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Salivary proteins of ticks can inhibit host hemostatic and inflammatory responses during the blood-sucking process of the parasites. A cDNA sequence, Hq021, was identified from a cDNA library of Haemaphysalis qinghaiensis. Hq021 encodes a mature protein containing 182 amino acids with a molecular mass of 20.5 kDa. The protein is rich in basic amino acids (lysine and arginine) at its C-terminus. Identification of no homologous proteins in databases suggested its novelty. A recombinant protein expressed with the cDNA in Escherichia coli could prolong the activated partial thromboplastin time (aPTT) of human plasma. We designated this basic tail-protein as Qinghaienin. Although Qinghaienin did not inhibit the amidolytic activities of some coagulation factors, it dose-dependently inhibited the generation of FXIIa, FXIa and kallikrein in plasma in the presence of silica-based aPTT reagent. By competing with FXII for the negatively-charged binding sites, Qinghaienin inhibited the activation of the zymogen and the coagulation processes. These findings suggest that Qinghaienin plays a critical role in both anticoagulation and anti-inflammatory processes during tick feeding and could serve as a potential candidate for the developing tick-derived anticoagulants., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gao Jinliang reports financial support was provided by National Natural Science Foundation of China (Contract No. 81760375). Gao Jinliang reports financial support was provided by National Natural Science Foundation of China (Contract No. 82360406). Gao Jinliang reports financial support was provided by Natural Science Foundation of Inner Mongolia Autonomous Region of China (Contract No. 2021MS08063). Chen Kaiting reports financial support was provided by Natural Science Foundation of Inner Mongolia Autonomous Region of China (Contract No. 2023QN08005). Gao Jinliang reports financial support was provided by the Science and Technology Innovation Guiding Project of Inner Mongolia (2021). He Xiaofei reports financial support was provided by Education Department of Inner Mongolia Autonomous Region of China (Contract No. S20231198Z). Gao Jinliang, She Zhanfei, Gao Dongbing, Chen Kaiting, He Xiaofei, Guang Hui, Cao Meina, Zhao Wenbin, Bai Mengdie, Yang Yinran, Su Muya and Ma Linyuan has patent #A tick-derived anticoagulation protein Qinghaienin and its encoding gene as well as its application in anticoagulation,Patent No. ZL 202310665559.5 licensed to Gao Jinliang, She Zhanfei, Gao Dongbing, Chen Kaiting, He Xiaofei, Guang Hui, Cao Meina, Zhao Wenbin, Bai Mengdie, Yang Yinran, Su Muya and Ma Linyuan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. While the presence of LPS in the bloodstream during infection is associated with disseminated intravascular coagulation, the mechanistic link between LPS and blood coagulation activation remains ill-defined. The contact pathway of coagulation-a series of biochemical reactions that initiates blood clotting when plasma factors XII (FXII) and XI (FXI), prekallikrein (PK), and high molecular weight kininogen interact with anionic surfaces-has been shown to be activated in Gram-negative septic patients. In this study, using an in vivo baboon model of Gram-negative Escherichia coli sepsis, we observed activation of the contact pathway including FXII, FXI, and PK. We examined whether E.coli LPS molecules could bind and activate contact pathway members by quantifying the interaction and activation of either FXII, FXI, or PK with each of the three chemotypes of LPS: O111:B4, O26:B6, or Rd2. The LPS chemotypes exhibited distinct physicochemical properties as aggregates and formed complexes with FXII, FXI, and PK. The LPS chemotype O26:B6 uniquely promoted the autoactivation of FXII to FXIIa and, in complex with FXIIa, promoted the cleavage of FXI and PK to generate FXIa and plasma kallikrein, respectively. Furthermore, in complex with the active forms of FXI or PK, LPS chemotypes were able to regulate the catalytic activity of FXIa and plasma kallikrein, respectively, despite the inability to promote the autoactivation of either zymogen. These data suggest that the procoagulant phenotype of E.coli is influenced by bacterial strain and the physicochemical properties of the LPS chemotypes., Competing Interests: Conflicts of interest C. U. L. and E. I. T. are employees of Aronora, Inc, a company that may have a commercial interest in the results of this research. J. J. S. serves as a medical consultant for Aronora, Inc. This potential conflict of interest has been reviewed and managed by conflict of interest in research committee of the Oregon Health & Science University. The remaining authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hereditary angioedema (HAE) is a rare genetic disorder characterized by local, self-limiting edema due to temporary increase in vascular permeability. HAE with normal C1 esterase inhibitor (C1INH) activity includes the form with mutations in the F12 gene encoding for coagulation factor XII (FXII-HAE) causing an overproduction of bradykinin (BK) leading to angioedema attack. BK binding to B2 receptors (BK2R) leads to an activation of phospholipase C (PLC) and subsequent generation of second messengers: diacylglycerols (DAGs) and possibly the endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and anandamide (AEA), and eCB-related N-acylethanolamines [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)]. To date, there are no data on the role of these lipid mediators in FXII-HAE., Methods: Here, we analyzed plasma levels of PLC, DAGs, and eCBs in 40 patients with FXII-HAE and 40 sex- and age-matched healthy individuals., Results: Plasma PLC activity was increased in FXII-HAE patients compared to controls. Concentrations of DAG 18:1-20:4, a lipid second messenger produced by PLC, were higher in FXII-HAE compared to controls, and positively correlated with PLC activity and cleaved high molecular kininogen (cHK). Also the concentrations of the DAG metabolite, 2-AG were altered in FXII-HAE. AEA and OEA were decreased in FXII-HAE patients compared to controls; by contrast, PEA, was increased. The levels of all tested mediators did not differ between symptomatic and asymptomatic patients. Moreover, C1INH-HAE patients had elevated plasma levels of PLC, which correlated with cHK, but the levels of DAGs and eCBs were the same as controls., Conclusions: BK overproduction and BKR2 activation are linked to alteration of PLCs and their metabolites in patients with FXII-HAE. Our results may pave way to investigations on the functions of these mediators in the pathophysiology of FXII-HAE, and provide new potential biomarkers and therapeutic targets., (© 2024 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

