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BACKGROUND Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2, causing vascular leakage. BK is inactivated by proteolysis by the plasma carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS Mice deficient in CPB2, CPN, or both (Cpb2-/- , Cpn-/- , and Cpb2-/- /Cpn-/- ) were compared with wild-type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments, mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS Skin irritation increased vascular leakage most in Cpb2-/- /Cpn-/- , less in Cpb2-/- and Cpn-/- , and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin complex, increased vascular leakage to the level seen in Cpb2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS Bradykinin is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma carboxypeptidases have more vascular leak than those lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.

BackgroundProthrombotic and inflammatory variables decrease after obesity surgery. The contact activation system may be a common denominator of these changes.ObjectiveTo characterize the contact system before and 6 months after Roux-en-Y gastric bypass (RYGB) and to evaluate associations with changes (post-surgery minus pre-surgery) in metabolic variables.MethodsWomen (n = 42) and men (n = 18) with obesity underwent RYGB, and measures of kallikrein generation, factor XII (FXII), prekallikrein, high molecular weight kininogen (HK), and C1 esterase inhibitor (C1-inh) were determined before and 6 months after surgery. Associations were evaluated using correlation and multivariate regression analyses.ResultsAfter RYGB, the endogenous kallikrein potential (EKP), peak kallikrein generation, FXII, and prekallikrein were reduced, and kallikrein generation lag time was prolonged (all p < 0.0005). Before and after RYGB, absolute values of EKP, lag time, and peak kallikrein generation correlated consistently with contact system proteins (range of correlation coefficients (rS): −0.43 to −0.28 and 0.24 to 0.45 (pre-surgery); −0.43 to −0.30 and 0.28 to 0.50 (post-surgery)). RYGB-associated changes in EKP correlated with C1-inh (rS = −0.29, p = 0.025), but also with triglycerides (rS = 0.34, p = 0.007) and cholesterol (rS = 0.28, p = 0.029), and independently associated with changes in C1-inh (β = −0.40) and triglycerides (β = 0.39). Changes in C1-inh associated with reductions in body weight (β = −0.39) and HbA1c (β = 0.38).ConclusionThe contact system was affected 6 months after RYGB. Absolute values of kallikrein generation before and after RYGB correlated with contact system proteins, whereas changes after RYGB associated with changes in C1-inh and metabolic variables.

Key Points Cleaved HK is observed in many patients with cancer, suggesting activation of the contact system.EVs from cancer cell lines or patients with cancer express polyphosphate, bind and activate FXII, and are prothrombotic., Visual Abstract, Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Angioedema is characterized by swelling of the skin or mucous membranes. Overproduction of the vasodilator bradykinin (BK) is an important contributor to the disease pathology, which causes rapid increase in vascular permeability. BK formation on endothelial cells results from high molecular weight kininogen (HK) interacting with gC1qR, the receptor for the globular heads of C1q, the first component of the classical pathway of complement. Endothelial cells are sensitive to blood-flow-induced shear stress and it has been shown that shear stress can modulate gC1qR expression. This study aimed to determine the following: (1) how BK or angioedema patients' (HAE) plasma affected endothelial cell permeability and gC1qR expression under shear stress, and (2) if monoclonal antibody (mAb) 74.5.2, which recognizes the HK binding site on gC1qR, had an inhibitory effect in HK binding to endothelial cells. Human dermal microvascular endothelial cells (HDMECs) grown on Transwell inserts were exposed to shear stress in the presence of HAE patients' plasma. Endothelial cell permeability was measured using FITC-conjugated bovine serum albumin. gC1qR expression and HK binding to endothelial cell surface was measured using solid-phase ELISA. Cell morphology was quantified using immunofluorescence microscopy. The results demonstrated that BK at 1 µg/mL, but not HAE patients' plasma and/or shear stress, caused significant increases in HDMEC permeability. The mAb 74.5.2 could effectively inhibit HK binding to recombinant gC1qR, and reduce HAE patients' plasma-induced HDMEC permeability change. These results suggested that monoclonal antibody to gC1qR, i.e., 74.5.2, could be potentially used as an effective therapeutic reagent to prevent angioedema.

The plasma contact system is the initiator of the intrinsic pathway of coagulation and the main producer of the inflammatory peptide bradykinin. When plasma is exposed to a negatively charged surface the two enzymes factor XII (FXII) and plasma prekallikrein (PK) bind to the surface alongside the co-factor high molecular weight kininogen (HK), where PK is non-covalently bound to. Here, FXII and PK undergo a reciprocal activation feedback loop that leads to full contact system activity in a matter of seconds. Although naturally occurring negatively charged surfaces have shown to be involved in the role of the contact system in thrombosis, such surfaces are elusive in the pathogenesis of bradykinin-driven hereditary angioedema (HAE). In this review, we will explore the molecular mechanisms behind contact system activation, their assembly on the endothelial surface, and their role in the HAE pathophysiology.

