Systemic inflammation biomarkers during angioedema attacks in hereditary angioedema.

Background: Hereditary angioedema (HAE) is a rare disease characterized by localized and self-limited angioedema (AE) attacks. A local increase of bradykinin (BK) mediates AE attacks in HAE, however the role of inflammation in HAE has been poorly explored We aim to analyze the role of inflammatory mediators in HAE patients during AE attacks. Methods: Patients with a confirmed HAE diagnosis due to C1 inhibitor deficiency (HAE-C1INH) or patients F12 gene mutations (HAE-FXII) attending to our outpatient clinic between November-2019 and May-2022 were included. Demographic and clinical characteristics were analyzed. Blood samples were collected both during symptom-free periods (baseline) and during HAE attacks, and acute phase reactants (APR), such as serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), D-Dimer and white blood cells were measured. Results: Seventy-eight patients were enrolled in the study, with a predominant representation of women (76%, n=59), and a mean age of 47.8 years (range 6-88). Among them, 67% (n=52) of patients had HAE-C1INH (46 classified as type 1 and 6 as type 2) while 33% (n=26) had HAE-FXII. During attack-free periods, the majority of patients exhibited normal levels of SAA, ESR, D-dimer, ACE and WCC. However, in a subset of patients (16% for SAA, 18% for ESR, and 14.5% for D-dimer), elevations were noted at baseline. Importantly, during HAE attacks, significant increases were observed in SAA in 88% of patients (p< 0.0001 vs. baseline), in ESR in 65% (p= 0.003 vs. baseline) and D-dimer in 71% (p=0.001 vs. baseline) of the patients. A comparison between baseline and acute attack levels in 17 patients revealed significant differences in SAA AA (p<0. 0001), ESR (p<0.0001) and D-dimer (p= 0.004). No significant differences were observed in CRP (p=0.7), ACE (p=0.67) and WCC (p=0.54). These findings remained consistent regardless of HAE type, disease activity or location of angioedema. Conclusion: The systemic increase in APR observed during HAE attacks suggests that inflammation extends beyond the localized edematous area. This finding underscores the potential involvement of inflammatory pathways in HAE and highlights the need for further investigation into their role in the pathophysiology of HAE.

[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.

Tackling the Root Cause of Surface-Induced Coagulation: Inhibition of FXII Activation to Mitigate Coagulation Propagation and Prevent Clotting.

Factor XII (FXII) is a zymogen present in blood that tends to adsorb onto the surfaces of blood-contacting medical devices. Once adsorbed, it becomes activated, initiating a cascade of enzymatic reactions that lead to surface-induced coagulation. This process is characterized by multiple redundancies, making it extremely challenging to prevent clot formation and preserve the properties of the surface. In this study, a novel modulatory coating system based on C1-esterase inhibitor (C1INH) functionalized polymer brushes, which effectively regulates the activation of FXII is proposed. Using surface plasmon resonance it is demonstrated that this coating system effectively repels blood plasma proteins, including FXII, while exhibiting high activity against activated FXII and plasma kallikrein under physiological conditions. This unique property enables the modulation of FXII activation without interfering with the overall hemostasis process. Furthermore, through dynamic Chandler loop studies, it is shown that this coating significantly improves the hemocompatibility of polymeric surfaces commonly used in medical devices. By addressing the root cause of contact activation, the synergistic interplay between the antifouling polymer brushes and the modulatory C1INH is expected to lay the foundation to enhance the hemocompatibility of medical device surfaces.

Factor XII Deficiency in Mexico: High Prevalence in the General Population and Patients with Venous Thromboembolic Disease.