The plaques associated with Alzheimer's disease are formed as a result of the aggregation of Aβ peptides, which vary in length from 38 to 43 amino acids. The 1-40 peptide is the most abundant, while the 1-42 peptide appears to be the most destructive to neurons and/or glial cells in a variety of assays. We have demonstrated that aggregated Aβ, a state prior to plaque formation, will activate the plasma bradykinin-forming pathway when tested in vitro. Aggregation is zinc-dependent, optimal at 25-50 µM, and the rate of aggregation is paralleled by the rate of activation of the bradykinin-forming pathway as assessed by plasma kallikrein formation. The aggregation of Aβ 1-38, 1-40, and 1-42 is optimal after incubation for 3 days, 3 h, and under 1 min, respectively. The cascade is initiated by the autoactivation of factor XII upon binding to aggregated Aβ; then, prekallikrein is converted to kallikrein, which cleaves high-molecular-weight kininogen (HK) to release bradykinin. Studies by a variety of other researchers have demonstrated the presence of each "activation-step" in either the plasma or spinal fluid of patients with Alzheimer's disease, including activated factor XII, kallikrein, and bradykinin itself. There is also evidence that activation is more prominent as dementia worsens. We now have medications that can block each step of the bradykinin-forming pathway as currently employed for the therapy of hereditary angioedema. Given the current state of therapy for Alzheimer's disease, which includes monoclonal antibodies that retard the rate of progression by 30% at most and have significant side effects, it seems imperative to explore prophylaxis using one of the long-acting agents that target plasma kallikrein or factor XIIa. There is a long-acting bradykinin antagonist in development, and techniques to target kallikrein mRNA to lower levels or knock out the prekallikrein gene are being developed.

This study examined the pathophysiological effects of venoms from neonate and adult specimens of the viperid snake Macrovipera lebetina obtusa , focusing on their ability to activate various blood clotting factors in human plasma. All venoms exhibited strong procoagulant properties. In concentration-response tests, the clotting potency of the neonate venoms fell within the range of their parents' maximum clotting velocities and areas under the curve. Intriguingly, females were more potent than males within each age group, but this requires a larger sample size to confirm. Antivenom neutralization efficacy was equipotent across age groups. The venoms potently activated Factor X (FX) robustly, consistent with previous knowledge of this genus. For the first time, the ability to activate Factors VII (FVII) and XII (FXII) was identified in this genus, with FXII exhibiting particularly strong activation. The study found no significant ontogenetic variation in procoagulant venom potency on human plasma, convergent with the Daboia genus, the other large-bodied lineage within the Palearctic viperid clade. However, the activation of FXII and FVII reveals previously undocumented pathways in the procoagulant activity of these venoms, contributing to the broader understanding of venom evolution and its clinical impacts. These findings have implications for venom biodiscovery and the development of antivenoms, highlighting the complexity of clotting factor activation beyond traditional investigations that have myopically focused upon FX and prothrombin pathways, thereby underscoring the importance of exploring additional clotting factors.

Hereditary angioedema (HAE) due to C1-inhibitor deficiency is a rare, debilitating, genetic disorder characterized by recurrent, unpredictable, attacks of edema. The clinical symptoms of HAE arise from excess bradykinin generation due to dysregulation of the plasma kallikrein-kinin system (KKS). A quantitative systems pharmacology (QSP) model that mechanistically describes the KKS and its role in HAE pathophysiology was developed based on HAE attacks being triggered by autoactivation of factor XII (FXII) to activated FXII (FXIIa), resulting in kallikrein production from prekallikrein. A base pharmacodynamic model was constructed and parameterized from literature data and ex vivo assays measuring inhibition of kallikrein activity in plasma of HAE patients or healthy volunteers who received lanadelumab. HAE attacks were simulated using a virtual patient population, with attacks recorded when systemic bradykinin levels exceeded 20 pM. The model was validated by comparing the simulations to observations from lanadelumab and plasma-derived C1-inhibitor clinical trials. The model was then applied to analyze the impact of nonadherence to a daily oral preventive therapy; simulations showed a correlation between the number of missed doses per month and reduced drug effectiveness. The impact of reducing lanadelumab dosing frequency from 300 mg every 2 weeks (Q2W) to every 4 weeks (Q4W) was also examined and showed that while attack rates with Q4W dosing were substantially reduced, the extent of reduction was greater with Q2W dosing. Overall, the QSP model showed good agreement with clinical data and could be used for hypothesis testing and outcome predictions., Competing Interests: Declarations. Competing interests: S. Juethner and A. Z. X. Zhu are full-time employees of Takeda and hold stocks/options in Takeda. D. Sexton and H. Q. Nguyen were employees of Takeda at the time of this analysis. H. Luo, Z. Zhang, and P. Jasper are full-time employees of the RES Group, which was contracted by Takeda to develop the QSP HAE model., (© 2024. Takeda Development Center Americas, Inc.)

