims to find an association between RPL and the Factor XIII Val34Leu polymorphism, as well as HPA-1 and HPA-3 in Sudanese women with RPL.MethodsThis case-control study conducted between June 2022 and December 2022 included 216 women, with 103 cases having minimum three abortions in the past, and 113 healthy controls with at least two full-term births and no abortion history. DNA was isolated from whole blood and the status of three genetic polymorphisms (HPA-1, HPA-3, and factor XIII) was done using a polymerase chain reaction (PCR). Data was analysed using the SPSS version 24 software.ResultsThe A/A genotype was found to be more prevalent in cases (79.6%) and controls (96.5%) regarding HPA-1. A significant difference was observed in overall allele frequency for B allele (97.0%) and expected frequency of A allele was (81.1%) using the Hardy-Weinberg distribution (p < 0.001). The genotype A/A was most common in these patients (90.3%) and controls (100%), while B/B genotype was only (9.7%) in patients regarding HPA-3. Furthermore, the frequency of Val/Val genotype was higher in cases (88.3%) as compared with controls (90.3%). The risk of RPL in patients was nearly the same in Val/Leu individuals and controls group but all these differences were not statistically significant (p > 0.05).ConclusionOur results indicate a link between Human Platelet Antigen-1 (HPA-1), Human Platelet Antigen-3 (HPA-3) and Factor XIII gene polymorphism with RPL.

New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants.

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure-activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases.

Triazol-1-yl Benzamides Promote Anticoagulant Activity via Inhibition of Factor XIIa.

Human factor XIIa (FXIIa) is a plasma serine protease that plays a significant role in several physiological and pathological processes. Animal models have revealed an important contribution of FXIIa to thromboembolic diseases. Remarkably, animals and patients with FXII deficiency appear to have normal hemostasis. Thus, FXIIa inhibition may serve as a promising therapeutic strategy to attain safer and more effective anticoagulation. Very few small molecule inhibitors of FXIIa have been reported. We synthesized and investigated a focused library of triazol-1-yl benzamide derivatives for FXIIa inhibition. We chemically synthesized, characterized, and investigated a focused library of triazol- 1-yl benzamide derivatives for FXIIa inhibition. Using a standardized chromogenic substrate hydrolysis assay, the derivatives were evaluated for inhibiting human FXIIa. Their selectivity over other clotting factors was also evaluated using the corresponding substrate hydrolysis assays. The best inhibitor affinity to FXIIa was also determined using fluorescence spectroscopy. Effects on the clotting times (prothrombin time (PT) and activated partial thromboplastin time (APTT)) of human plasma were also studied. We identified a specific derivative (1) as the most potent inhibitor in this series. The inhibitor exhibited nanomolar binding affinity to FXIIa. It also exhibited significant selectivity against several serine proteases. It also selectively doubled the activated partial thromboplastin time of human plasma. Overall, this work puts forward inhibitor 1 as a potent and selective inhibitor of FXIIa for further development as an anticoagulant.

Antibodies against non-catalytic B subunit of factor XIII inhibit activation of factor XIII and fibrin crosslinking.

Coagulation factor XIII (FXIII) is a proenzyme of plasma transglutaminase. It comprises two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIII-B). We previously reported that alloantibodies against FXIII-B could promote FXIII clearance in a patient with congenital FXIII-B deficiency who had received infusions of plasma-derived human FXIII (A2B2 heterotetramer). We aimed to investigate whether anti-FXIII-B antibodies affect the catalytic function of FXIII. FXIII activation and fibrin crosslinking were examined in the presence of patient plasma, isolated patient IgG, or rat anti-FXIII-B monoclonal antibodies. Alloantibody levels were increased by repeated infusions of plasma-derived A2B2 heterotetramer, which enhanced binding to the functionally important FXIII-B Sushi domains. The patient plasma strongly inhibited cleavage of the FXIII-A activation peptide, amine incorporation, and fibrin crosslinking in normal plasma. Further, anti-FXIII-B alloantibodies blocked the formation of the complex of FXIII-B with FXIII-A, and fibrinogen. Rat monoclonal antibodies against the 10th Sushi domain of FXIII-B inhibited the incorporation of FXIII-B to fibrin, FXIII activation (i.e., cleavage of FXIII-A activation peptide), and ultimately fibrin crosslinking in normal plasma, independent of their effect on heterotetramer assembly with FXIII-A. Alloantibody binding to the A2B2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A2B2 heterotetramer and FXIII-B, but not to FXIII-A. Anti-FXIII-B antibodies binding to the A2B2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its non-catalytic subunit (FXIII-B).

