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Background Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. Objective The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. Methods A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. Results These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times. Conclusion These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.

Key Points Hemolysis releases 5.8S rRNA and activates blood coagulation in human and zebrafish via FXII and Hgfac, respectively.Only the 3'-end 26 nucleotides of 5.8S rRNA were necessary and sufficient for this activation., Visual Abstract, Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. It has previously been shown that the RNA released from damaged blood cells activates clotting. However, the nature of the RNA released from hemolysis is still elusive. We found that after hemolysis, red blood cells from both zebrafish and humans released RNA that contained mostly 5.8S ribosomal RNA (5.8S rRNA), This RNA activated coagulation in zebrafish and human plasmas. By using both natural and synthetic 5.8S rRNA and its truncated fragments, we found that the 3'-end 26-nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor, blocked 3'-26 RNA–mediated coagulation activation in the plasma of both zebrafish and humans. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA– and 3'-26 RNA–mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activated normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via an FXII-like protein. Because zebrafish have no FXII and because hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. To summarize, we identified that an rRNA released in hemolysis activates clotting in human and zebrafish plasma. Furthermore, we showed that fish Hgfac plays a role in rRNA-mediated activation of coagulation.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI., Blood coagulation is started by contact to surfaces and this is the principle for a commonly used diagnostic clotting test, aPTT. Here, the authors identify the structure in coagulation factor XII that initiates surface-driven coagulation and use the information to develop improved aPTT assays.

Coagulation is controlled by a delicate balance of prothrombotic and antithrombotic mechanisms, to prevent both excessive blood loss from injured vessels and pathologic thrombosis. The liver plays a pivotal role in hemostasis through the synthesis of plasma coagulation factors and their inhibitors that, in addition to thrombosis and hemostasis, orchestrates an array of inflammatory responses. As a result, impaired liver function has been linked with both hypercoagulability and bleeding disorders due to a pathologic balance of pro- and anticoagulant plasma factors. At sites of vascular injury, thrombus propagation that finally may occlude the blood vessel depends on negatively charged biopolymers, such as polyphosphates and extracellular DNA, that provide a physiological surface for contact activation of coagulation factor XII (FXII). FXII initiates the contact system that drives both the intrinsic pathway of coagulation, and formation of the inflammatory mediator bradykinin by the kallikrein–kinin system. Moreover, FXII facilitates receptor-mediated signalling, thereby promoting mitogenic activities, angiogenesis, and neutrophil stimulation with implications for liver diseases. Here, we summarize current knowledge on the FXII-driven contact system in liver diseases and review therapeutic approaches to target its activities during impaired liver function.

The effect of anabolic–androgenic steroid (AAS) abuse on the contact activation system (CAS) is not known in detail. We hypothesized that current AAS abuse reduces the kallikrein-generating capacity of CAS significantly and investigated the impact of AAS on the proteins and capacity of CAS in current and former AAS abusers and healthy age-matched controls. Men 18 to 50 years of age were included as current AAS abusers, former AAS abusers, or controls. Blood samples were collected after overnight fasting. Kallikrein generation (lag time, peak height, and endogenous kallikrein potential [EKP]), coagulation factor XII (FXII), prekallikrein, high-molecular-weight kininogen (HK), and Complement C1 esterase inhibitor (C1inh) were assessed. Groups were compared by analysis of variance or Kruskal–Wallis test and probabilities were corrected for multiple comparisons. Associations were evaluated by linear regression models. The EKP was significantly reduced in current (n = 37) AAS abusers (984 ± 328 nmol/L × min) compared with former (n = 33) abusers (1,543 ± 481 nmol/L × min) and controls (n = 30) (1,521 ± 339 nmol/L × min), p