Background: The kallikrein-kinin system (KKS) is a complex biochemical pathway that plays a crucial role in regulating several physiological processes, including inflammation, coagulation, and blood pressure. Dysregulation of the KKS has been associated with several pathological conditions such as hereditary angioedema (HAE), hypertension, and stroke. Developing an accurate quantitative model of the KKS may provide a better understanding of its role in health and disease and facilitate the rapid and targeted development of effective therapies for KKS-related disorders., Objectives: Here, we present a novel, detailed mechanistic model of the plasma KKS, elucidating the processes of Factor XII (FXII) activation, the kallikrein feedback loop, cleavage of high molecular weight kininogen leading to bradykinin (BK) production, and the impact of inhibitors., Methods: The model incorporates both surface and solution-phase reactions of all proteins in the KKS, describing how binding site concentration affects the rate of surface reactions. The model was calibrated and validated using a variety of published and in-house experimental datasets, which encompass a range of dextran sulphate (DXS) concentrations to initiate contact activation and various KKS inhibitors to block bradykinin production., Results: Our mathematical model showed that a trace amount of activated FXII is required for subsequent FXII activation. The model also reveals a bell-shaped curve relationship between the activation of the KKS and the number of DXS surface binding sites. Simulations of BK generation in healthy and HAE plasma demonstrated the impact of C1 esterase inhibitor (C1inh) deficiency via increased peak BK levels and accelerated formation in HAE plasma. The efficacy of KKS inhibitors, such as CSL312, ecallantide, and C1inh, was also evaluated, with CSL312 showing the most potent inhibition of BK generation., Conclusions: The present model represents a valuable framework for studying the intricate interactions within the plasma KKS and provides a better understanding of the mechanism of action of various KKS-targeted therapies., Competing Interests: I have read the journal’s policy, and the authors of this manuscript have the following competing interests: AR, HL, MP, CP, and IM are employees of CSL Limited. AR, HL, SD, MP, CP, and IM hold shares in CSL Limited., (Copyright: © 2024 Rezvani-Sharif et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Factor XII (FXII), the zymogen of the protease FXIIa, contributes to pathologic processes such as bradykinin-dependent angioedema and thrombosis through its capacity to convert the homologs prekallikrein and factor XI to the proteases plasma kallikrein and factor XIa. FXII activation and FXIIa activity are enhanced when the protein binds to a surface. Here, we review recent work on the structure and enzymology of FXII with an emphasis on how they relate to pathology. FXII is a homolog of pro-hepatocyte growth factor activator (pro-HGFA). We prepared a panel of FXII molecules in which individual domains were replaced with corresponding pro-HGFA domains and tested them in FXII activation and activity assays. When in fluid phase (not surface bound), FXII and prekallikrein undergo reciprocal activation. The FXII heavy chain restricts reciprocal activation, setting limits on the rate of this process. Pro-HGFA replacements for the FXII fibronectin type 2 or kringle domains markedly accelerate reciprocal activation, indicating disruption of the normal regulatory function of the heavy chain. Surface binding also enhances FXII activation and activity. This effect is lost if the FXII first epidermal growth factor (EGF1) domain is replaced with pro-HGFA EGF1. These results suggest that FXII circulates in blood in a "closed" form that is resistant to activation. Intramolecular interactions involving the fibronectin type 2 and kringle domains maintain the closed form. FXII binding to a surface through the EGF1 domain disrupts these interactions, resulting in an open conformation that facilitates FXII activation. These observations have implications for understanding FXII contributions to diseases such as hereditary angioedema and surface-triggered thrombosis, and for developing treatments for thrombo-inflammatory disorders., Competing Interests: D.G. is a consultant for pharmaceutical companies (Anthos Therapeutics; Aronora, Inc.; Bayer Pharma; Bristol-Myers Squibb; Ionis Pharmaceuticals; Janssen, Pharmaceuticals) with interests in targeting factor XI, factor XII, and prekallikrein for therapeutic purposes. The other authors do not have conflicts of interest to declare., (Thieme. All rights reserved.)

Background and Purpose: Psoriasis is an autoimmune inflammatory skin disease, featuring microvascular abnormalities and elevated levels of bradykinin. Contact activation of Factor XII can initiate the plasma kallikrein-kinin cascade, producing inflammation and angioedema. The role of Factor XII in psoriasis is unknown., Experimental Approach: The effects of deficiency of Factor XII or its enzymatic substrate, prekallikrein, were examined in the imiquimod-induced mouse model of psoriasis. Skin microcirculation was assessed using intravital confocal microscopy and laser Doppler flowmeter. A novel antibody blocking Factor XII activation was evaluated for psoriasis prevention., Key Results: Expression of Factor XII was markedly up-regulated in human and mouse psoriatic skin. Genetic deletion of Factor XII or prekallikrein, attenuated imiquimod-induced psoriatic lesions in mice. Psoriatic induction increased skin microvascular blood perfusion, causing vasodilation, hyperpermeability and angiogenesis. It also promoted neutrophil-vascular interaction, inflammatory cytokine release and enhanced Factor XII / prekallikrein enzymatic activity with elevated bradykinin. Factor XII or prekallikrein deficiency ameliorated these microvascular abnormalities and abolished bradykinin increase. Antagonism of bradykinin B 2 receptors reproduced the microvascular protection of Factor XII / prekallikrein deficiency, attenuated psoriatic lesions, and prevented protection by Factor XII / prekallikrein deficiency against psoriasis. Furthermore, treatment of mice with Factor XII antibody alleviated experimentally induced psoriasis and suppressed microvascular inflammation., Conclusion and Implications: Activation of Factor XII promoted psoriasis via prekallikrein-dependent formation of bradykinin, which critically mediated psoriatic microvascular inflammation. Inhibition of contact activation represents a novel therapeutic strategy for psoriasis., (© 2024 British Pharmacological Society.)

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders., (© 2024. The Author(s).)

Coagulation factor XII (FXII) conveys various functions as an active protease that promotes thrombosis and inflammation, and as a zymogen via surface receptors like urokinase-type plasminogen activator receptor (uPAR). While plasma levels of FXII are increased in diabetes mellitus and diabetic kidney disease (DKD), a pathogenic role of FXII in DKD remains unknown. Here we show that FXII is locally expressed in kidney tubular cells and that urinary FXII correlates with kidney dysfunction in DKD patients. F12-deficient mice (F12 -/- ) are protected from hyperglycemia-induced kidney injury. Mechanistically, FXII interacts with uPAR on tubular cells promoting integrin β1-dependent signaling. This signaling axis induces oxidative stress, persistent DNA damage and senescence. Blocking uPAR or integrin β1 ameliorates FXII-induced tubular cell injury. Our findings demonstrate that FXII-uPAR-integrin β1 signaling on tubular cells drives senescence. These findings imply previously undescribed diagnostic and therapeutic approaches to detect or treat DKD and possibly other senescence-associated diseases., (© 2024. The Author(s).)