Molecular docking and dynamics simulation analysis of the human FXIIa with compounds from the Mcule database.

The human factor XIIa is a serine protease enzyme that is implicated in the pathological thrombosis. This coagulation factor represents an interesting molecular target to design safer antithrombotic agents without adversely influencing physiological hemostasis. Therefore, it is of interest to virtually screen the human factor XIIa crystal with millions of compounds in Mcule database in order to identify potential inhibitors. For this purpose, both molecular docking and dynamics simulation were employed to identify potential hits. Also, various predictive approaches were utilized to estimate chemical, pharmacokinetics and toxicological features for the top hits. As such, we report here that compound 4 (1-(4-benzylpiperazin-1-yl)-2-[5-(3,5-dimethylpyrazol-1-yl)-1,2,3, 4-tetrazol-2-yl]ethanone) may be a potential ligand against the human factor XIIa for further consideration in the design and development of novel antithrombotic agents.

Ethacrynic acid is an inhibitor of human factor XIIIa

BackgroundEthacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques.MethodsA series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA’s effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis.ResultsFXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa.ConclusionsOverall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.

Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic β-amino acids

Peptides that contain β-amino acids display stable secondary structures, such as helices and sheets, and are often referred to as foldamers. Cyclic β2,3-amino acids (cβAAs), such as 2-aminocyclohexanecarboxylic acid (2-ACHC), are strong helix/turn inducers due to their restricted conformations. Here we report the ribosomal synthesis of foldamer peptides that contain multiple, up to ten, consecutive cβAAs via genetic code reprogramming. We also report the de novo discovery of macrocyclic cβAA-containing peptides capable of binding to a protein target. As a demonstration, potent binders with low-to-subnanomolar KD values were identified for human factor XIIa (hFXIIa) and interferon-gamma receptor 1, from a library of their 1012 members. One of the anti-hFXIIa macrocyclic peptides that exhibited a high inhibitory activity and serum stability was co-crystallized with hFXIIa. The X-ray structure revealed that it adopts an antiparallel β-sheet structure induced by a (1S,2S)-2-ACHC residue via the formation of two γ-turns. This work demonstrates the potential of this platform to explore the previously inaccessible sequence space of cβAA-containing peptides.

A totally recombinant fibrin matrix for mesenchymal stem cell culture and delivery.

Mesenchymal stem cells (MSCs) have been widely studied for tissue engineering and treating diseases in laboratories, clinical trials, and clinics. Fibrin matrices are often used to culture MSCs or increase the retention of MSCs at the injection site. However, fibrins made with the human plasma derived fibrinogen have high cost and risk of human pathogen transmission. In this article, we studied if fibrin matrices made with recombinant human fibrinogen, recombinant human thrombin, and recombinant human factor XIII could be used to culture and deliver MSCs. We systematically investigated the relationships between the fibrin matrix formulation, its nanostructure, and the behaviors of the cells in the matrix including the cell morphology, viability, and growth. We found that the fibrinogen concentration significantly affected the matrix structure and cell behaviors. We then used an optimized fibrin matrix to deliver human MSCs into mice subcutaneously. We found that the matrix could significantly enhance the retention of MSCs at the injection site. To our best knowledge, this is the first study on using fibrin matrices made with entirely recombinant proteins for culturing and delivering MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3135-3142, 2018.