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30 days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Összefoglaló. Bevezetés: Egy új, számítógép által segített betegminta-asszociációs analízis eredménye szerint a COVID–19 tüneteinek kialakításában kiemelt tényezőként jelenik meg a bradikinin. Eszerint a bradikinin lebontása lelassul az angiotenzinkonvertáló enzim aktivitásának csökkenése miatt, ami jelentősen megemelkedő bradikininszinthez vezet a tüdőben. Nem merült fel azonban a véralvadási faktorok lehetséges szerepe a „bradikininviharban”, annak ellenére, hogy az idősebb cardiovascularis betegekben aktiválódó XII-es faktor és a C1-észteráz-inhibitor (C1INH) alacsony szintje nagy mennyiségű bradikinin képződéséhez vezethet. Módszer: Átfogó irodalmi áttekintés. Eredmények: 1) A vírus által fertőzött, sérült endotheliumsejtek felülete az a hely, amellyel érintkezve elindulhat a XII-es véralvadási faktor aktivációja – ez serkenti a prekallikrein/kallikrein/kinin rendszert, és bradikininképződést okoz. Ez a folyamat megtörténik a súlyos vese- és tüdőkárosodást okozó hantavírus-fertőzésekben. 2) Idős betegekben az atherosclerosis miatt többszörösen sérült, merev, „stiff” erek endotheliumfelszínein jóval magasabb lehet a XII-es faktor kontakt úton történő aktivációja, mint a fiatal egyének ereiben. Ez a tény egyik oka lehet az idős, cardiovascularis betegek körében tapasztalt magasabb halálozásnak. Következtetés: Az aktivált XII-es véralvadási faktor célzott gátlása újabb gyógyítási lehetőség lehet a SARS-CoV-2-fertőzött idős betegekben. Jelenleg már hatásosnak bizonyult a bradikininképzést gátló C1INH-nak, továbbá a bradikininreceptor-gátlóknak az adása is. Orv Hetil. 2020; 161(50): 2099–2103. Summary. Introduction: Bradykinin was implicated in a new complex model of pathomechanism leading to the symptoms of COVID-19 created by a computer-assisted association analysis. According to this model, the decrease in angiotensin-converting enzyme expression leads to impaired bradykinin elimination and subsequent enrichment in the lungs. However, there is no mentioning of the importance of blood coagulation factor XII in increased bradykinin production, in spite of its age-dependent activation and the lower level of C1-esterase inhibitor (C1INH). Activated factor XII may be an important contributor to the “bradykinin storm” in elder cardiovascular patients. Method: Literature review. Results: 1) Activation of the coagulation factor XII on the surface of SARS-CoV-2 infected endothelial cells may trigger the prekallikrein/kallikrein/kinin system producing bradykinin. Such process is taking place in hantavirus infections causing severe lung and kidney damages. 2) The endothelial system is dysregulated in elderly patients, resulting in potentially higher factor XII activities on the surface of damaged endothelial cells in the stiffened arteries. This can contribute to the higher mortality rates in the elderly. Conclusion: The targeted inhibition of activated blood coagulation factor XII may represent a new therapeutic target for COVID-19, especially for elder patients. Recently, beneficial results have already been observed by the clinical applications of recombinant C1INH and bradykinin receptor antagonists. Orv Hetil. 2020; 161(50): 2099–2103.

INTRODUCTION: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. OBJECTIVE: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. METHODS: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. RESULTS: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. CONCLUSION: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

Background Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. Objective To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. Methods Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. Results and conclusions We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+ -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.

International audience; Sex-dependent differences in immunity and coagulation play an active role in the outcome of community-acquired pneumonia (CAP). Contact phase proteins act at the crossroads between inflammation and coagulation thus representing a point of convergence in host defense against infection. Here, we measured the levels of factor XII (FXII), FXIIa-C1 esterase inhibitor (C1INH) complexes, and high-molecular-weight kininogen (HK) in plasma of patients with CAP and correlated them to clinical disease severity. Levels of FXIIa-C1INH/albumin ratio were elevated, irrespective of sex, in plasma of patients with CAP ( n = 139) as compared with age-matched donors ( n = 58). No simultaneous decrease in FXII levels, indicating its consumption, was observed. Stratification by sex revealed augmented FXII levels in plasma of women with CAP as compared with sex-matched donors yet no apparent differences in men. This sex-specific effect was, however, attributable to lower FXII levels in female donors relative to men donors. Plasma estradiol levels mirrored those for FXII. Levels of HK/albumin ratio were decreased in CAP plasma as compared with donors, however, after stratification by sex, this difference was only observed in women and was related to higher HK/albumin values in female donors as opposed to male donors. Finally, strong negative correlation between plasma levels of HK/albumin ratio and CAP severity, as assessed by CRB65 score, in males and females was observed. Our study identifies sex-dependent differences in plasma levels of the contact phase proteins in elderly subjects that may contribute to specific clinical outcomes in CAP between men and women.