Background: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis., Objectives: To unravel and prevent EGF-1-mediated FXII surface-binding with a variable domain of heavy chain-only antibody (V H H)., Methods: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain-specific V H Hs were raised to inhibit EGF-1-mediated FXII contact activation while preserving quiescence., Results: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1-specific V H Hs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V H H "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence., Conclusion: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1-specific V H Hs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device-induced thrombosis., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of competing interests There are no competing interests to disclose.

Background: Factor XII (FXII) is a multifunctional protease capable of activating thrombotic and inflammatory pathways. FXII has been linked to thrombosis in extracorporeal membrane oxygenation (ECMO), but the role of FXII in ECMO-induced inflammatory complications has not been studied. We used novel gene-targeted FXII- deficient rats to evaluate the role of FXII in ECMO-induced thromboinflammation., Methods: FXII-deficient (FXII -/- ) Sprague-Dawley rats were generated using CRISPR/Cas9. A minimally invasive venoarterial (VA) ECMO model was used to compare wild-type (WT) and FXII -/- rats in 2 separate experimental cohorts: rats placed on ECMO without pharmacologic anticoagulation and rats anticoagulated with argatroban. Rats were maintained on ECMO for 1 hour or until circuit failure occurred. Comparisons were made with unchallenged rats and rats that underwent a sham surgical procedure without ECMO., Results: FXII -/- rats were maintained on ECMO without pharmacologic anticoagulation with low resistance throughout the 1-hour experiment. In contrast, WT rats placed on ECMO without anticoagulation developed thrombotic circuit failure within 10 minutes. Argatroban provided a means to maintain WT and FXII -/- rats on ECMO for the 1-hour time frame without thrombotic complications. Analyses of these rats demonstrated that ECMO resulted in increased neutrophil migration into the liver that was significantly blunted by FXII deficiency. ECMO also resulted in increases in high molecular weight kininogen cleavage and complement activation that were abrogated by genetic deletion of FXII., Conclusions: FXII initiates hemostatic system activation and key inflammatory sequelae in ECMO, suggesting that therapies targeting FXII could limit both thromboembolism and inopportune inflammatory complications in this setting., (Copyright © 2023 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Conflict of Interest Statement The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.

It is accepted that the contact activation complex of the intrinsic pathway of blood coagulation cascade produces active enzymes that lead to plasma coagulation following biomaterial contact. In this study, FXII was activated through contact with hydrophilic glass beads and hydrophobic octadecyltrichlorosilane-modified glass beads from neat buffer solutions. These FXII contact activation products generated from material interaction were found to suppress the procoagulant activity of exogenous αFXIIa, and this inhibition was dependent on surface wettability and the concentration of exogenous αFXIIa. Higher relative inhibition rates were generally observed at low concentrations of αFXIIa (1-2 μg/mL) while both hydrophobic and hydrophilic materials showed similar inhibition levels (~39%) at high concentrations of αFXIIa (20 μg/mL). The presence of prekallikrein in the activation system increased the amount of FXIIa produced during FXII contact activation, and also suppressed the apparent levels of inhibitors on hydrophilic surfaces, while having no effect on apparent levels of inhibitors on hydrophobic surface. The combination of FXII contact activation products and activator surfaces was found to dramatically increase inhibition of αFXIIa activity compared to the activation products alone, regardless of activator surface wettability and the presence of prekallikrein. This finding of inhibitors in the suite of proteins generated by contact activation provides additional knowledge into the complex series of interactions that occur when plasma comes into contact with material surfaces., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31 + ) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

Abstract: Plasma kallikrein (PKa) is an important activator of factor XII (FXII) of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin-generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway-initiated TG compared with that of wild-type controls and was comparable with that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared with wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway-initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway-initiated TG in the complex milieu of mouse WB, and a component of this contribution occurs in an FXII-independent manner., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., Competing Interests: JG-S has received support for attending meetings from CSL Behring and Takeda. ML-H has received honoraria for educational purposes and/or support for attending meetings from CSL Behring and Takeda. PG-B has received honoraria for educational purposes and/or support for attending meetings from CSL Behring and Takeda. AS-C has received consulting fees, honoraria for educational purposes and/or support for attending meetings from CSL Behring, Kalvista and Takeda. VC has received support for attending meetings from BioCryst. MG has received consulting fees, honoraria for educational purposes and/or support for attending meetings from Biocryst, Biomarin, CSL Behring, Kalvista, Pharming, Pharvaris and Takeda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2024 Gil-Serrano, Labrador-Horrillo, Galvan-Blasco, Sala-Cunill, Bigas, Pereira-González, Luengo, Cardona and Guilarte.)

Background: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices., Objectives: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis., Methods: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions., Results: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions., Conclusion: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices., Competing Interests: Declaration of competing interests M.W. and E.I.T. are employees of Aronora Inc, and they as well as Oregon Health & Science University may have a financial interest in the results of this study. This potential conflict of interest has been reviewed and managed by Oregon Health & Science University. The remaining authors declare no competing financial interests., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg -/- ) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12 -/- ) and HRG deficiency (by siRNA or Hrg -/- ), there is further thrombosis reduction compared to F12 -/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa., (© 2024. The Author(s).)

Objectives: In eosinophilic granulomatosis with polyangiitis (EGPA) a prothrombotic state, including formation of denser fibrin networks with reduced lysability has been observed. Little is known about the intrinsic pathway in EGPA. We investigated whether coagulation factors (F)XI and FXII are associated with eosinophil-driven prothrombotic state., Methods: In 34 consecutive EGPA patients with remission we assessed FXI and FXII levels along with plasma fibrin clot permeability (Ks), fibrin clot morphology using scanning electron microscopy, and efficiency of fibrinolysis, expressed as lysis time (t50%) and maximum rate of increase in D-dimer levels (D-Drate)., Results: Increased FXI level (>130%, the upper reference limit) was found in 8 (23.5%) patients. Compared to patients with FXI levels ≤130%, those with increased FXI had higher eosinophil count (+365%) and reduced percentage of neutrophils (-20.4%), along with reduced Ks (-20.5%). In patients with FXI>130% clots were composed of thinner fibrin fibers (-17.5%). FXI was not associated with C-reactive protein and fibrinogen levels or anti-neutrophil cytoplasmic antibodies titers. There were no correlations between FXI and FXII levels as well as between FXII and eosinophil count (all p>0.05)., Conclusions: To our knowledge, this study is the first to show association between FXI and a prothrombotic state in EGPA. Given clinical trials on FXI inhibition as an antithrombotic option, our findings suggest that this therapeutic approach could be useful in diseases with hypereosinophilia.