Structures of human plasma β–factor XIIa cocrystallized with potent inhibitors

AbstractActivated factor XIIa (FXIIa) is a serine protease that has received a great deal of interest in recent years as a potential target for the development of new antithrombotics. Despite the strong interest in obtaining structural information, only the structure of the FXIIa catalytic domain in its zymogen conformation is available. In this work, reproducible experimental conditions found for the crystallization of human plasma β-FXIIa and crystal growth optimization have led to determination of the first structure of the active form of the enzyme. Two crystal structures of human plasma β-FXIIa complexed with small molecule inhibitors are presented herein. The first is the noncovalent inhibitor benzamidine. The second is an aminoisoquinoline containing a boronic acid–reactive group that targets the catalytic serine. Both benzamidine and the aminoisoquinoline bind in a canonical fashion typical of synthetic serine protease inhibitors, and the protease domain adopts a typical chymotrypsin-like serine protease active conformation. This novel structural data explains the basis of the FXII activation, provides insights into the enzymatic properties of β-FXIIa, and is a great aid toward the further design of protease inhibitors for human FXIIa.

Influence of cryoprecipitate, Factor XIII, and fibrinogen concentrate on hyperfibrinolysis.

Hyperfibrinolysis is a potentially life-threatening condition associated with poor clot integrity and excessive bleeding. Although antifibrinolytics are an effective treatment, more liberal use of these drugs may lead to a prothrombotic risk, and an earlier and potentially safer treatment option would be desirable. Hyperfibrinolysis has been shown to be attenuated by in vitro supplementation of purified human Factor (F)XIII concentrate. Cryoprecipitate represents an alternative source of FXIII and the only approved source of concentrated FXIII in some countries. The aim of this study was to investigate whether cryoprecipitate, FXIII, and fibrinogen concentrate mitigate hyperfibrinolysis. Ten citrate blood specimens from healthy subjects were spiked with tissue plasminogen activator (t-PA) and subsequently supplemented with cryoprecipitate, FXIII, fibrinogen concentrate, and ɛ-aminocaproic acid (EACA). Thromboelastometry tests were performed at baseline, after t-PA, and after supplementation. Hyperfibrinolysis was assessed using the clot lysis index at 60 minutes (LI60; reciprocal of maximum lysis). The LI60 was significantly improved (fibrinolysis attenuated) after cryoprecipitate supplementation compared to t-PA alone and compared to FXIII and fibrinogen concentrate. Hyperfibrinolysis was only fully reversed using EACA. In addition, cryoprecipitate demonstrated the least variability in the attenuation of hyperfibrinolysis among 10 healthy subjects, compared to FXIII and fibrinogen concentrate. This is the first study to show that cryoprecipitate is able to significantly mitigate hyperfibrinolysis in an in vitro model. Further investigations are warranted to determine whether cryoprecipitate may have a previously unrecognized benefit and should be administered earlier in resuscitation protocols.

Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.

Nonclinical analysis of the safety, pharmacodynamics, and pharmacokinetics of plasma-derived human FXIII concentrate in animals.

Factor XIII (FXIII) is a coagulation protein which plays a major role in hemostasis by covalently cross‐linking fibrin molecules, thereby stabilizing the blood clot and increasing resistance to fibrinolysis. FXIII deficiency, either congenital or acquired, is associated with spontaneous bleeding, increased bleeding time, and poor wound healing. Purified plasma‐derived human FXIII concentrate (pd hFXIII) has been available since 1993 for therapeutic use in congenital FXIII deficiency. This set of nonclinical investigations aimed to evaluate the pharmacodynamic effects and assess the safety profile of pd hFXIII. The efficacy and safety of pd hFXIII were evaluated by pharmacodynamic, pharmacokinetic, and toxicity studies in mice and rats, safety pharmacology studies in dogs, neoantigenicity study, local tolerance, and thrombogenicity tests in rabbits. Administration of pd hFXIII resulted in the correction of deficits in clot formation kinetics and strength as measured by thromboelastometry, and was not associated with thrombus formation up to 350 IU/kg in FXIII knockout mice. There was no production of neoantigens resulting from the viral elimination manufacturing steps detected, and no adverse reactions were observed in toxicity studies with single doses up to 3550 IU/kg in mice and 1420 IU/kg in rats; nor from repeat doses of 350 IU/kg in rats. In addition, local tolerance tests revealed a good tolerability profile in rabbits. Overall, this data showed that pd hFXIII was well tolerated and pharmacodynamically active in preclinical animal models, supporting pd hFXIII as a therapy for FXIII deficiency.