Chronic urticaria (CU) is a chronic inflammatory mast cell-driven disorder. Endothelial cells (ECs) contribute importantly to key features of CU. Several markers of EC (dys)function in CU have been reported, but have not yet been systematically reviewed. In this study, we systematically reviewed and categorized all published markers of EC functions in CU through a comprehensive search in Pubmed, The Cochrane Library, Web of Science, and SCOPUS using the following Mesh terms: CU AND pathogenesis AND (vasculopathy OR microangiopathy OR ECs OR marker). In total, 79 articles were selected and the identified biomarkers were categorized according to EC (dys)function in CU. The most frequent and consistently reported upregulated biomarkers in CU skin were adhesion molecules, TF, and P-selectin. The most frequently reported upregulated and reliable biomarkers in sera of CU patients were F1+2 for coagulation cascade involvement, D-dimers for fibrinolysis, and MMP-9 for vascular permeability. Emerging biomarkers described in the selected articles were endostatin, heat shock proteins, cleaved high molecular weight kininogen, and adipokines. This systematic review contributes to the pool of growing evidence for vascular involvement in CU where EC dysfunction is present in different aspects of cell survival, maintenance of vascular structure, and coagulation/fibrinolysis balance., SCOPUS: re.j, info:eu-repo/semantics/published

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

An activated plasma contact system is an abnormality observed in many Alzheimer’s disease (AD) patients. Since mild cognitive impairment (MCI) patients often develop AD, we analyzed the status of contact system activation in MCI patients. We found that kallikrein activity, high molecular weight kininogen cleavage, and bradykinin levels— measures of contact system activation— were significantly elevated in MCI patient plasma compared to plasma from age- and education-matched healthy individuals. Changes were more pronounced in MCI patients with impaired short-term recall memory, indicating the possible role of the contact system in early cognitive changes.

BACKGROUND: Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein-kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation. OBJECTIVE: We hypothesize that HK contributes to the hypercoagulable and pro-inflammatory state that causes end-organ damage and early mortality in sickle mice. METHODS: We evaluated the role of HK in the Townes mouse model of SCD. RESULTS/CONCLUSIONS: We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK-deficient background. We found that short-term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long-term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.

Purpose of review Angioedema without urticaria is composed of an increasing subtype's variety and presents a challenging diagnosis. This review summarizes the subtypes recently described and subsequent new findings helpful within their classification. Recent findings New methods to measure cleaved high molecular weight kininogen and activated plasma kallikrein have emerged as potential biochemical tests to identify bradykinin-mediated angioedema. Three new subtypes of hereditary angioedema (HAE) with normal C1 inhibitor were described in the past two years: HAE due to mutation in plasminogen gene, in kininogen gene, and in angiopoietin-1 gene; implicating the fibrinolytic and contact systems, and the regulation of vasculature, respectively. The understanding of some mechanisms in angioedema has been improved, compatible to the dominant-negative for some C1 inhibitor variants; furthermore, the increased activation of truncated F12 mutants by plasma kallikrein; and the diminished binding of angiopoietin-1 to its receptor. Summary The validation of biomarkers for the contact system activation could be beneficial in differentiating bradykinin - from histaminergic-mediated angioedema. Currently, the available laboratorial tests are still somewhat restricted to the evaluation of the complement activation and the mediators of nonhistaminergic and nonbradykinin-mediated angioedema remain to be identified.

Hereditary angioedema (HAE) with C1 inhibitor deficiency is a rare disorder characterized by unpredictable, potentially life-threatening recurrent angioedema attacks. Lanadelumab is a fully human monoclonal antibody with selective binding to active plasma kallikrein, and prevents the formation of cleaved high molecular weight kininogen (cHMWK) and bradykinin, thereby preventing HAE attacks. The clinical pharmacology of lanadelumab was characterized following subcutaneous administration in 257 subjects (24 healthy subjects and 233 patients with HAE). The pharmacokinetics of lanadelumab were described using a one-compartment model with first-order rate of absorption and linear clearance, showing slow absorption and a long half-life (14.8 days). A covariate analysis retained body weight and health status on apparent clearance (CL/F) and body weight on volume of distribution (V/F). Population estimates of CL/F and V/F were 0.0249 L/hour (0.586 L/day) and 12.8 L, respectively. An indirect-response Imax model showed 53.7% maximum suppression in cHMWK formation with a low potential for interactions with concomitant medications (analgesic, anti-inflammatory, and antirheumatic medications). A 300 mg dose administered Q2W was associated with a mean steady-state minimum concentration (Cmin,ss ; 25.4 μg/mL) that was ~ 4.5-fold higher than the half-maximal inhibitory concentration for cHMWK reduction (5.71 μg/mL). Exposure-response analyses suggest that 300 mg Q2W dosing was associated with a significantly reduced HAE attack rate, prolonged time to first attack after treatment initiation, and lower need for concomitant medications. The response was comparable across patient body weight groups. Findings from this analysis support the dosing rationale for lanadelumab to prevent attacks in patients with HAE.