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., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Ethnopharmacological Relevance: Bombyx batryticatus is traditionally used to treat patients with stroke, but its mechanism remains unclear., Aim of the Study: To explore the interventional effect of Bombyx batryticatus extract as an activator of FⅫ on angiogenesis of rats with cerebral ischemia/reperfusion injury., Materials and Methods: Firstly, the mechanism of Bombyx batryticatus interfering with IS was predicted by systematic pharmacology method, and then it was further verified by animal experiments. The effects of Bombyx batryticatus extract on plasma coagulation were detected, and the activation of coagulation factor Ⅻ (FⅫ) and its downstream substrate kallikrein kinase (KK) was detected in vitro. The brain morphology and expressions of FXII, KK, vascular endothelial growth factors (VEGF), CD31, Brdu/von Willebrand Factor (vWF) were detected. The morphological changes, cell proliferation and VEGF expression of brain microvascular endothelial cells were detected by oxygen glucose deprivation model. The pharmacodynamic substances of Bombyx batryticatus extract were identified by Liquid Chromatography - Mass Spectrometry (LC-MS)., Results: The results of systematic pharmacology found that the treatment of IS by Bombyx batryticatus may be related to blood coagulation and other processes. In vitro, Bombyx batryticatus extract prolonged the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) (P < 0.05), activated FⅫ and promoted the production of downstream substrate KK, with dose-dependent (P < 0.05). Bombyx batryticatus extract improved the neuronal damage of rats, activated FXII and increased the production of KK and the expressions of VEGF, CD31, Brdu/vWF (P < 0.05). Bombyx batryticatus extract also increased the proliferation of brain microvascular endothelial cells and expression of VEGF in rats (P < 0.05). A total of 809 metabolites in Bombyx batryticatus extract were identified by LC-MS., Conclusion: Bombyx batryticatus extract may ameliorate the injury of nerve function in rats with cerebral ischemia/reperfusion injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Anticoagulation therapy stands as a key treatment for thrombotic diseases. The consequential bleeding risk tied to existing anticoagulation methods significantly impacts patient prognosis. In the intensive care unit (ICU), patients often necessitate organ support, leading to the inevitable placement of artificial devices in blood vessels, thereby requiring anticoagulation treatment to avert clot formation that might impede organ support. Nevertheless, these patients commonly encounter a heightened risk of bleeding. Hemophilia B, identified in 1953, manifests as a deficiency in coagulation factor XI (FXI), which focused people's perspective on the endogenous coagulation pathway, that is, the contact pathway. Upon interaction between the surface of artificial devices and FXII, FXII activates, subsequently triggering FXI and initiating the "coagulation cascade" within the contact pathway. Inhibitors targeting the contact pathway encompass two primary categories: FXII inhibitors and FXI inhibitors, capable of impeding this process. This article reviews the role of FXII and FXI in activating the contact pathway, seeking to illuminate their contributions to thrombus formation. By listing the relatively mature drugs and their indications, clinicians are familiar with this new anticoagulant.

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., (Copyright © 2023. Published by Elsevier Inc.)

Recent studies have pointed to the role of Krüpple-like factor 12 (KLF12) in cancer-associated processes, including cancer proliferation, apoptosis, and metastasis. However, the role of KLF12 in tumor immunity remains obscure. Here, we found that KLF12 expression was significantly higher in non-small cell lung cancer (NSCLC) cells with higher programmed death-ligand 1 (PD-L1) expression. Additionally, a positive correlation between KLF12 and PD-L1 was observed in clinical patient tumor tissues. By chromatin immunoprecipitation (ChIP) analysis, KLF12 was identified to bind to the CACCC motif of the PD-L1 promoter. Overexpression of KLF12 promoted PD-L1 transcription, whereas silencing of KLF12 inhibited PD-L1 transcription. Furthermore, signal transducer and activator of transcription 1 (STAT1)- and STAT3-triggered PD-L1 transcription was abolished in the absence of KLF12, and KLF12 knockdown weakened the binding of STAT1 and STAT3 to the PD-L1 promoter. Mechanistically, KLF12 physically interacted with P300, a histone acetyltransferase. In addition, KLF12 silencing reduced P300 binding to the PD-L1 promoter, which subsequently caused decreased acetylation of histone H3. PD-L1 transcription driven by KLF12 overexpression was eliminated by EP300 silencing. In immunocompetent mice, KLF12 knockout inhibited tumor growth and promoted infiltration of CD8 + T cells. However, this phenomenon was not observed in immunodeficient mice. Overall, this study reveals KLF12-mediated transcriptional regulation of PD-L1 in NSCLC; targeting KLF12 may be a potential therapeutic strategy for NSCLC., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Hereditary angioedema (HAE) is a rare disorder characterized by cutaneous and submucosal swelling caused mostly by excessive local bradykinin production. Bradykinin is a vasoactive peptide generated by the limited proteolysis of high molecular weight kininogen (HMWK) by plasma kallikrein via the contact activation system. The contact activation system occurs not only in solution but also on the cell surface. Factor XII (FXII), prekallikrein, and HMWK are assembled on the endothelial cell surface via several proteins, including a trimer of a receptor for globular C1q domain in a Zn 2+ -dependent manner, and the reciprocal activation on the cell surface is believed to be physiologically important in vivo. Thus, the contact activation system leads to the activation of coagulation, complement, inflammation, and fibrinolysis. C1-inhibitor (C1-INH) is a plasma protease inhibitor that is a member of the serpin family. It mainly inhibits activated FXII (FXIIa), plasma kallikrein, and C1s. C1-INH hereditary deficiency induces HAE (HAE-C1-INH) due to excessive bradykinin production via the incomplete inhibition of plasma kallikrein and FXIIa through the low C1-INH level. HAE is also observed in patients with normal C1-INH (HAEnCI) who carry pathogenic variants in genes of factor XII, plasminogen, angiopoietin 1, kininogen, myoferlin, and heparan sulfate 3-O-sulfotransferase 6, which are associated with bradykinin production and/or vascular permeability. HAE-causing pathways triggered by pathogenic variants in patients with HAE-C1-INH and HAEnCI are reviewed and discussed., (Copyright © 2023 Japanese Society of Allergology. Published by Elsevier B.V. All rights reserved.)

