[Pedigree Analysis of Hereditary Coagulation Factor XII Deficiency Caused by Compound Heterozygous Mutation p.Gly175Cys and p.Gly542Ser of F12 Gene].

OBJECTIVE: To analyze the clinical phenotype and gene mutation of a genetic coagulation factor XII (FXII) deficiency pedigree and explore the molecular pathogenesis. METHODS: The activated partial thromboplastin time (APTT) and FXII activity (FXII:C) were detected by clotting method. The FXII antigen (FXII:Ag) was tested with ELISA. All exons and flanks of F12 gene were determined by Sanger sequencing. ClustalX-2.1-win, PROVEAN and Swiss-Pdb Viewer software were used to analyze the conservatism of amino acids at the mutant site, forecast whether the mutant amino acids were harmful and confirm the influence of the mutation on protein structure. RESULTS: The APTT of the proband prolonged to 71.3 s. The FXII:C and FXII:Ag were decreased to 5% and 6%, respectively. There were two heterozygous missense mutations c.580G>T and c.1681G>A detected in exon 7 and exon 14 of F12 gene, resulting in p.Gly175Cys and p.Gly542Ser, severally. Proband's father carried the p.Gly175Cys heterozygous mutation, while mother, brother and daughter had the p.Gly542Ser heterozygous mutation. Software analysis showed that both Gly175 and Gly542 were conserved, the two mutations were harmful and when mutations had occurred, the corresponding sites affected the protein local structure. CONCLUSION: The p.Gly175Cys and p.Gly542Ser compound heterozygous mutations are the molecular pathogenesis of the hereditary coagulation FXII deficiency pedigree. The p.Gly175Cys mutation has been detected for the first time in the world.

Hypocoagulability in severe yellow fever infection is associated with bleeding: results from a cohort study.

Background: Severe yellow fever infection (YFI) may be complicated by a hemorrhagic diathesis. However, the hemostasis profile of YFI has rarely been reported. Objectives: The aim of this study was to characterize the hemostatic features of YFI by using a rotational thromboelastometry (ROTEM). Methods: We evaluated clinical, laboratory, and ROTEM parameters in adults with severe YFI and their correlation with hemostatic variables according to bleeding and death. Results: A total of 35 patients were included (median age, 49 years). ROTEM was performed in 22 patients, of whom 21 (96%) presented bleeding and 4 (18%) died. All patients who died had major bleeding. Patients who died presented prolonged clotting time (CT; median, 2326 seconds; IQR, 1898-2986 seconds) and reduced alpha angle (median, 12°; IQR, 12°-15°) in comparison with patients who had minor (median CT, 644 seconds; IQR, 552-845 seconds and alpha angle, 47°; IQR, 28°-65°) and major (median CT, 719 seconds; IQR, 368-1114 seconds and alpha angle, 43°; IQR, 32°-64°) bleeding who survived. In patients who had bleeding, CT showed a strong negative correlation with factor (F)V (r = -.68), FIX (r = -.84), and FX (r = -.63) as well as alpha angle showed a strong negative correlation with FIX (r = -.92). In patients who died, the correlations were even stronger. A total of 19/21 (90%) patients presented hypocoagulability assessed by ROTEM. Conclusion: Hypocoagulabitity is the hallmark of the bleeding diathesis of severe YFI. Abnormal CT and alpha angle associated with death and could be used as potential predictors of adverse outcome in severe YFI.

Novel Thiourea and Oxime Ether Isosteviol-Based Anticoagulants: MD Simulation and ADMET Prediction.

Activated blood coagulation factor X (FXa) plays a critical initiation step of the blood-coagulation pathway and is considered a desirable target for anticoagulant drug development. It is reversibly inhibited by nonvitamin K antagonist oral anticoagulants (NOACs) such as apixaban, betrixaban, edoxaban, and rivaroxaban. Thrombosis is extremely common and is one of the leading causes of death in developed countries. In previous studies, novel thiourea and oxime ether isosteviol derivatives as FXa inhibitors were designed through a combination of QSAR studies and molecular docking. In the present contribution, molecular dynamics (MD) simulations were performed for 100 ns to assess binding structures previously predicted by docking and furnish additional information. Moreover, three thiourea- and six oxime ether-designed isosteviol analogs were then examined for their drug-like and ADMET properties. MD simulations demonstrated that four out of the nine investigated isosteviol derivatives, i.e., one thiourea and three oxime ether ISV analogs, form stable complexes with FXa. These derivatives interact with FXa in a manner similar to Food and Drug Administration (FDA)-approved drugs like edoxaban and betrixaban, indicating their potential to inhibit factor Xa activity. One of these derivatives, E24, displays favorable pharmacokinetic properties, positioning it as the most promising drug candidate. This, along with the other three derivatives, can undergo further chemical synthesis and bioassessment.