High-molecular-weight kininogen (HMWK) deficiency is a very rare hereditary disorder caused by a defect of Kininogen-1 gene (KGN1). A 67-year-old asymptomatic male with an isolated prolonged activated partial thromboplastin time (aPTT) was recognized to have HMWK deficiency. The propositus had less than 1% HMWK procoagulant activity. The plasma HMWK procoagulant activities of his 2 younger sisters were 1.1% and less than 1%, respectively. Prekallikrein (PK) activity was also reduced in the propositus and two of his younger sisters with severe HMWK deficiency. Genetic testing to identify the KGN1 mutation provides a precise diagnosis for the patient and other family members. This Chinese family has a novel KGN1 nonsense variant, C to T, at nucleotide position 1456 leading to a stop codon in position 486 (p. Gln486*).

Infection with SARS-CoV-2 triggers the simultaneous activation of innate inflammatory pathways including the complement system and the kallikrein-kinin system (KKS) generating in the process potent vasoactive peptides that contribute to severe acute respiratory syndrome (SARS) and multi-organ failure. The genome of SARS-CoV-2 encodes four major structural proteins – the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. However, the role of these proteins in either binding to or activation of the complement system and/or the KKS is still incompletely understood. In these studies, we used: solid phase ELISA, hemolytic assay and surface plasmon resonance (SPR) techniques to examine if recombinant proteins corresponding to S1, N, M and E: (a) bind to C1q, gC1qR, FXII and high molecular weight kininogen (HK), and (b) activate complement and/or the KKS. Our data show that the viral proteins: (a) bind C1q and activate the classical pathway of complement, (b) bind FXII and HK, and activate the KKS in normal human plasma to generate bradykinin and (c) bind to gC1qR, the receptor for the globular heads of C1q (gC1q) which in turn could serve as a platform for the activation of both the complement system and KKS. Collectively, our data indicate that the SARS-CoV-2 viral particle can independently activate major innate inflammatory pathways for maximal damage and efficiency. Therefore, if efficient therapeutic modalities for the treatment of COVID-19 are to be designed, a strategy that includes blockade of the four major structural proteins may provide the best option.

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

Background Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. Objectives and methods We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+ , to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. Results The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. Conclusions Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

Sickle cell disease (SCD) is an inherited monogenic red blood cell disorder affecting millions worldwide. SCD causes vascular occlusions, chronic hemolytic anemia, and cumulative organ damage such as nephropathy, pulmonary hypertension, pathologic heart remodeling, and liver necrosis. Coagulation system activation, a conspicuous feature of SCD that causes chronic inflammation, is an important component of SCD pathophysiology. The key coagulation factor, thrombin (factor IIa [FIIa]), is both a central protease in hemostasis and thrombosis and a key modifier of inflammation. Pharmacologic or genetic reduction of circulating prothrombin in Berkeley sickle mice significantly improves survival, ameliorates vascular inflammation, and results in markedly reduced end-organ damage. Accordingly, factors both upstream and downstream of thrombin, such as the tissue factor–FX complex, fibrinogen, platelets, von Willebrand factor, FXII, high-molecular-weight kininogen, etc, also play important roles in SCD pathogenesis. In this review, we discuss the various aspects of coagulation system activation and their roles in the pathophysiology of SCD.

