Factor XII blockade inhibits aortic dilatation in angiotensin II-infused apolipoprotein E-deficient mice

Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Chronic inflammation and excessive matrix remodelling are considered important in AAA pathogenesis. Kinins are bioactive peptides important in regulating inflammation. Stimulation of the kinin B2 receptor has been previously reported to promote AAA development and rupture in a mouse model. The endogenous B2 receptor agonist, bradykinin, is generated from the kallikrein-kinin system following activation of plasma kallikrein by Factor XII (FXII). In the current study whole-body FXII deletion, or neutralisation of activated FXII (FXIIa), inhibited expansion of the suprarenal aorta (SRA) of apolipoprotein E-deficient mice in response to angiotensin II (AngII) infusion. FXII deficiency or FXIIa neutralisation led to decreased aortic tumor necrosis factor-alpha-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-alpha-converting enzyme) (ADAM-17)) activity, plasma kallikrein concentration, and epithelial growth factor receptor (EGFR) phosphorylation compared with controls. FXII deficiency or neutralisation also reduced Akt1 and Erk1/2 phosphorylation and decreased expression and levels of active matrix metalloproteinase (Mmp)-2 and Mmp-9. The findings suggest that FXII, kallikrein, ADAM-17, and EGFR are important molecular mediators by which AngII induces aneurysm in apolipoprotein E-deficient mice. This could be a novel pathway to target in the design of drugs to limit AAA progression

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12–/– mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

 A superconducting pair in high-temperature superconductivity structure was supposed to have an electrical quadrupole moment. It was shown, that under certain conditions, at the part of acoustic phonons, conduction electrons in high-temperature superconductivity structure can form superconducting pairs, when the interaction energy of the quadrupole moment of a superconducting pair with the structure electric field exceeds the energy of Coulomb repulsion between electrons. The electrical field parameters in a CuO2 plane of high-temperature superconductivity structure have been obtained by the method of effective point charges. The distance between electrons in superconducting pair in a CuO2 plane have been estimated in ~ 75 ÷ 130 Å. Within the framework of the suggested model it was shown that high-temperature superconductivity IR-spectra, which were observed at 500 cm-1, can be caused by the quadrupole resonance of superconducting pairs. Выдвинуто предположение, что электрический квадрупольный момент куперовской пары в структуре высокотемпературных сверхпроводников отличен от нуля. Показано, что при определенных условиях при участии акустических фононов электроны проводимости в структуре высокотемпературных сверхпроводников могут образовывать куперовские пары, когда энергия взаимодействия квадрупольного момента куперовской пары превышает энергию кулоновского отталкивания между электронами. Параметры электрического поля в плоскости CuO2 в высокотемпературных сверхпроводниках рассчитаны методом эффективных точечных зарядов. Расстояние между электронами в куперовской паре в плоскости CuO2 равно ~ 75 ÷ 130 Å. В соответствии с предлагаемой моделью инфракрасные спектры, наблюдаемые в районе 500 см-1 для высокотемпературных сверхпроводников, могут быть обусловлены квадрупольным резонансом куперовских пар. Висунуто припущення, що електричний квадрупольний момент куперівської пари в структурі високо-температурних надпровідників відмінний від нуля. Показано, що за певних умов за участю акустичних фононів електрони провідності в структурі високотемпературних надпровідників можуть утворювати куперівські пари, коли енергія взаємодії квадрупольного моменту куперівської пари перевищує енергію кулонівського відштовхування між електронами. Параметри електричного поля в площині CuО2 у високотемпературних надпровідниках розраховані методом ефективних точкових зарядів. Відстань між електронами в куперівській парі в площині CuО2 становить 75–130 Å. Відповідно до запропонованої моделі показано, що інфрачервоні спектри, які спостерігаються в районі 500 см-1 для високотемпера-турних надпровідників, можуть бути обумовлені квадрупольним резонансом куперівських пар

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

Background and Purpose Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. Methods For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. Results Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. Conclusions With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury

HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure

ABSTRACTHageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (β-FXIIa)

Generation of a humanized FXII knock-in mouse-A powerful model system to test novel anti-thrombotic agents

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

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins

Prophylactic rHA-Infestin-4 application is still active 24 h after tMCAO in rats.

<p>Prophylactic rHA-Infestin-4 application is still active 24 h after tMCAO in rats.</p