INTRODUCTION Coagulation factor XII (FXII) plays a role in thrombin generation, fibrinolysis, inflammation, angiogenesis, chemotaxis and diapedesis. FXII deficiency is not associated with bleeding risk unlike other coagulation factors. MATERIALS/METHODS We investigated thrombin generation assay (TGA) profile modification in FXII deficiency and the correlation with TGA and deficiency severity. TGA was performed in platelet poor plasma (PPP) with tissue factor (1 pmol/L) and phospholipid (4 µmol/L) standardized concentration. Thrombin generation profiles were compared in 54 patients with FXII deficiency, 25 healthy controls and 23 patients with hemophilia A (factor VIII (FVIII) deficiency. Patients with FXII deficiency were classified in three groups based on FXII activity (30-50%, 10-29%

OBJECTIVES Extracorporeal membrane oxygenation provides large surface exposure to human blood leading to coagulation activation. Only limited clinical data are available on contact activation and coagulation factor XII activity in extracorporeal membrane oxygenation patients. DESIGN Prospective cohort study. SETTING Three medical ICUs at the Medical University of Vienna. PATIENTS Adult patients receiving venovenous or venoarterial extracorporeal membrane oxygenation. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS The primary outcome was the change in coagulation factor XII activity in response to extracorporeal membrane oxygenation. Secondary outcomes included the prevalence of reduced coagulation factor XII activity (< 60%) among patients receiving extracorporeal membrane oxygenation and association of coagulation factor XII activity with thromboembolic and bleeding complications. An exploratory endpoint was the association of coagulation factor XII activity and activated partial thromboplastin time in heparinase-treated samples in vitro. Fifty-one patients with a total of 117 samples were included in the study between July 2018 and February 2020. Fifty patients (98%) had reduced coagulation factor XII activity at any timepoint during extracorporeal membrane oxygenation. Median coagulation factor XII activity during extracorporeal membrane oxygenation treatment was 30% (interquartile range, 21.5-41%) and increased after discontinuation (p = 0.047). Patients with thromboembolic complications had higher median coagulation factor XII activity during extracorporeal membrane oxygenation (34% vs 23%; p = 0.023). The odds of a thromboembolic event increased by 200% per tertile of median coagulation factor XII activity (crude odds ratio, 3.034; 95% CI, 1.21-7.63). No association with bleeding was observed. In heparinase-treated samples, coagulation factor XII activity correlated well with activated partial thromboplastin time (r = -0.789; p = 0.007). CONCLUSIONS We observed a high prevalence of reduced coagulation factor XII activity in adult patients on extracorporeal membrane oxygenation, which may confound activated partial thromboplastin time measurements and limit its clinical usefulness for monitoring and titrating anticoagulation with unfractionated heparin. Lower coagulation factor XII activity was associated with less thromboembolic complications, which may highlight the potential of coagulation factor XII to serve as a target for anticoagulation in extracorporeal membrane oxygenation.

Traumatic brain injury (TBI) is a major health problem for over 3.17 million people in the US, attracting increasing public attentions. Understanding the underlying mechanism of TBI is urgent for better diagnosis and treatment. Here, we examined the hypothesis that cerebral hemorrhagic coagulation and subsequent immune cells infiltration causes the progressive mechanisms of brain injury in moderate fluid percussion injury model. This represents a subdural hematoma and hemorrhagic head injury. We found increased hemorrhagic lesions and infarct volume in the injured brain with increment of pressure. The extent of hemorrhage was also validated by the bio-distribution of fluorescent tracer in cerebrospinal fluid (CSF) pathway after the injury. Bio-distribution of tracer was specifically diminished at the site of hemorrhage resulting from coagulation, which blocked the interstitial and CSF movement of the tracer. Increased expression of coagulation factor XII and necrotic cell death in and around the impact site confirmed the reason for this blockade. Different biomarkers, including immune cells accumulation and neuronal death showed that blood-brain barrier disruption played an important role for induction of neuroinflammation and neurodegeneration around the impact site. Our results suggest that instant hemorrhagic injury resulting from rupturing the brain blood vessels intertwined with coagulation causes onsite perivascular inflammation and neurodegeneration. Understanding of this sequential event should be valuable for development of therapeutic treatment in TBI. Graphical Abstract Underlying mechanisms in moderate/severe blunt TBI: hemorrhage following cerebrovascular disruption results in coagulation, thrombotic necrosis, and acute immune cell infiltration.