Safety of recombinant activated factor VII for treatment of breakthrough bleeds in patients with congenital haemophilia A and inhibitors receiving emicizumab prophylaxis: Review of the real-world evidence.

BACKGROUND: Emicizumab is used as a subcutaneous prophylaxis for prevention of bleeding episodes in patients with haemophilia A (HA) with and without inhibitors. While low bleeding rates were observed in clinical trials, patients still experience breakthrough bleeds (BTBs) with emicizumab in the real-world. Current guidelines recommend use of recombinant activated factor VII (rFVIIa) for treatment of BTBs in patients with inhibitors. Due to thrombotic events observed in the HAVEN 1 study, activated prothrombin complex concentrate (aPCC) should be used with caution. OBJECTIVES: The objective of this review is to identify and discuss real-world data on the frequency of BTBs and the safety of concomitant rFVIIa use in patients with inhibitors on emicizumab prophylaxis. METHODS: A search of the following databases was conducted on 15 July 2022: BIOSIS Previews® , Current Contents Search® , Embase® , MEDLINE® . Search terms included 'real world', 'haemophilia A', and 'emicizumab'. RESULTS AND CONCLUSIONS: Eleven relevant publications were identified (seven original research articles and four congress abstracts). The frequency of BTBs specifically for HA patients with inhibitors was described in three publications with 5%-56% patients on emicizumab reporting ≥1 bleeding episode. Treatment of these BTBs appeared to be managed according to relevant guidelines. Importantly, no thrombotic complications occurred during concomitant rFVIIa use. Due to the nature of real-world studies, direct comparison of the results between studies is limited. However, real-world data show that BTBs in inhibitor patients during emicizumab prophylaxis can be safely treated with rFVIIa.

Molecular Dynamics Simulation Study of the Selective Inhibition of Coagulation Factor IXa over Factor Xa.

Thromboembolic disorders, arising from abnormal coagulation, pose a significant risk to human life in the modern world. The FDA has recently approved several anticoagulant drugs targeting factor Xa (FXa) to manage these disorders. However, these drugs have potential side effects, leading to bleeding complications in patients. To mitigate these risks, coagulation factor IXa (FIXa) has emerged as a promising target due to its selective regulation of the intrinsic pathway. Due to the high structural and functional similarities of these coagulation factors and their inhibitor binding modes, designing a selective inhibitor specifically targeting FIXa remains a challenging task. The dynamic behavior of protein-ligand interactions and their impact on selectivity were analyzed using molecular dynamics simulation, considering the availability of potent and selective compounds for both coagulation factors and the co-crystal structures of protein-ligand complexes. Throughout the simulations, we examined ligand movements in the binding site, as well as the contact frequencies and interaction fingerprints, to gain insights into selectivity. Interaction fingerprint (IFP) analysis clearly highlights the crucial role of strong H-bond formation between the ligand and D189 and A190 in the S1 subsite for FIXa selectivity, consistent with our previous study. This dynamic analysis also reveals additional FIXa-specific interactions. Additionally, the absence of polar interactions contributes to the selectivity for FXa, as observed from the dynamic profile of interactions. A contact frequency analysis of the protein-ligand complexes provides further confirmation of the selectivity criteria for FIXa and FXa, as well as criteria for binding and activity. Moreover, a ligand movement analysis reveals key interaction dynamics that highlight the tighter binding of selective ligands to the proteins compared to non-selective and inactive ligands.

Comparison of coagulation parameters associated with fibrinogen concentrate and cryoprecipitate for treatment of bleeding in patients undergoing cytoreductive surgery for pseudomyxoma peritonei: Subanalysis from a randomized, controlled phase 2 study.