Factor XII (FXII) becomes a serine protease when blood is exposed to artificial medical surfaces or when pathologic surfaces arise in disease states leading to its autoactivation. Initiation of the blood coagulation cascade was the first recognized activity of FXIIa. Blocking FXIIa activity formed on artificial medical surfaces should reduce induced blood coagulation leading to thrombosis. In contrast to FXII enzymatic activities, less is known about zymogen FXII functions. Studies show that zymogen FXII has biologic activity in various cells in vivo. In endothelium, FXII stimulates cell growth and proliferation and, in vivo, neoangiogenesis after injury. In fibroblasts, transforming growth factor‐β increases FXII expression, which in turn stimulates fibroblast proliferation, contributing to tissue fibrosis. In neutrophils, FXII stimulates Akt2 to initiate neutrophil adhesion, migration, and chemotaxis, priming events leading to NETosis. Factor FXII deficiency leads to decreased neutrophil recruitment and improved wound healing. In dendritic cells, FXII contributes to neuroinflammation, and its deficiency or pharmacologic inhibition renders mice less susceptible to autoimmune encephalomyelitis. These combined studies indicate that FXII also contributes to multiple components of the inflammatory response. In sum, targeting FXII's biologic activities may provide novel approaches to reduce thrombosis and the inflammatory response in various disease states.

The kallikrein/kinin system, an intravascular biochemical pathway that includes several proteins involved in the contact activation system of coagulation, renin–angiotensin activation and inflammation, may or may not play a role in venous thromboembolism (VTE) occurrence. Within a large prospective population-based study in the United States, we conducted a nested case–cohort study to test the hypothesis that higher plasma levels of high molecular weight kininogen (HK) or prekallikrein are associated with greater VTE incidence. We related baseline enzyme-linked immunosorbent assay measures of HK and prekallikrein in 1993 to 1995 to incidence VTE of the lower extremity (n = 612) through 2015 (mean follow-up = 18 years). We found no evidence that plasma HK or prekallikrein was associated positively with incident VTE. HK, in fact, was associated inversely and significantly with VTE in most proportional hazards regression models. For example, the hazard ratio of VTE per standard deviation higher HK concentration was 0.88 (95% confidence interval = 0.81, 0.97), after adjustment for several VTE risk factors. Our findings suggest that plasma levels of these factors do not determine the risk of VTE in the general population.

Activated-mast cells (MCs) within gingival-tissue of chronic-periodontitis (CP) patients, release various inflammatory-factors. Bradykinin is a nine-amino-acid peptide and pro-inflammatory mediator, produced through factor-XII-cascade or tryptase-cascade. The ability of MC-chymase in bradykinin generation has not been discussed yet. This study investigated the salivary levels of MC-chymase, high molecular weight kininogen (HMWK) and bradykinin of CP patients; examined the potential of MC-proteases in bradykinin production using biochemistry-models; and explored the effects of bradykinin on gingival fibroblasts (GFs). Saliva-samples were collected; MC-protease activities were detected; HMWK cleavage was assessed by western-blot and SDS-PAGE; bradykinin levels were measured using immunoassay. Primary GFs were extracted and cultured with or without bradykinin; cell-viability, gelatine-zymography and flow-cytometry were applied. Immunocytochemistry and western-blot were used to detect intracellular protein expressions of bradykinin-stimulated GFs. The data showed that the salivary-levels of MC-proteases, bradykinin, HMWK, and lactoferrin of CP-patients were increased. HMWK was cleaved by MC-chymase in-vitro, resulting in bradykinin generation. Bradykinin promoted cell proliferation, cell cycle and matrix-metalloproteinase-2(MMP-2) activity, and increased intracellular expressions of nuclear-factor-kappa-B(NF-κB), focal-adhesion-kinase(FAK), transforming-growth-factor-β(TGF-β), P38, P53 of GFs. MC-chymase promotes bradykinin production to stimulate GFs and to continue inflammation during CP development. A new BK-generation cascade found in this study provides a new basis for the pathogenesis of CP and the mechanism of continuous inflammation. The activation of MC-chymase/bradykinin-generation cascade depends on HMWK level and MC-chymase activity under inflammatory condition. MC-chymase contributes to bradykinin production, mediating the cross-talks between MCs and GFs. MC-chymase can be used as a therapeutic target and a salivary biomarker in this case.

An important manifestation of severe COVID-19 is the ARDS-like lung injury that is associated with vascular endothelialitis, thrombosis, and angiogenesis. The intravascular innate immune system (IIIS), including the complement, contact, coagulation, and fibrinolysis systems, which is crucial for recognizing and eliminating microorganisms and debris in the body, is likely to be involved in the pathogenesis of COVID-19 ARDS. Biomarkers for IIIS activation were studied in the first 66 patients with COVID-19 admitted to the ICU in Uppsala University Hospital, both cross-sectionally on day 1 and in 19 patients longitudinally for up to a month, in a prospective study. IIIS analyses were compared with biochemical parameters and clinical outcome and survival. Blood cascade systems activation leading to an overreactive conjunct thromboinflammation was demonstrated, reflected in consumption of individual cascade system components, e.g., FXII, prekallikrein, and high molecular weight kininogen and in increased levels of activation products, e.g., C4d, C3a, C3d,g, sC5b-9, TAT, and D-dimer. Strong associations were found between the blood cascade systems and organ damage, illness severity scores, and survival. We show that critically ill COVID-19 patients display a conjunct activation of the IIIS that is linked to organ damage of the lung, heart, kidneys, and death. We present evidence that the complement and in particular the kallikrein/kinin system is strongly activated and that both systems are prognostic markers of the outcome of the patients suggesting their role in driving the inflammation. Already licensed kallikrein/kinin inhibitors are potential drugs for treatment of critically ill patients with COVID-19., publishedVersion