Background: Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy., Methods: We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl 3 -induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro., Results: We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe ( P =0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe ( P =0.0008) and healthy lungs from mice injected with 3F7-NIR ( P =0.021)., Conclusions: Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo., Competing Interests: Disclosures J.F. Arthur, I.L. Muir, V. Rayzman, and C. Panousis are employees of CSL, Ltd, Melbourne, Australia. The other authors report no conflicts.

Background: During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates prekallikrein and factor XI (FXI). Recently, we showed that the FXII first epidermal growth factor-1 (EGF1) domain is required for normal activity when polyphosphate is used as a surface., Objectives: The aim of this study was to identify amino acids in the FXII EGF1 domain required for polyphosphate-dependent FXII functions., Methods: FXII with alanine substitutions for basic residues in the EGF1 domain were expressed in HEK293 fibroblasts. Wild-type FXII (FXII-WT) and FXII containing the EGF1 domain from the related protein Pro-HGFA (FXII-EGF1) were positive and negative controls. Proteins were tested for their capacity to be activated, and to activate prekallikrein and FXI, with or without polyphosphate, and to replace FXII-WT in plasma clotting assays and a mouse thrombosis model., Results: FXII and all FXII variants were activated similarly by kallikrein in the absence of polyphosphate. However, FXII with alanine replacing Lys 73 , Lys 74 , and Lys 76 (FXII-Ala 73,74,76 ) or Lys 76 , His 78 , and Lys 81 (FXII-Ala 76,78,81 ) were activated poorly in the presence of polyphosphate. Both have <5% of normal FXII activity in silica-triggered plasma clotting assays and have reduced binding affinity for polyphosphate. Activated FXIIa-Ala 73,74,76 displayed profound defects in surface-dependent FXI activation in purified and plasma systems. FXIIa-Ala 73,74,76 reconstituted FXII-deficient mice poorly in an arterial thrombosis model., Conclusion: FXII Lys 73 , Lys 74 , Lys 76 , and Lys 81 form a binding site for polyanionic substances such as polyphosphate that is required for surface-dependent FXII function., Competing Interests: Declaration of competing interests D.G. receives consultant fees from pharmaceutical companies with an interest in the inhibition of contact activation and the kallikrein-kinin system for therapeutic purposes. All other authors have no conflicts to report., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Titanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered compatible with blood, they appear to support plasma contact activation and may be thrombogenic., Objectives: The objective of this study was to compare Ti and titanium nitride (TiN) with known activators of contact activation (kaolin and silica) in plasma-clotting assays and to assess binding and activation of factor XII, (FXII), factor XI (FXI), prekallikrein, and high-molecular-weight kininogen (HK) with Ti/TiN., Methods: Ti-based nanospheres and foils were compared with kaolin, silica, and aluminum in plasma-clotting assays. Binding and activation of FXII, prekallikrein, HK, and FXI to surfaces was assessed with western blots and chromogenic assays., Results: Using equivalent surface amounts, Ti and TiN were comparable with kaolin and superior to silica, for inducing coagulation and FXII autoactivation. Similar to many inducers of contact activation, Ti and TiN are negatively charged; however, their effects on FXII are not neutralized by the polycation polybrene. Antibodies to FXII, prekallikrein, or FXI or coating Ti with poly-L-arginine blocked Ti-induced coagulation. An antibody to FXII reduced FXII and PK binding to Ti, kallikrein generation, and HK cleavage., Conclusion: Titanium compounds induce contact activation with a potency comparable with that of kaolin. Binding of FXII with Ti shares some features with FXII binding to soluble polyanions but may have unique features. Inhibitors targeting FXII or FXI may be useful in mitigating Ti-induced contact activation in patients with titanium-based implants that are exposed to blood., Competing Interests: Declaration of competing interest statement D.G. is a consultant for pharmaceutical companies (Anthos Therapeutics; Aronora, Inc.; Bayer Pharma; Bristol-Myers Squibb; Ionis Pharmaceuticals; Janssen, Pharmaceuticals) with interests in targeting factor XI, factor XII, and prekallikrein for therapeutic purposes. M.W. and E.I.T are employees of Aronora, and they, as well as Oregon Health and Sciences University, may have financial interest in the results of this study. EPF is an employee of KalVista Pharmaceuticals Inc. The other authors have no conflicts to report., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes., Competing Interests: None declared., (Thieme. All rights reserved.)

Hageman 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).

Anticoagulants have been in use for nearly a century for the treatment and prevention of venous and arterial thromboembolic disorders. The most dreaded complication of anticoagulant treatment is the occurrence of bleeding, which may be serious and even life-threatening. All available anticoagulants, which target either multiple coagulation factors or individual components of the tissue factor (TF) factor VIIa or the common pathways, have the potential to affect hemostasis and thus to increase bleeding risk in treated patients. While direct oral anticoagulants introduced an improvement in care for eligible patients in terms of safety, efficacy, and convenience of treatment, there remain unmet clinical needs for patients requiring anticoagulant drugs. Anticoagulant therapy is sometimes avoided for fear of hemorrhagic complications, and other patients are undertreated due to comorbidities and the perception of increased bleeding risk. Evidence suggests that the contact pathway of coagulation has a limited role in initiating physiologic in vivo coagulation and that it contributes to thrombosis more than it does to hemostasis. Because inhibition of the contact pathway is less likely to promote bleeding, it is an attractive target for the development of anticoagulants with improved safety. Preclinical and early clinical data indicate that novel agents that selectively target factor XI or factor XII can reduce venous and arterial thrombosis without an increase in bleeding complications., (Copyright © 2022 by The American Society of Hematology.)