Background and Aims: The FORMA-05 study compared the efficacy and safety of human fibrinogen concentrate (HFC) versus cryoprecipitate for hemostasis in bleeding patients undergoing cytoreductive surgery for pseudomyxoma peritonei (PMP). This subanalysis explores coagulation parameters in the FORMA-05 patients, with a focus on the seven patients who developed thromboembolic events (TEEs). Methods: FORMA-05 was a prospective, randomized, controlled phase 2 study in which patients with predicted blood loss ≥2 L received HFC (4 g) or cryoprecipitate (two pools of five units), repeated as needed. Plasma fibrinogen, platelet count, factor (F) XIII, FVIII, von Willebrand Factor (VWF) antigen and ristocetin cofactor activity levels, EXTEM A20, FIBTEM A20, and endogenous thrombin potential (ETP) were measured perioperatively. Results: Fibrinogen, platelet count, EXTEM and FIBTEM A20, FXIII, FVIII, VWF levels, and ETP were maintained throughout surgery in both the HFC group (N = 21) and the cryoprecipitate group (N = 23). Seven TEEs were observed in the cryoprecipitate group. The two patients developing deep vein thromboses (DVT) appeared to have a procoagulant status preoperatively, with distinctively higher fibrinogen level, FIBTEM A20, and platelet levels, all of which persisted perioperatively. The five patients developing pulmonary embolism (PE) had slightly higher VWF levels preoperatively, with a disproportionate increase intraoperatively (postcryoprecipitate administration) and postoperatively. Conclusions: Patients treated with HFC versus cryoprecipitate showed broad overlaps in coagulation parameters. Patients with PE experienced a disproportionate VWF rise following cryoprecipitate administration, whereas patients developing DVT displayed a procoagulant status before and following surgery. Preoperative testing may allow these patients to be identified.

Gene Therapy Approaches for the Treatment of Hemophilia B.

In contrast to the standard enzyme-replacement therapy, administered from once per 7-14 days to 2-3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.

Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling.

BACKGROUND: Blood coagulation factor XIII (FXIII) promotes cross-linking between fibrin molecules at the final stage of the blood coagulation cascade. However, its expression in cells or tissues and function, particularly factor XIII subunit B (FXIII-B), remains controversial. Hemorrhagic FXIII deficiency following anti-interleukin-6 (IL-6) receptor antibody treatment has been reported in patients with rheumatoid arthritis (RA). Patients receiving this biologics have reduced FXIII activity when compared to the activity in those treated with other biologics. The relationship between pro-inflammatory cytokines and FXIII expression remains unknown. METHODS: To investigate the expression pattern of FXIII in synovial tissues, immunohistochemistry, RT-qPCR, and western blotting were performed. FXIII-A expressed monocyte-derived macrophages were treated with recombinant IL-6 and anti-IL-6 receptor antibody. RNA sequencing of FXIII-B-overexpressing cells was performed to clarify the function of FXIII-B. RESULTS: The immunohistochemical analysis of synovial tissues revealed that factor XIII subunit A (FXIII-A) was expressed in M2 macrophages, and FXIII-B was expressed in fibroblast-like synoviocytes. IL-6 stimulation upregulated FXIII-A expression in IL-4-induced monocyte-derived macrophages, and the anti-IL-6 receptor antibody suppressed FXIII-A expression. FXIII-B was more abundantly secreted in the supernatant of fibroblast-like synoviocytes compared with that of other cells. RNA sequencing showed that FXIII-B elevated the expression of genes associated with anti-apoptotic molecules and chemokines. CONCLUSIONS: Our findings highlight that synovial tissue is one of the sources of FXIII production. We also have demonstrated IL-6-dependent FXIII-A expression and the novel potential functions of FXIII-B.

The influence of lower-leg injury and knee arthroscopy on natural anticoagulants and fibrinolysis.

BACKGROUND: Patients with lower-leg injuries and those undergoing knee arthroscopy are at increased risk of developing venous thromboembolism. The mechanism is unknown, including the influence of lower-leg injury and knee arthroscopy on natural anticoagulant factors and fibrinolysis. OBJECTIVES: To study the effect of lower-leg injury and knee arthroscopy on plasma levels of anticoagulant and fibrinolytic factors. METHODS: We applied the following 2 designs to investigate this effect: a cross-sectional study for lower-leg trauma and a before-and-after study for knee arthroscopy. Plasma samples of POT-CAST- and POT-KAST-randomized clinical trial participants (collected shortly after lower-leg trauma or before or after arthroscopy) were analyzed for clot lysis time and levels of antithrombin, tissue factor pathway inhibitor, protein C, free protein S, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, antiplasmin, thrombin activatable fibrinolysis inhibitor, plasmin-antiplasmin, and D-dimer. For the effect of lower-leg injury, samples of 289 patients were compared with preoperative samples of 293 arthroscopy patients, acting as controls using linear regression and adjusting for age, sex, body mass index, comorbidities, and diurnal variation. For the effect of knee arthroscopy, mean changes were calculated for 277 patients using linear mixed models adjusted for diurnal variation. Parameters other than CLT and D-dimer were measured in smaller subsets. RESULTS: In lower-leg injury patients, most parameters were stable, whereas D-dimer increased. After arthroscopy, most parameters decreased (especially clot lysis time, D-dimer, plasminogen, and anticoagulant factors), whereas tissue plasminogen activator and thrombin activatable fibrinolysis inhibitor slightly increased. CONCLUSION: In contrast to lower-leg injury, knee arthroscopy was associated with decreased natural anticoagulant factor levels. Neither lower-leg injury nor knee arthroscopy affected in vivo fibrinolysis.

Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII.

Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production.

Case Report: Use of endobronchial Watanabe spigot and coagulation factor XIII supplementation in the treatment of persistent pneumothorax due to pneumocystis pneumonia with human immunodeficiency virus infection.

Pneumocystis jiroveceii pneumonia is one of the most common opportunistic infections associated with human immunodeficiency virus. Endobronchial Watanabe spigot has been recommended for refractory pneumothorax, even with persistant air leak despite continuous negative pressure control via thoracic drainage. Moreover, coagulation factor XIII is considered effective in wound healing.

Detection and Gene Mutation Analysis of Three Variations in Two Unrelated Chinese Hereditary Coagulation Factor XI Deficiency Families.

INTRODUCTION: Three variations including a novel F11 gene variation were detected in two unrelated Chinese families with coagulation factor XI deficiency, and their possible pathogenesis was elucidated. METHODS: The genomic DNA of the probands' pedigrees was extracted, and all exons and flanking sequences of F11 gene were subjected to PCR amplification and Sanger sequencing. ClustalX-2.1-win, Mutation Taster, and Swiss-Pdb Viewer software were used to analyze the conservation and impact of the variations on protein function and structure. RESULTS: DNA sequencing showed that the proband one had p.Gly350Glu and p.Trp501stop complex heterozygous variations, while the proband two took p.Pro338Leu and p.Trp501stop compound heterozygous variations. Conservation, structural, and functional analysis of variant amino acids indicated that these three variations were harmful and probably affected the structure and function of the variable protein. CONCLUSIONS: Three variations including p.Pro338Leu, p.Gly350Glu, and p.Trp501stop responsible for the reduction of the FXI activities were herein detected. Notably, the p.Pro338Leu variation was discovered for the first time in the world. Furthermore, the p.Gly350Glu was first reported in China.

Befovacimab, an anti-tissue factor pathway inhibitor antibody: Early termination of the multiple-dose, dose-escalating Phase 2 study due to thrombosis.

INTRODUCTION: Befovacimab (formerly BAY 1093884) is a fully human monoclonal antibody able to bind to tissue factor pathway inhibitor (TFPI) and developed as a non-replacement therapy for individuals with haemophilia A/B, with or without inhibitors. AIM: To assess the safety of multiple escalating doses of befovacimab in individuals with severe haemophilia A/B with or without inhibitors. METHODS: In this non-randomised, open-label Phase 2 study (NCT03597022), adult males with <1% factor VIII or <2% factor IX and ≥4 bleeds in the previous six months were enrolled in three dose cohorts (100/225/400 mg). Participants received befovacimab subcutaneously once weekly. The primary endpoint was safety; secondary endpoints included annualised bleeding rate (ABR) and pharmacokinetics/pharmacodynamics (PK/PD) of befovacimab. RESULTS: A total of 24 participants (n = 8 in each dose cohort) were treated for 2-47 weeks. Patients treated with 100 mg and 225 mg doses of befovacimab demonstrated improved bleeding control compared with pre-study bleeding rates, with a dose-dependent effect. Dosing was suspended and the study prematurely terminated following three drug-related thrombotic serious adverse events (SAEs): two at the 225 mg dose and one at the 400 mg dose. These occurred in the absence of bleeding episodes or concomitant use of replacement/bypass therapies. No laboratory abnormalities were observed, and PK/PD data did not show correlation between SAE occurrence and levels of circulating befovacimab or free TFPI. CONCLUSION: Despite favourable initial results from preclinical and clinical studies, a positive safety profile of befovacimab was not confirmed. The lack of SAE-related laboratory abnormalities or differentiating PK/PD characteristics in participants experiencing SAEs raises concerns about the predictability of thrombosis following befovacimab treatment and emphasises the need for further investigation into the therapeutic window of anti-TFPI treatment.