Catridecacog: First drug to treat rare genetic blood clotting disorder

Catridecacog, or coagulation factor XIII A-subunit, recombinant (Tretten—Novo Nordisk), a minimally invasive option for treating rare congenital factor XIII A-subunit deficiency, has received FDA approval. According to Novo Nordisk, catridecacog helps to prevent bleeding in children and adults who have the coagulation factor XIII A-subunit deficiency, an extremely rare genetic disorder. Catridecacog is the first drug to receive FDA approval for this indication. FDA also granted catridecacog orphan drug status because it treats a rare disease. Factor XIII is a terminal enzyme in the blood coagulation cascade that stimulates cross-linking of fibrin and other proteins in the fibrin clot to promote hemostasis. In human plasma, factor XIII circulates as a heterotetramer of two functional subunit proteins—two factor XIII A-subunits and two factor XIII B-subunits. The factor XIII A-subunit acts as the enzyme, while factor XIII B-subunit acts as a carrier for the factor XIII A-subunit. During coagulation, the factor XIII is activated by thrombin and calcium; the factor XIII A-subunit dissociates from the factor XIII B-subunit to become active and help cross-link fibrin to enhance platelet and clot adhesion to injured tissue. Patients with the factor XIII A-subunit deficiency have a significantly lower level of the active subunit, thus resulting in less stable clotting for injured tissue. Catridecacog binds to the free human factor XIII B-subunit, as factor XIII A-subunit, to strengthen fibrin clots, impede fibinolysis, and enhance platelet adhesion to the injury site. Give patients the Médication Guide and review it with them carefully. Verify the diagnosis, since catridecacog should not be used for prophylactic treatment of bleeding in patients with congenital factor XIII B-subunit deficiency. Advise patients that breakthrough bleeding may be a sign and symptom of inhibitor formation, and they may have to see their health care provider to measure antibody concentrations. Tell patients to inform their health care provider of all the medications they may be taking. Patients with the factor XIII A-subunit deficiency have a significantly lower level of the active subunit, thus resulting in less stable clotting for injured tissue. During clinical development, researchers studied the effectiveness of catridecacog in 77 patients with coagulation factor XIII A-subunit deficiency. Catridecacog was effective in preventing bleeding in 90% of the patients when given the recommended dose of 35 IU/ kg. The mean age of study participants was approximately 26 years (63.4% > 18 years), and the male to female ratio was 3:2. The most common related adverse events (occurring in >1% of patients treated with catridecacog) were headache, pain in the extremities, injection site pain, and an increase in nonneutralizing antibodies. Use of catridecacog is contraindicated in patients who have known hypersensitivity to the active substance or to any of the excipients. Because increased levels of rFXIII may increase the risk of thrombosis, health professionals should exercise caution in patients with a predisposition to conditions of thrombosis. Incorrect storage of the product after reconstitution may result in loss of sterility and increased levels of activated rFXIII. Catridecacog, coagulation factor XIII A-subunit, recombinant (Tretten)Manufacturer: Novo NordiskDrug class: Blood coagulation factorsIndication: Treatment of congenital factor XIII A-subunit deficiencyDosage: 35 IU/kg once monthly■To administer, reconstitute with sterile water for injection with a 10-mL syringe.■A dose recommendation cannot be made for older adults, pregnant patients, or children younger than age 6 years due to lack of data.Of note: Treatment should be initiated under the supervision of a physician experienced in the treatment of rare bleeding disorders. Do not use vial if solid matter and discoloration are present in the reconstituted product. Administer at a rate not exceeding 1–2 mL per minute. Do not administer as a drip or with other infusion solutions. Manufacturer: Novo Nordisk Drug class: Blood coagulation factors Indication: Treatment of congenital factor XIII A-subunit deficiency Dosage: 35 IU/kg once monthly■To administer, reconstitute with sterile water for injection with a 10-mL syringe.■A dose recommendation cannot be made for older adults, pregnant patients, or children younger than age 6 years due to lack of data. Of note: Treatment should be initiated under the supervision of a physician experienced in the treatment of rare bleeding disorders. Do not use vial if solid matter and discoloration are present in the reconstituted product. Administer at a rate not exceeding 1–2 mL per minute. Do not administer as a drip or with other infusion solutions.