INTRODUCTION: Thrombosis is one of the leading causes of morbidity and mortality worldwide. Venous thromboembolic disease (VTD) is considered a new epidemic. FXII deficiency is supposed to be a cause of thrombosis. To search for unknown causes of thrombosis in our population, our aim was to determine if FXII deficiency can be considered a risk factor for VTD. METHODS: Young adult Mexican patients with at least one VTD episode and healthy controls were included in this prospective, observational, controlled study. Liver and renal function tests, blood cytometry, and blood coagulation assays were performed. Plasma FXII activity and its concentration were evaluated. RESULTS: Over a two-year period, 250 patients and 250 controls were included. FXII activity was significantly lower in the control group compared to patients with VTD (p = 0.005). However, percentage of patients and controls with FXII deficiency was 8.8 and 9.2%, respectively (p = 1.000). No significant association was found between FXII deficiency and VTD (p = 1.0). FXII plasma concentration was lower in controls vs. patients with VTD: 4.05 vs. 6.19 ng/mL (p <0.001). Percentage of patients with low FXII plasma concentration was 1.6% and 6.0% in patients and controls, respectively (p = 0.010). CONCLUSIONS: FXII deficiency is a frequent finding in patients with VTD and controls in Mexico. Some patients with FXII deficiency had normal APTT result, an effect not described above. FXII plasma concentration was lower in patients with low activity.

Homozygous missense variant F12 (Gly506Asp) associated with severe factor XII deficiency: a case report.

BACKGROUND: Factor XII deficiency can be related to either homozygous or compound heterozygous pathogenic variants in the F12 gene. The disease is commonly known as Hageman trait and is inherited in both autosomal recessive or dominant patterns. Clinically, factor XII deficiency is not associated with bleeding but conversely has been linked to thrombotic events, recurrent pregnancy loss, and hereditary angioedema. Molecular data of F12 deficiency are scarce and have revealed varying results between cases. However, most of the reported variants are missense mutations, gross deletions, or small insertion. Factor XII deficiency has been reported in the Saudi population in several studies, either as isolated case reports or included within the studies of rare bleeding factors deficiency. However, molecular data are lacking as no case report of genetic studies related to factor XII deficiency has been published in our local population, to the best of our knowledge. CASE REPORT: Herein we describe a homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) in a 36-year-old Saudi multiparous female referred from the surgical clinic with significantly high activated partial thromboplastin time during preoperative assessment for sleeve gastrectomy. The patient had no history of bleeding episodes during the previous deliveries nor any tooth extractions. She had single event of spontaneous abortion during the 15th week of gestation without any bleeding complication. There was no history of thrombosis or skin manifestations, and she was not taking any medicines. There was no family history of bleeding or thrombosis. Family history revealed consanguinity as the parents are first-degree cousins. Physical examination was unremarkable. Upon investigation, the prolonged activated partial thromboplastin time was fully corrected by a 1:1 mixing study with normal pool plasma while lupus anticoagulant tests were negative. Factor assays and von Willebrand factor tests are all within normal ranges except for factor XII, which was severely deficient. A homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) was identified. CONCLUSION: F12 (5:176,830,269 G>A; p.Gly506Asp) variant is likely to be a pathogenic variant among homozygous factor XII-deficient patients. Genetic counseling and management of the patients and families should be based on clinical evaluation.

Analysis of an Inherited FXII Deficiency Pedigree Associated with Double Heterozygous Mutations in the F12 Gene.

This article is intended to identify the potential mutations of the FXII gene (F12) in an inherited FXII deficiency pedigree and illuminate the pathogenesis of the disease. The coagulation FXII activity (FXII:C) and FXII antigen (FXII:Ag) were inspected by one-stage clotting assay and enzyme-linked immunosorbent assay(ELISA), respectively. Polymerase chain reaction amplification (PCR) was performed and the F12 gene was sequenced directly. A molecular model of FXIIprotein was established for further analysis. ClustalX-2.1-win and online bioinformatic software were used to estimate the conservatism and possible impact of the protein change. The proband presented prolonged APTT(180 s) and extreme low FXII:C and FXII:Ag (both < 1%, reference range:72-113%). A compound heterozygous were found by the direct sequencing of the F12 gene. One was a deletion mutation c.1792_1796delGTCTA, which is a novel mutation; the other was an insertion mutation, c.1092_1093insC. Bioinformatic and modeling analyses indicated that the the two frameshift mutations may be deleterious and possibly alter the structure and the function of the protein. The mutations c.1792_1796delGTCTA and c.1092_1093insC could be the main causes of reducing FXII in this pedigree, and c.1792_1796delGTCTA mutation was the first report in the world.