Coagulation factor inhibitors in COVID-19: From SARS-CoV-2 vaccination to infection.

Background: Recent reports have highlighted patients with COVID-19 and vaccine recipients diagnosed with coagulation factor inhibitors. This is challenging. as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been identified as a prothrombotic risk factor, with heparin treatment decreasing mortality. However, both infection and vaccination have been associated with immune-mediated hematologic abnormalities, including thrombocytopenia, further rendering these groups at risk for both hemorrhagic and thrombotic events. Objectives: We sought to characterize the incidence and clinical findings of coagulation factor inhibitors in patients with COVID-19 and vaccine recipients. Methods: We queried the US Centers for Disease Control and Prevention's Vaccine Adverse Event Reporting System (VAERS), a publicly accessible database, for reports of potential bleeding episodes or coagulation disturbances associated with SARS-CoV-2 vaccination. We performed an additional comprehensive literature review to identify reports of SARS-CoV-2 infection or vaccination-associated coagulation factor inhibitors. Results: VAERS data showed 58 cases of coagulation factor inhibitors, suggesting a rate of 1.2 cases per 10 million doses. A total of 775 articles were screened and 15 were suitable for inclusion, with six reports of inhibitors after vaccination and nine reports of inhibitors after infection. Inhibitor specificity for factor VIII was most common. Among reported cases, two patients expired due to hemorrhage, one following infection and one following vaccination. Conclusion: The incidence of coagulation factor inhibitors in patients with SARS-CoV-2 vaccination and infection appears similar to the general population. Nonetheless, given the importance of heparin therapy in treating hospital patients, recognition of inhibitors is important.

Analysis of current status of antithrombotic therapy in 136 patients with nonvalvular atrial fibrillation / 中国基层医药

Objective:To analyze the current status and effectiveness of different antithrombotic regimens in patients with non-valvular atrial fibrillation.Methods:The clinical data of 136 patients with non-valvular atrial fibrillation who received treatment in The Second People's Hospital of Yongkang from May 2018 to May 2019 were retrospectively analyzed. According to the treatment plan, they were divided into no antithrombosis group ( n = 32), rivaroxaban group ( n = 41), warfarin group ( n = 42), and aspirin group ( n = 21). Based on treatment of primary disease and complications, patients in the no antithrombosis group were not given anticoagulation or antiplatelet therapy, those in the rivarxaban group were given rivarxaban (10 mg/d), those in the warfarin group were given warfarin (2.5 mg/d), and those in the aspirin group were given aspirin (0.1 g/d). The incidence of thromboembolism and bleeding, all-cause mortality and readmission rate within 1 year were compared among groups. Results:There were significant differences in age, type of atrial fibrillation, coronary heart disease, heart failure, and hypertension among groups (all P < 0.05). There were no significant differences in sex, history of stroke/transient ischemic attack, and the percentage of patients developing diabetes mellitus and hyperlipidemia among groups (all P > 0.05). The incidence of thromboembolic events within 1 year in the no antithrombosis, rivaroxaban, warfarin, and aspirin groups were 21.87% (7/32), 7.32% (3/41), 2.38% (1/42), and 19.05% (4/21), respectively, and there were significant differences among groups ( χ2 = 8.98, P < 0.05). The 1-year incidence of bleeding events in the no antithrombosis, rivaroxaban, warfarin, and aspirin groups were 18.75% (6/32), 29.27% (12/41), 4.76% (2/42), 4.76% (61/21), respectively, and there were significant differences among groups ( χ2 =11.77, P < 0.05). There were no significant differences in the 1-year incidence of thromboembolism events and bleeding events among patients aged < 65 years, 65-75 years, and > 75 years (all P > 0.05), but there were significant difference in all-cause mortality and readmission rate ( χ2 = 6.76, 7.56, both P < 0.05). Conclusion:Early antithrombotic therapy is very important for patients with non valvular atrial fibrillation. The treatment regimens should be individualized, and the risk of death increases with age.

A Structure Based Study of Selective Inhibition of Factor IXa over Factor Xa.

Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein-ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than -8.0 kcal/mol in the FIXa-grids and higher than -7.5 kcal/mol in the FXa-grids.

A Case of Traumatic Cerebrospinal Fluid Rhinorrhea Successfully Treated Using Intravenous Factor XIII Administration.