A novel mutation in the transglutaminase-1 gene in an autosomal recessive congenital ichthyosis patient.

Structure-function implication on a novel homozygous Trp250/Gly mutation of transglutaminase-1 (TGM1) observed in a patient of autosomal recessive congenital ichthyosis is invoked from a bioinformatics analysis. Structural consequences of this mutation are hypothesized in comparison to homologous enzyme human factor XIIIA accepted as valid in similar structural analysis and are projected as guidelines for future studies at an experimental level on TGM1 thus mutated.

A totally recombinant human fibrin sealant

BackgroundApplications of plasma-derived human fibrin sealants (pdhFS) have been limited because of cost, limited supply of pathogen-screened plasma, the need for bioengineering improvements, and regulatory issues associated with federal approval. We describe a totally recombinant human fibrin sealant (rhFS), which may engender an abundant, safe, and cost-effective supply of efficacious fibrin sealant. Materials and methodsA first-generation rhFS made from recombinant human fibrinogen (rhFI; produced in the milk of transgenic cows), activated recombinant human factor XIII (rhFXIIIa; produced in yeast), and recombinant human thrombin (rhFIIa; purchased, made in animal cell culture) was formulated using thromboelastography (TEG). The hemostatic efficacy of rhFS versus commercial pdhFS was compared in a nonlethal porcine hepatic wedge excision model. ResultsThe maximal clot strength of rhFS measured in vitro by TEG was not statistically different than that of pdhFS. TEG analysis also showed that the rhFS gained strength more quickly as reflected by a steeper α angle; however, the rhFS achieved this clot strength with a 5-fold lower factor I content than the pdhFS. When these fibrin sealants were studied in a porcine hepatic wedge excision model, the hemostatic scores of the rhFS were equivalent or better than that of the pdhFS. ConclusionsThe bioengineered rhFS had equivalent or better hemostatic efficacy than the pdhFS in a nonlethal hemorrhage model, despite the factor I concentration in the rhFS being about one-fifth that in the pdhFS. Because the rhFS is amenable to large-scale production, the rhFS has the potential to be more economical and abundant than the pdhFS, while having a decreased risk of blood-borne pathogen transmission.

Characterization of Anopheles gambiae Transglutaminase 3 (AgTG3) and Its Native Substrate Plugin