Human kallikrein-kinin system (KKS) is a proteolytic cascade with two serine-protease zymogen couples (Factor XII and prekallikrein (PK) and their activated forms, FXIIa, PKa, respectively), releasing bradykinin by cleavage of native high-molecular-weight kininogen (nHK) into cleaved HK. For KKS investigation in human plasma, this cascade is usually triggered on ice eventually by mixing with purified proteins. It has been established that purified FXIIa, PK, and nHK required a fixed order and timing for mixing protein on ice to ensure reproducibility of testing, we investigated the activation kinetics of both enzymes. The activation process of this in vitro minimal reconstitution of KKS was studied by progress curve analysis, in condition of high enzyme/substrate ratio and by using on natural rather than peptide substrates. FXIIa and PKa were found five-times less active on ice than at 37°C: kcat = 0.133 ± 0.034 and 0.0119 ± 0.0027 s-1, KM = 672 ± 150 and 115 ± 24 nM, respectively. The progress curve analysis of our in vitro KKS reconstitutions differed from a Michaelis-Menten mathematical simulation by a faster initial rate and a slower late rate. These two features were also observed ex vivo by using dextran sulfate-activated plasma and could reinforce the hypothesis of a maximal local effect (bradykinin release) and a minimal systemic consequence (PK preservation) in KKS activation process. Analyzing the complete curve of cold KKS activation would provide valuable information for ex vivo investigation of KKS in samples from patients presenting with hereditary angioedema and other inflammatory conditions., (© 2022 The Author(s).)

Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ultrasmall gold nanoparticles (usNPs) and nanoclusters are an emerging class of nanomaterials exhibiting distinctive physicochemical properties and in vivo behaviors. Although understanding the interactions of usNPs with blood components is of fundamental importance to advance their clinical translation, currently, little is known about the way that usNPs interact with the hemostatic system. This study describes the effects of a model anionic p -mercaptobenzoic acid-coated usNP on the coagulation cascade, with particular emphasis on the contact pathway. It is found that in a purified system, the anionic usNPs bind to and activate factor XII (FXII). The formed usNP-FXII complexes are short-lived (residence time of ∼10 s) and characterized by an affinity constant of ∼200 nM. In human plasma, the anionic usNPs activate the contact pathway and promote coagulation. The usNPs also exhibit anticoagulant activity in plasma by interfering with the thrombin-mediated cleavage of fibrinogen. Taken together, these findings establish that anionic usNPs can disturb the normal hemostatic balance, which in turn may hinder their clinical translation. Finally, it is shown that usNPs can be designed to be nearly inert in plasma by surface coating with the natural peptide glutathione.

Purpose of Review: Factor XII (FXII), the precursor of the protease FXIIa, contributes to pathologic processes including angioedema and thrombosis. Here, we review recent work on structure-function relationships for FXII based on studies using recombinant FXII variants., Recent Findings: FXII is a homolog of pro-hepatocyte growth factor activator (Pro-HGFA). We prepared FXII in which domains are replaced by corresponding parts of Pro-HGA, and tested them in FXII activation and activity assays. In solution, FXII and prekallikrein undergo reciprocal activation to FXIIa and kallikrein. The rate of this process is restricted by the FXII fibronectin type-2 and kringle domains. Pro-HGA replacements for these domains accelerate FXII and prekallikrein activation. When FXII and prekallikrein bind to negatively charged surfaces, reciprocal activation is enhanced. The FXII EGF1 domain is required for surface binding., Summary: We propose a model in which FXII is normally maintained in a closed conformation resistant to activation by intramolecular interactions involving the fibronectin type-2 and kringle domains. These interactions are disrupted when FXII binds to a surface through EGF1, enhancing FXII activation and prekallikrein activation by FXIIa. These observations have important implications for understanding the contributions of FXII to disease, and for developing therapies to treat thrombo-inflammatory disorders., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Vontz, Kallenberger, Xu, Ploscariu, Ramyar, Garcia, Ghebrehiwet and Geisbrecht.)

Zinc (II), the second most abundant transition metal in blood, binds to the initiator of the contact pathway, factor XII (FXII). This binding induces conformational changes in the structure of FXII eventually leading to its activation. Despite many in vitro and in vivo studies on zinc-mediated activation of FXII, its molecular mechanism remains elusive mainly due to absence of a full-length structural model of FXII. To this end, this study investigated the role of zinc in the structure and dynamics of the full-length structure FXII that was obtained through molecular modeling. We have used four structural templates covering more than 70% of the FXII sequence and the remaining interconnecting regions were built by loop modeling. The resulting full-length structure of FXII contained disordered regions, but in comparison to the AlphaFold (AF) prediction, our full-length model represented a more realistic structure because of the disordered regions which were modeled to yield a more compact full-length structure in our model than the AF structure. Other than the disordered regions, our model and AF prediction were highly similar. The resulting full-length FXII structure was used to generate different systems representing the zinc-bound form (holo). Further to assess the contribution of the disulfide bridges, we also analyzed the apo and holo FXII structures with oxidized or reduced cysteine side-chains. Simulations suggested zinc binding conferred rigidity to the structure, particularly to the light chain of FXII. Zinc binding alone was sufficient to limit the backbone flexibility while 15 disulfide bonds, which were scattered throughout the structure, made a less significant contribution to the backbone rigidity. Altogether our results provide insights into the first realistic full-length structure of FXII focusing on the impact of structural zinc and disulfide bridges in the dynamics of this structure., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Previously, we showed that histidine-rich glycoprotein (HRG) binds factor (F) XIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Therefore, HRG downregulates the contact pathway in vitro and in vivo., Objective: To identify the domains on HRG responsible for contact pathway inhibition., Methods: Recombinant HRG domain constructs (N-terminal [N1, N2, and N1N2], proline-rich regions, histidine-rich region [HRR], and C-terminal) were expressed and purified. The affinities of plasma-derived HRG, HRG domain constructs, and synthetic HRR peptides for FXII, FXIIa, β-FXIIa, and polyphosphate (polyP) were determined using surface plasmon resonance, and their effects on polyP-induced FXII autoactivation, FXIIa-mediated activation of FXI and prekallikrein, the activated partial thromboplastin time (APTT), and thrombin generation were examined., Results: HRG and HRG domain constructs bind FXIIa, but not FXII or β-FXII. HRR, N1, and N1N2 bind FXIIa with affinities comparable with that of HRG, whereas the remaining domains bind with lower affinity. Synthetic HRR peptides bind FXIIa and polyP with high affinity. HRG and HRR significantly inhibit FXII autoactivation and prolong the APTT. Like HRG, synthetic HRR peptides inhibit FXII autoactivation, attenuate FXIIa-mediated activation of prekallikrein and FXI, prolong the APTT, and attenuate thrombin generation., Conclusion: The interaction of HRG with FXIIa and polyP is predominantly mediated by the HRR domain. Like intact HRG, HRR downregulates the contact pathway and contributes to HRG-mediated down regulation of coagulation., (© 2021 International Society on Thrombosis and Haemostasis.)