Traumatic cerebrospinal fluid (CSF) rhinorrhea occurs around 2% of severe head trauma. We should find the fistula and surgically seal it or perform conservative therapy with bed rest with/without lumbar spinal CSF drainage. However, the fistula may not be identified, and treatment may sometimes be challenging. Blood coagulation factor XIII (factor XIII) is one of the blood coagulation factors. It also promotes fibroblast proliferation during the wound healing process. We herein reported a traumatic CSF rhinorrhea patient who was successfully treated using intravenous (IV) factor XIII administration. This report would contribute to the effectiveness of factor XIII administration in the treatment of traumatic CSF rhinorrhea. A 58-year-old man fell from a height of 1.5 meters and hit his forehead. He presented with numbness in both upper limbs but no paresis. Neck magnetic resonance imaging (MRI) revealed cervical spinal cord injury without a cervical vertebral or cranial fracture. He was conservatively treated and discharged after three months. He had been aware of rhinorrhea since the trauma but was treated as allergic rhinitis. A year after the trauma, he was diagnosed with traumatic CSF rhinorrhea. We confirmed a bit of rhinorrhea despite the seven-day bedrest, so we intravenously administered 240 international units of factor XIII every day for 10 days. After 10 days, there was no rhinorrhea at all, and the patient was discharged on the 28th day. He has had no recurrence of rhinorrhea after a three-month follow-up. Factor XIII administration might be useful to treat traumatic CSF rhinorrhea.

Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach.

BACKGROUND: Up to 30% of hemophilia A patients develop inhibitory antibodies against the infused factor VIII (FVIII). The development of a deimmunized FVIII is an unmet high medical need. Although improved recombinant FVIII (rFVIII) products evolved within the last years, the immunogenicity has not been solved. A deimmunized FVIII could reduce the probability of inhibitor development, providing safer therapy. OBJECTIVE: To develop a deimmunized FVIII molecule by modifying major histocompatibility complex (MHC) class II presentation, leading to a functional but less immunogenic molecule. METHODS: We performed (1) in silico prediction of potentially immunogenic T cell epitopes and their modification by amino acid substitutions in the FVIII sequence, (2) evaluation of functional and structural similarity of the modified rFVIII to unmodified FVIII and registered products, and (3) confirmation of the reduced immunogenicity by in vitro testing. RESULTS: A partially deimmunized fully functional FVIII molecule incorporating 19 amino acid substitutions was generated. The substitutions led to a reduction of the immunogenicity score, indicating a reduced immunogenicity based on in silico calculations. This was confirmed in an in vitro dendritic cell (DC)--T cell assay. Using this assay, cells from healthy donors proved the significantly reduced immunogenicity of the modified FVIII variant by revealing less proliferation of T helper cells to this variant than to the unmodified FVIII. CONCLUSION: In silico predictions resulted in a partially deimmunized FVIII. This FVIII is fully functional and was demonstrated to be less immunogenic in in vitro testing. This approach may result in a reduction of the inhibitor risk for patients with hemophilia A.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Šresolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Case Report: An Adult Patient With Deficiency of Adenosine Deaminase 2 Resembled Unilateral Frosted Branch Angiitis.

Purpose: Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive systemic autoinflammatory disorder. We describe a rare case of an adult patient with DADA2 who presented with unilateral frosted branch angiitis (FBA) combined with branch retinal vein occlusion and panuveitis. Method: This paper is a clinical case report. Results: A 31-year-old male patient complained of blurred vision in his right eye for 2 days. His fundus examination showed FBA combined with branch retinal vein occlusion and panuveitis. He had a medical history of intermittent and recurrent fever, skin rash and aphthous ulcer for 5 years, and lacunar infarction for 1 month. Laboratory examinations showed hypogammaglobulinemia and mild prolonged activated partial thromboplastin time (APTT). Brain magnetic resonance imaging (MRI) revealed old lacunar infarction in the right basal ganglia and the lateral ventricle and fresh lacunar infarction in the right pons, respectively. The perivascular sheathing of FBA and macular edema were resolved after steroid administration and treatment of intravitreal anti-VEGF injection. During the period of follow-up, the patient subsequently suffered from recurrence of strokes, abnormality of coagulation function, sudden hearing loss of the left ear, and diplopia. His gene sequencing results demonstrated several deletion mutations in ADA2, and the diagnosis of DADA2 was eventually confirmed. Conclusions: FBA represents a very rare ocular feature of DADA2 and may in some cases be the presenting manifestation. Therefore, ophthalmologists need to be aware of this rare autoinflammatory disease.