Myeloid lineage cells evince distinct steady-state level of certain gene groups in dependence on hereditary angioedema severity.

Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity. SERPING1 showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members CXCL8, 10 and 11 were downregulated, while other genes such as FCGR1A, or long non-coding RNA NEAT1 were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.

In vitro evaluation of blood plasma coagulation responses to four medical-grade polyurethane polymers.

Segmented polyurethane (PU) block copolymers are widely used in implantable cardiovascular medical devices due to their good biocompatibility and excellent mechanical properties. More specifically, PU Biospan MS/0.4 was used in ventricular assist devices over the past decades. However, this product is being discontinued and it has become necessary to find an alternative PU biomaterial for application in cardiovascular devices. One important criterion for assessing cardiac biomaterials is blood compatibility. In this study, we characterized the surface properties of four medical-grade PU biomaterials: Biospan MS/0.4, BioSpan S, BioSpan 2F, and CarboSil 20 80A, including surface chemistry, topography, microphase separation structure and wettability, and then measured the blood plasma coagulation responses using bovine and human blood plasma. Results showed that BioSpan 2F contains high amounts of fluorine and has the lowest surface free energy while the other materials have surfaces with silicone present. An in vitro coagulation assay shows that these materials demonstrated improved blood coagulation responses compared to the polystyrene control and there were no significant differences in coagulation time among all PU biomaterials. The chromogenic assay showed all PU materials led to low FXII contact activation, and there were no significant differences in FXII contact activation, consistent with plasma coagulation responses.

An Emerging Role for Type I Interferons as Critical Regulators of Blood Coagulation.

Type I interferons (IFNs) are central mediators of anti-viral and anti-bacterial host defence. Detection of microbes by innate immune cells via pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and cGAS-STING, induces the expression of type I IFN-stimulated genes. Primarily comprising the cytokines IFN-α and IFN-ß, type I IFNs act via the type I IFN receptor in an autocrine or exocrine manner to orchestrate rapid and diverse innate immune responses. Growing evidence pinpoints type I IFN signalling as a fulcrum that not only induces blood coagulation as a core feature of the inflammatory response but is also activated by components of the coagulation cascade. In this review, we describe in detail recent studies identifying the type I IFN pathway as a modulator of vascular function and thrombosis. In addition, we profile discoveries showing that thrombin signalling via protease-activated receptors (PARs), which can synergize with TLRs, regulates the host response to infection via induction of type I IFN signalling. Thus, type I IFNs can have both protective (via maintenance of haemostasis) and pathological (facilitating thrombosis) effects on inflammation and coagulation signalling. These can manifest as an increased risk of thrombotic complications in infection and in type I interferonopathies such as systemic lupus erythematosus (SLE) and STING-associated vasculopathy with onset in infancy (SAVI). We also consider the effects on coagulation of recombinant type I IFN therapies in the clinic and discuss pharmacological regulation of type I IFN signalling as a potential mechanism by which aberrant coagulation and thrombosis may be treated therapeutically.

Factor XII deficiency: a clinical and molecular genetic study.