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 ( bz1728 ) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro , for rescued root growth. We focused here on nobiro6 , which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6 / taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

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.

Blood coagulation is essential to maintain the integrity of a closed circulatory system (hemostasis), but also contributes to thromboembolic occlusion of vessels (thrombosis). Thrombosis may cause deep vein thrombosis, pulmonary embolism, myocardial infarction, peripheral artery disease, and ischemic stroke, collectively the most common causes of death and disability in the developed world. Treatment for the prevention of thromboembolic diseases using anticoagulants such as heparin, coumarins, thrombin inhibitors, or antiplatelet drugs increase the risk of bleeding and are associated with an increase in potentially life-threatening hemorrhage, partially offsetting the benefits of reduced coagulation. Thus, drug development aiming at novel targets is needed to provide efficient and safe anticoagulation. Within the last decade, experimental and preclinical data have shown that some coagulation mechanisms principally differ in thrombosis and hemostasis. The plasma contact system protein factors XII and XI, high-molecular-weight kininogen, and plasma kallikrein specifically contribute to thrombosis, however, have minor, if any, role in hemostatic coagulation mechanisms. Inherited deficiency in contact system proteins is not associated with increased bleeding in humans and animal models. Therefore, targeting contact system proteins provides the exciting opportunity to interfere specifically with thromboembolic diseases without increasing the bleeding risk. Recent studies that investigated pharmacologic inhibition of contact system proteins have shown that this approach provides efficient and safe thrombo-protection that in contrast to classical anticoagulants is not associated with increased bleeding risk. This review summarizes therapeutic and conceptual developments for selective interference with pathological thrombus formation, while sparing physiologic hemostasis, that enables safe anticoagulation treatment., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The plasma protein factor XII (FXII) is the liver-derived zymogen of the serine protease FXIIa that initiates an array of proteolytic cascades. Zymogen activation, enzymatic FXIIa activity and functions are regulated by interactions with cell receptors, negatively charged surfaces, other serine proteases, and serpin inhibitors, which bind to distinct protein domains and regions in FXII(a). FXII exerts mitogenic activity, while FXIIa initiates the pro-inflammatory kallikrein-kinin pathway and the pro-thrombotic intrinsic coagulation pathway, respectively. Growing evidence indicates that FXIIa-mediated thrombo-inflammation plays a crucial role in various pathological states besides classical thrombosis, such as endothelial dysfunction. Consistently, increased FXIIa levels are associated with hypercholesterolemia and hypertriglyceridemia. In contrast, FXII deficiency protects from thrombosis but is otherwise not associated with prolonged bleeding or other adverse clinical manifestations. Here, we review current concepts for FXII(a)-driven vascular inflammation focusing on endothelial hyperpermeability, receptor signaling, atherosclerosis and immune cell activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction : Blood coagulation factor XII (FXII) is an emerging and potentially safe drug target, which dysregulation is associated with thrombosis, hereditary angioedema, and (neuro)inflammation. At the same time, FXII-deficiency is practically asymptomatic. Industrial and academic institutions have developed a number of potential therapeutic agents targeting either FXII zymogen or its active form FXIIa for the treatment of thrombotic and inflammatory conditions associated with the activity of this enzyme. Areas covered : A short overview of the FXII(a) structure and function, underlining its suitability as a drug target, is given. The article reviews patents reported over the last three decades on FXII(a)-targeting therapeutic agents. These agents include small molecules, proteins, peptides, oligonucleotides, siRNAs, and monoclonal antibodies. Expert opinion : The performed analysis of patents revealed that many FXII(a) inhibitors are in the early preclinical stage, while several already showed efficacy in vivo animal models of thrombosis, sepsis, hereditary angioedema, and multiple sclerosis. Two anti-FXIIa agents namely tick protein Ir-CPI and monoclonal antibody CSL312 are currently in human clinical trials. The results of these trials and further studies of FXII(a) pathophysiological functions will encourage the development of new FXII(a) inhibitors.

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  < 0.001. Current abusers had higher levels of FXII and C1inh and lower levels of prekallikrein and HK than controls, p ≤ 0.025. Stepwise regression analysis showed that EKP was associated with C1inh and prekallikrein in current AAS abusers, R 2  = 0.70, p  < 0.001. We conclude that current AAS abuse reduces the kallikrein-generating capacity of CAS by increasing the concentration of C1inh and reducing the concentration of prekallikrein. These changes may contribute to the anti-inflammatory effect of testosterone., Competing Interests: None declared., (Thieme. All rights reserved.)

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., (© 2021. The Author(s).)

Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated., (Copyright © 2021. Published by Elsevier Ltd.)