Male Anopheles mosquitoes coagulate their seminal fluids via cross-linking of a substrate, called Plugin, by the seminal transglutaminase AgTG3. Formation of the "mating plug" by cross-linking Plugin is necessary for efficient sperm storage by females. AgTG3 has a similar degree of sequence identity (~30%) to both human Factor XIII (FXIII) and tissue transglutaminase 2 (hTG2). Here we report the solution structure and in vitro activity for the cross-linking reaction of AgTG3 and Plugin. AgTG3 is a dimer in solution and exhibits Ca(2+)-dependent nonproteolytic activation analogous to cytoplasmic FXIII. The C-terminal domain of Plugin is predominantly α-helical with extended tertiary structure and oligomerizes in solution. The specific activity of AgTG3 was measured as 4.25 × 10(-2) units mg(-1). AgTG3 is less active than hTG2 assayed using the general substrate TVQQEL but has 8-10× higher relative activity when Plugin is the substrate. Mass spectrometric analysis of cross-linked Plugin detects specific peptides including a predicted consensus motif for cross-linking by AgTG3. These results support the development of AgTG3 inhibitors as specific and effective chemosterilants for A. gambiae.

A Retrospective Review of the Use of Autologous Platelet Gels for Rhytidectomy

Tissue sealants exist in 2 forms: platelet-rich plasma (PRP) and platelet-rich fibrin matrix (PRFM). They both result in activation of fibrinogen to fibrin, but PRFM contains a higher concentration of fibrinogen. PRFM products, otherwise known as fibrin sealants, make use of pooled human donor fibrinogen, factor XIII, and fibronectin. When they are mixed with thrombin, calcium chloride, and aprotinin, a fibrin clot is formed. Fibrin sealants are available as single-use kits. As an example, Evicel (Ethicon 360) comes in a triluminal catheter. One lumen contains the fibrinogen solution; one contains the thrombin solution; and one is filled with air to allow the spray application. On spraying, the fibrinogen and thrombin solutions are mixed, and a fibrin clot is created within seconds. In 2001, our facility compared the severity of edema and ecchymosis with and without the use of autologous PRP in 8 healthy women after standard deep-plane rhytidectomy. Unilateral application of the PRP was performed on closure of the superficial musculoaponeurotic system and skin flaps (Figure). The conclusion from this prospective, randomized, controlled pilot study was that there was a trend toward decreased postoperative ecchymosis and edema on the side of PRP application. The trend was more apparent early in the postoperative period (days 3-12) and more so for ecchymosis than edema. Because of the limited power of the study, however, a statistically significant difference could not be elucidated. In 2009, Lee et al published their results using the fibrin sealant Crosseal (Ethicon Inc) in 9 patients who were undergoing rhytidectomy. The sealant was applied unilaterally at random, and the contralateral side served as the control. The severity of ecchymosis for each side was scored 1 (minimal) to 10 (severe) by 5 blinded observers at postoperative days 3 and 7. The authors found a statistically lower level of ecchymosis for the fibrin sealant side (4.5) as compared with the control (6.2). Zoumalan and Rizk investigated hematoma occurrence with fibrin glue in both deep-plane and lateral superficial musculoaponeurotic system rhytidectomies. In their study, 459 patients underwent skin flap closure with fibrin glue, and 146 patients underwent standard closure. The authors concluded that there was a statistically significant reduction in hematoma rate when fibrin glue was applied to the skin flap. The hematoma rate was 3.4% in the control group and 0.4% in the fibrin sealant group (P=.01). Based on these studies, it seems likely that tissue sealants reduce the severity of ecchymosis in the acute postoperative period and may also minimize hematoma occurrence. It should be noted, however, that many other studies have not been able to corroborate these findings. Perhaps one of the more important and less frequently discussed advantages of fibrin sealants is the reduced operative time to achieve hemostasis and wound closure. Clearly, patients stand to benefit from reduced anesthesia time. However, the reduced facility and anesthesia fees must be weighed against the product expense. The unit cost for 2 mL of fibrin sealant plus the applicator is $200. In our experience, the use of fibrin sealants saves approximately 5 minutes of operative time during rhytidectomy. Because the product costs exceed the reduced facility costs, our facility no longer uses PRP or PRFM. Unfortunately, in our opinion, the potential benefits of these sealants have not been adequately substantiated to justify their costs in rhytidectomy. Figure. Application of platelet-rich plasma to the skin flap during closure of a deep-plane face-lift (reprinted with permission from Powell et al2).