Factor XII deficiency is a rare inherited disorder caused by clotting factor XII (FXII, F12) deficiency. It is often asymptomatic but can have both thrombotic and haemorrhagic symptoms. The aim of this study was to describe the spectrum of F12 gene mutations in a Russian population and learn more about the relationship between F12 variants and clinical phenotypes. We obtained and analysed genetic and clinical data from 33 apparently unrelated patients with FXII plasma levels below 60% and genetic data from 26 healthy controls with no history of FXII deficiency. Forty mutant alleles and six different deleterious substitutions were identified. Of these substitutions, three were major in the Russian population (c.-62C > T, c.-57G > C and c.1532-1G > A, total frequency 92.5%) and the three others (p.615 del C, c.1180_1181delCA, and CD218 TAT- > CAT p.Tyr218His) were rare and novel in the world population. Eight patients with mild FXII deficiency were found to be homozygous for a hypomorphic variant of functional polymorphism C46T and have no other deleterious substitutions in the F12 gene. Contrary to data in the literature, our study showed that mild haemorrhagic manifestations are common among patients with FXII deficiency.

Oral FXIIa inhibitor KV998086 suppresses FXIIa and single chain FXII mediated kallikrein kinin system activation.

Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC50s of these compounds linearly correlated for rFXIIa and rFXII-T (R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC50 = 7.2 nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage (p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases.

gC1qR/C1qBP/HABP-1: Structural Analysis of the Trimeric Core Region, Interactions With a Novel Panel of Monoclonal Antibodies, and Their Influence on Binding to FXII.

The protein gC1qR/C1qBP/HABP-1 plays an essential role in mitochondrial biogenesis, but becomes localized at the cellular surface in numerous pathophysiological states. When this occurs on endothelial cells, surface-exposed gC1qR activates the classical pathway of complement. It also promotes assembly of a multi-protein complex comprised of coagulation factor XII (FXII), pre-kallikrein (PK), and high-molecular weight kininogen (HMWK) that activates the contact system and the kinin-generating system. Since surface-exposed gC1qR triggers intravascular inflammatory pathways, there is interest in identifying molecules that block gC1qR function. Here we further that objective by reporting the outcome of a structure/function investigation of gC1qR, its interactions with FXII, and the impact of a panel of monoclonal anti-gC1qR antibodies on FXII binding to gC1qR. Although deletion mutants have been used extensively to assess gC1qR function, none of these proteins have been characterized structurally. To that end, we determined a 2.2 Å resolution crystal structure of a gC1qR mutant lacking both of its acidic loops, but which retained nanomolar-affinity binding to FXII and FXIIa. This structure revealed that the trimeric gC1qR assembly was maintained despite loss of roughly thirty residues. Characterization of a novel panel of anti-gC1qR monoclonal antibodies identified several with biochemical properties distinct from previously described antibodies, as well as one which bound to the first acidic loop of gC1qR. Intriguingly, we found that each of these antibodies could partly inhibit binding of FXII and FXIIa to gC1qR. Based on these results and previously published studies, we offer new perspectives for developing gC1qR inhibitors.

Pregnancy in Patients With Hereditary Angioedema and Normal C1 Inhibitor.

Background: HAE with normal C1 inhibitor (HAE-nC1-INH) has been identified as a bradykinin mediated angioedema. Estrogens are one of the main trigger factors. Pregnancy in HAE with C1 inhibitor deficiency showed variable course, however, few reports are available for HAE-nC1-INH. We evaluated the course of pregnancies in women diagnosed with HAE-nC1-INH. Methods: Women with diagnosis of HAE-nC1-INH according to the following criteria: clinical manifestations similar to HAE-C1-INH, normal biochemical evaluation and family history were included. A questionnaire about pregnancies was applied after consent. Genetic evaluation for known mutations was performed in all patients. Results: A total of 45 pregnancies occurring in 26 HAE-nC1-INH patients were evaluated (7/26 patients with F12 variant). Spontaneous abortion was reported in 8/45 (17.8%) pregnancies. Onset of attacks started before the pregnancy in 18/26 patients; during the pregnancy in 2/26; and after the pregnancy in 6/26. HAE attacks occurred in 24/37 pregnancies (64,7%): during the 1st trimester in 41.7%; 2nd trimester in 12.5%; 3rd trimester in 20.8%; 1st and 3rd trimesters in 4.2% and during the whole pregnancy in 20.8%. Among 15/18 patients who had attacks before pregnancy, symptoms persisted with worsening in 9/15; improvement in 4/15; no change in 1/15, and no response in 1/15. Conclusions: The occurrence of abortion in HAE-nC1-INH was similar to the expected for not affected women. The 1st trimester of the pregnancy was more symptomatic for HAE-nC1-INH women. Considering the strong relevance of estrogens in HAE-nC1-INH, pregnancy could worsen the course of disease.