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

Coagulation factor XII (FXII) is synthesized in the liver and secreted into the circulation, where it initiates the contact activation system. Although typically thought to be restricted to the circulation, FXII protein has been found in the brain of Alzheimer's disease (AD) and multiple sclerosis patients. Moreover, activation of the contact system has been detected in the cerebrospinal fluid of these patients as well as in the brain of healthy and AD individuals. While FXII protein has been detected in the brain, its source and its potential role in brain physiology and/or pathology have not been elucidated. Using in situ hybridization, we show that a shorter FXII mRNA isoform is expressed by neurons in human brain and in the brain of FXII humanized mice, with the highest expression observed in pyramidal neurons. This shorter FXII transcript contains an open reading frame coding for the portion of FXII that spans its proline-rich and catalytic domains (FXII297-596). We show that a recombinant version of this shorter FXII protein is activated by plasma kallikrein, reciprocally activates prekallikrein, and converts pro-hepatocyte growth factor (HGF) to active HGF in vitro. HGF-Met signaling plays a role in neuronal development and survival, and its dysregulation has been implicated in neurodevelopmental disorders and neurodegeneration. Taken together, our results show that a short isoform of FXII mRNA is expressed in the brain and raise the possibility that brain-derived FXII may be involved in HGF-Met signaling in neurons.

Coagulation factor XII (FXII) plays a crucial role in thrombosis. Moreover, deficiencies in FXII are not associated with excessive bleeding, and its depletion exhibits satisfactory protective effect on thrombus formation. Several strategies targeting FXII have been applied to inhibit thrombosis formation. In this study, C57BL/6 mice were injected with adeno-associated virus (AAV) to identify the role of short hairpin RNA (shRNA) in thrombosis. Differences in liver FXII, coagulation function, and thrombus formation were detected. The potential side effects of FXII were then evaluated through analysis of tail bleeding, biochemical indices, and pathological sections. Results showed that shRNAs, especially shRNA2, carried by AAV, effectively reduced the expression of FXII. Furthermore, only shRNA2 demonstrated an anti-thrombosis effect on multiple models without hemorrhage and side effects. Hence the novel approach of AAV-based shRNA is specific and safe for inhibiting FXII and thrombosis.

Hereditary angioedema (HAE) is a life-threatening disease characterized by recurrent episodes of subcutaneous and mucosal swellings and abdominal cramping. Corticosteroids and antihistamines, which are usually beneficial in histamine-induced acquired angioedema, are not effective in HAE. Therefore, diagnosing HAE correctly is crucial for affected patients. We report a family from Northern Germany with six individuals suffering from recurrent swellings, indicating HAE. Laboratory tests and genetic diagnostics of the genes SERPING1, encoding C1 esterase inhibitor (C1-INH), and F12, encoding coagulation factor XII, were unremarkable. In three affected and one yet unaffected member of the family, we were then able to identify the c.988A > G (also termed c.1100A > G) mutation in the plasminogen (PLG) gene, which has recently been described in several families with HAE. This mutation leads to a missense mutation with an amino acid exchange p.Lys330Glu in the kringle 3 domain of plasminogen. There was no direct relationship between the earlier described cases with this mutation and the family we report here. In all affected members of the family, the symptoms manifested in adulthood, with swellings of the face, tongue and larynx, including a fatal case of a 19 year-old female individual. The frequency of the attacks was variable, ranging between once per year to once a month. In one individual, we also found decreased serum levels of plasminogen as well as coagulation factor XII. As previously reported in patients with PLG defects, icatibant proved to be very effective in controlling acute attacks, indicating an involvement of bradykinin in the pathogenesis. Open Access Publikationsfonds 2019

To identify potential mutations of F11 gene in a family with hereditary coagulation factor XI (FXI) deficiency and explore the molecular pathogenesis. The FXI activity and FXI antigen were tested with clotting assay and ELISA, respectively. The FXI gene was amplified by PCR with direct sequencing. Three bioinformatics softwares were used to study the conservatism and harm of the mutation. The proband had a prolonged activated partial thromboplastin time (84.2 s), whose FXI activity and FXI antigen were 3.0 and 8.6%. Gene sequencing revealed that the propositus carried a heterozygous nonsense mutation c.738G>A in exon 7 resulting in a p.Trp228stop and deletions mutation c.1325delT in exon 12 resulting in a p.Leu424Cys. Two bioinformatics softwares all were indicated the mutation had affected the function of the protein. The c.738G>A heterozygous nonsense variation and the c.1325delT heterozygous deletion variation are associated with decreased FXI levels in this family, which is the first reported in the world.

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.

To identify potential mutations of the FXII gene (The proband was a 58-year-old male who had chronic gastritis. He was found to have a significantly prolonged activated partial thromboplastin time (APTT) at 101.0s (reference range, 29.0-43.0 s) beforeThe coagulation factor XII activity (FXII:C) and FXII antigen (FXII:Ag) were measured by one-stage clotting assay and enzyme-linked immunosorbent assay, respectively. TheThe proband had a prolonged APTT (101.0 s), whose FXII:C and FXII:Ag were obviously reduced, both at 1.0% (normal range, 72-113%). Gene sequencing revealed that he carried a homozygous missense mutation of Met527Ile. Family study showed that his mother, son and daughter carried a heterozygous Met527Ile. Bioinformatics and model analysis of the mutation indicated that Met527Ile may be detrimental and potentially alters the structure and the function of the protein.The novel mutation Met527Ile could potentially account for the reduced activity of FXII in this family.

Background Factor XII (FXII) is a serine protease that participates in the intrinsic coagulation pathway. Several studies have shown that plasmatic FXII exert a deleterious role in cerebral ischemia and traumatic brain injury by promoting thrombo-inflammation. Nevertheless, the direct impact of FXII on neuronal cell fate remains unknown.Methods We investigated whether FXII influenced neuronal death induced in vivo by stereotaxic injection of N-methyl-D-Aspartate (NMDA) and in vitro by serum deprivation of cultured neurons.Results We found that FXII reduced brain lesions induced in vivo and protected cultured neurons from apoptosis through a growth factor-like effect. This mechanism was triggered by direct interaction with epidermal growth factor (EGF) receptor, activation of this receptor and engagement of anti-apoptotic intracellular pathways. Interestingly, the “proteolytically” active and two-chain form of FXII, αFXIIa, exerted additional protective effects by converting the pro-form of hepatocyte growth factor (HGF) into its mature form, which in turn activated HGF receptor (HGFR/c-Met) pathway. Lastly, the use of non-proteolytic FXII (αFXIIa-PPACK) unveiled an alternative EGFR and HGFR co-activation pathway, through co-receptor transphosphorylation. Conclusion This study describes novel mechanisms of action of FXII and discloses neurons as target cells for the protective effects of single and double-chain forms of FXII.

Streptococcus oralis (S. oralis) has been recognized as a fatal pathogen to cause multiorgan failure by contributing to the formation of microthrombus. Coagulation and fibrinolysis systems have been found under the control of circadian clock genes. This study aimed to explore the correlation between BMAL1 and coagulation factor biosynthesis in S. oralis infection. Mice were administered S. oralis to induce sepsis, and HepG2 cells were also infected by S. oralis. The expression of BMAL1 of hepatocytes was downregulated in the S. oralis infection group, leading to the downregulation of coagulation factor VII (FVII) and the upregulation of the coagulation factor XII (FXII) in vitro and in vivo. Furthermore, we confirmed that the deficiency of BAML1 contributed to the elevation of FVII and the decline in FXII by constructing BMAL1-deficiency (Bmal1−/−) mice. The current result showed that BMAL1 regulates FVII directly. Thus, a novel insight into the coagulation abnormality in S. oralis infection was gained that may optimize the treatment of sepsis by rescuing the expression of BMAL1 in the liver.

Background Hereditary angioedema due to the Thr328Lys variant in the coagulation factor XII (HAE-FXII) affects mainly women in whom the symptomatology is dependent on high estrogen levels. Clinical variability and incomplete penetrance are challenging features that hinder the diagnosis and management of HAE-FXII. The c.-4T>C Kozak polymorphism is the only common variation accounting for FXII plasma levels and was previously shown to modify the course of HAE due to C1-Inhibitor deficiency. Objectives To assess the influence of the c.-4T>C polymorphism on disease expression in 39 Spanish HAE-FXII index patients. Methods The c.-4T>C polymorphism was sequenced by the standard Sanger method, and HAE severity was calculated according to the score by Cumming et al. (2003) The activation of the contact system was quantified by the kallikrein-like activity of plasma in chromogenic assays upon activation with high-molecular-weight dextran sulfate. Results The c.-4CC genotype was overrepresented in the studied cohort: 82% were CC-homozygous (expected frequency = 59%) and 18% were CT-heterozygous (expected frequency = 39%) (p = 0.001). Patients with a c.-4CC genotype exhibited higher kallikrein-like activity (0.9659 ± 0.1136) than those with a c.-4TC genotype (0.7645 ± 0.1235) (p = 0.024) or healthy donors. Moreover, the polymorphism influenced HAE-FXII severity score (c.-4CC = 4.43 ± 2.28 vs c.-4TC = 2.0 ± 1.15; p = 0.006) but not the degree of estrogen dependence or time until remission. Conclusion The c.-4T>C polymorphism is overrepresented in a Spanish HAE-FXII cohort and significantly influences the degree of contact system activation and the clinical severity of the disease.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.