C1 esterase inhibitor-mediated immunosuppression in COVID-19: Friend or foe?

From asymptomatic to severe, SARS-CoV-2, causative agent of COVID-19, elicits varying disease severities. Moreover, understanding innate and adaptive immune responses to SARS-CoV-2 is imperative since variants such as Omicron negatively impact adaptive antibody neutralization. Severe COVID-19 is, in part, associated with aberrant activation of complement and Factor XII (FXIIa), initiator of contact system activation. Paradoxically, a protein that inhibits the three known pathways of complement activation and FXIIa, C1 esterase inhibitor (C1-INH), is increased in COVID-19 patient plasma and is associated with disease severity. Here we review the role of C1-INH in the regulation of innate and adaptive immune responses. Additionally, we contextualize regulation of C1-INH and SERPING1, the gene encoding C1-INH, by other pathogens and SARS viruses and propose that viral proteins bind to C1-INH to inhibit its function in severe COVID-19. Finally, we review the current clinical trials and published results of exogenous C1-INH treatment in COVID-19 patients.

Mutant plasminogen in hereditary angioedema is bypassing FXII/kallikrein to generate bradykinin.

Hereditary angioedema (HAE) is characterized by recurrent localized edema in various organs, which can be potentially fatal. There are different types of hereditary angioedema, which include genetic deficiency of C1 inhibitor (C1-INH) and hereditary angioedema with normal C1-INH (HAEnCI). In HAEnCI patients mutations have been identified in the F12, PLG, KNG1, ANGPT1, MYOF, and HS3ST6 genes. The release of bradykinin from kininogen via the kallikrein-kinin system (KKS) has been shown to be the main mediator in HAE-FXII, but for HAE-PLG there are only first indications how the PLG mutations can result in bradykinin release. Here we identified in a multi-generation HAE-PLG family an additional F12 mutation, resulting in the loss of one F12 allele. There were no differences in the clinical presentation between HAE-PLG patients with and without the additional F12 mutation, thus we concluded that the kallikrein-kinin system is bypassed in HAE-PLG. Structural modeling and in vitro assays using purified proteins confirmed the PLG mutation c.988A>G; p.K330E to be a gain of function mutation resulting in an increased bradykinin release by direct cleavage of high molecular weight kininogen (HMWK). Thus, we can provide clinical and experimental evidence that mutant plasminogen in HAE-PLG is bypassing FXII/kallikrein to generate bradykinin.

The Standard Point-of-Care Hemochron Jr. ACT+ Test in Monitoring Heparin Administration for Cardiopulmonary Bypass in Severe Factor XII Deficiency.

Coagulation factor XII (FXII) is a plasma serine protease that belongs to the contact activation complex responsible for initiating the intrinsic coagulation pathway. FXII deficiency is a rare congenital disorder that is not associated with an increased tendency for bleeding. However, as contact activation is impaired in FXII deficiency, both the celite- and kaolin-initiated activated clotting time (ACT) measurements are prolonged markedly, which poses a challenge for anticoagulation monitoring in patients undergoing cardiac surgery. The authors successfully have used the standard Hemochron Jr. ACT+ test, which is activated by silica and phospholipid in addition to kaolin, to monitor anticoagulation for cardiopulmonary bypass in two patients with severe FXII deficiency. The ACT+ test showed low baseline values, increased adequately in response to heparin, and decreased to baseline after protamine. Importantly, there was no abnormal intra- or postoperative bleeding nor any thrombotic complications. Furthermore, in vitro dose-response ACT+ testing of FXII-deficient blood with increasing heparin concentrations supports the use of ACT+ in FXII deficiency.

Coagulation factor-XII induces interleukin-6 by primary lung fibroblasts: a role in idiopathic pulmonary fibrosis?

The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Plasma levels of FXII were measured by ELISA in patients with IPF and in age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and Western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Compared with 35 healthy donors, plasma levels of FXII were not higher in patients with IPF (n = 27, P > 0.05). Tissue FXII was elevated in IPF (n = 11) and increased numbers of FXII+ cells were found in IPF (n = 8) lung tissue compared with nondiseased controls (n = 6, P < 0.0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII induced IL-6 production via PAR-1 and NF-κB. FXII induced migration of fibroblasts in a concentration-dependent manner. FXII is normally confined to the circulation but it leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis.

Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation.

BACKGROUND: Coagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. METHODS: We performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced FXII activation using a chromogenic substrate assay. FINDINGS: FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasmaaac acafajföeFXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. INTERPRETATION: Collectively, our study supports that the NET/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both NETs and FXIIa may offer a potential novel therapeutic strategy. FUNDING: This study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

Hormonal Effects on Urticaria and Angioedema Conditions.

Women appear to be more frequently affected with urticaria and angioedema. Sex hormones are believed to have an important mechanistic role in regulating pathways involved in these conditions. This effect is likely nonspecific for chronic spontaneous urticaria (CSU) or many forms of angioedema (AE), because many other chronic diseases such as asthma are also affected by sex hormones. The role of sex hormones has been better elucidated for hereditary AE, because they have been shown to have multiple effects including upregulation of FXII, an important activator of the kallikrein pathway. However, their role in the underlying pathogenesis for CSU is less clear. Autoimmunity is clearly linked to CSU, which is more common in women. This suggests that sex hormones could act as adjuvants in activating or upregulating autoimmune pathways. The purpose of this review is to discuss in detail the role of sex hormones in CSU and AE and how a better understanding of the impact hormones has on these conditions might lead to new treatment advancements with better clinical outcomes.

Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant.

Hereditary angioedema due to pathogenic FXII variants (HAE-FXII) is a rare dominant disease caused by increased activation of the plasma contact system. The most prevalent HAE-FXII variant, c.1032C > A p.Thr309Lys (FXII309Lys), results in a smaller FXII protein with increased sensitivity to fluid-phase activation by poorly understood mechanisms. We aimed to investigate the functionality of the FXII309Lys variant in 33 HAE-FXII patients, 25 healthy controls and 46 patients with congenital disorders of glycosylation (CDG). Activation of the plasma contact system was assessed by western blot and amidolytic assay in basal conditions or after treatment with either artificial or physiological activators. Recombinant wild-type and FXII309Lys variants were expressed in S2 insect (Drosophila) cells. Amidolytic and fibrin generation assays were performed in fresh plasma samples. FXII309Lys samples exhibited an increased electrophoretic mobility comparable with N-glycan-deficient FXII from CDG patients and asialo-FXII generated by neuraminidase treatment. They presented increased sensitivity to activation by dextran sulphate and silica which resulted in the generation of an aberrant 37-kDa heavy chain. We did not observe increased susceptibility of FXII309Lys to proteolysis by exogenous or tPA-generated plasmin. However, both exogenous and endogenous thrombin cleaved the FXII309Lys variant, releasing a 37-kDa fragment and resulting in enhanced proteolytic activation on the fluid phase. This model supports a sequential proteolytic activation process involving thrombin priming of FXII309Lys, followed by kallikrein cleavage and generation of active ßFXIIa. The present results and the observation that angioedema episodes in HAE-FXII patients occur predominantly during hypercoagulable situations suggest a key role for thrombin.