Deep vein thrombosis (DVT) is a common peripheral vascular disease, which may result in pulmonary embolism and is accompanied by endothelial injury. However, the pathogenesis of DVT remains unclear. Coagulation factor XII (FXII), as an important coagulation factor, has been reported to be closely associated with thrombosis. However, the association between FXII protein and DVT formation is not yet fully understood. The present study examined the effects of FXII protein on DVT formation and aimed to reveal the underlying mechanism. In the present study, histological characterization of the femoral vein tissue was examined by hematoxylin and eosin staining. The damage to the femoral vein tissue was examined by TUNEL assay. Superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were examined using ELISA. Tumor necrosis factor (TNF)‑α, interleukin (IL)‑6, IL‑8 and phosphoinositide 3‑kinase (PI3K)/AKT signaling were determined by ELISA, immunohistochemical staining and western blot analysis. The results demonstrated that thrombosis, FXII protein, cell apoptosis and the SOD concentrations were decreased, while the MDA concentrations were increased in mice with DVT compared with the control or sham groups. TNF‑α, IL‑6, IL‑8 and PI3K/AKT signaling was also upregulated in the mice with DVT. Furthermore, the knockdown of FXII significantly upregulated the SOD concentrations and downregulated thrombosis and cell apoptosis, as well as the MDA concentrations in mice with DVT. The knockdown of FXII also significantly downregulated the protein expression of TNF‑α, IL‑6 and IL‑8, and the activation of PI3K/AKT signaling. Additionally, LY294002 pre‑treatment markedly downregulated thrombosis and cell apoptosis and the MDA content, whereas it upregulated the SOD concentrations in mice with DVT. LY294002 pre‑treatment also significantly downregulated the TNF‑α, IL‑6 and IL‑8 protein levels. Taken together, the present study demonstrates that FXII protein promotes DVT via the activation of PI3K/AKT signaling by inducing an inflammatory response. Targeting FXII protein may thus prove to be a potential approach for the treatment of DVT.

BACKGROUND: The complement and coagulation systems share an evolutionary origin with many components showing structural homology. Certain components, including complement factor H (FH) and coagulation factor XII (FXII), have separately been shown to have auxiliary activities across the two systems.OBJECTIVES: The interaction between FXII and FH was investigated.METHODS: Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) complex formation between different FXII forms and FH was investigated. The presence of α-FXIIa:FH complexes upon contact activation in plasma was evaluated by ELISA and immunoprecipitation.RESULTS: We identified and characterized a direct interaction between the components and demonstrated that among different forms of FXII, only the activated α-FXIIa formed complexes with FH, with an apparent binding strength K d of 34 ± 9 nmol/L. The complex formation involved the kringle domain of the heavy chain of FXII. C1-inhibitor induced inhibition of α-FXIIa did not alter the binding of α-FXIIa toward FH. We further demonstrated the presence of α-FXIIa:FH complexes in normal human plasma upon contact activation, indicating formation of α-FXIIa:FH complexes as a consequence of α-FXIIa generation. Complex formation between α-FXIIa and FH was also assessed in hereditary angioedema (HAE) patients with C1-inhibitor deficiency as well as rheumatoid arthritis (RA) patients with high levels of anti-cyclic citrullinated peptide (anti-CCP) upon contact activation. We observed elevated levels of α-FXIIa:FH complexes in HAE patients, and equal levels of complexes in RA patients and healthy individuals upon contact activation. CONCLUSION: A direct interaction between α-FXIIa and FH is demonstrated. Our findings represent a new crosstalk between these systems, potentially important in the onset and pathology of inflammatory vascular diseases.

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1β (IL-1β) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation., Systemic autoinflammatory syndromes such as cryopyrin-associated periodic syndrome (CAPS) are rare and often involve genes related to the inflammasome. Here, the authors report a syndrome characterised by systemic inflammation and cold-induced urticarial rash associated with a Factor XII-activating mutation.