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.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

The future therapies for hereditary angioedema will likely involve the development of oral agents as alternatives to parenteral administration of drugs, specific targeting of proteins and/or enzymes that are not yet possible (e.g., factor XIIa), new agents that target the β2receptor with sustained action properties, testing of products to determine whether the β1receptor contributes significantly to attacks of angioedema, disrupting protein synthesis by using RNA technology as an alternative to enzyme inhibition, and, finally, gene therapy to attempt to cure the disease. Complete inhibition of attacks may well require sustained blood levels of C1 inhibitor that exceed 85% of normal, and it may be possible to delete the prekallikrein gene (analogous to familial prekallikrein deficiency), which is the one factor that might alleviate bradykinin formation, even by factor XII‐independent initiating mechanisms, with the possible exception of Mannose Associated Serine Protease 1 (MASP-1) cleavage of high molecular weight kininogen (HK). Deletion of the light chain of high-molecular-weight kininogen would eliminate all possibilities for bradykinin formation, except tissue kallikrein cleavage of low-molecular-weight kininogen to support normal physiologic function to at least 50%.

Bacterial products including endotoxin have been administered to humans over the past century as therapeutic, diagnostic, and experimental agents. The acute phase response is the early immediate host response to infection or tissue injury and results in fever, leukocytosis, changes in vascular permeability, and altered metabolic responses in various organs. Humoral defense mechanisms are rapidly activated after endotoxin administration. The contact system composed of Factors XII, XI, prekallikrein, and high molecular weight kininogen amplifies several host inflammatory responses including the initiation of coagulation via the intrinsic pathway, activation of plasminogen and neutrophils, as well as the generation of bradykinin, a potent vasodilator. Cytokines serve as important links to amplify the inflammatory response initiated by endotoxin. Hepatic synthetic function is rapidly upregulated in response to endotoxin. Endotoxin has been implicated in the pathogenesis of gram-negative bacterial pneumonia as well as the development of acute and chronic lung inflammation due to occupational dust exposure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a newly identified coronavirus which has spread from China to the rest of the world causing the pandemic coronavirus disease 19 (COVID-19). It has fatality rate that floats from 5 to 15% and the symtoms are fever, cough, myalgia and/or fatigue up to dyspnea, responsible for hospitalization and in most of the cases of artificial oxygenation. In the attempt to understand how the virus spreads and how to pharmacologically abolish it, it was highlighted that SARS-CoV2 infects human cells by means of angiotensin converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2) and 3-chymotrypsin-like protease (3CLpro), also known as Mpro. Once bound to its receptor ACE2, the other two proteases, in concert with the receptor-mediated signaling, allow virus replication and spread throughout the body. Our attention has been focused on the role of ACE2 in that its blockade by the virus increases Bradykinin and its metabolites, well known to facilitate inflammation in the lung (responsible for cough and fever), facilitate both the coagulation and complement system, three mechanisms that are typical of angioedema, cardiovascular dysfunction and sepsis, pathologies which symptoms occur in COVID-19 patients. Thus, we propose to pharmacologically block the kallicrein-kinin system upstream bradykinin and the ensuing inflammation, coagulation and complement activation by means of lanadelumab, which is a clinically approved drug for hereditary angioedema.

Development of synthetic antibodies for early-stage cancer diagnosis is a pursued goal in materials research. Molecular imprinting has shown advantages for this purpose, whereas preparation of molecularly imprinted polymer (MIP) for peptide/protein recognition is still a challenge. In the present study, a new MIP as an artificial antibody for biomarker analysis was synthesized by epitope and surface-confined imprinting approaches. The target peptides (K-1944 and K-2209) were the amino acids 440–456 and 438–456 fragments of high molecular weight kininogen that have sensitivity and specificity for the diagnosis of gastric, colorectal and liver cancers. For molecular imprinting, a heptapeptide, as an epitope for recognition, was selected as a template and immobilized on silica. Metal coordination between Cu(II) and template residues (His and Asp) was employed to create the binding sites. 4-Vinylpyridine was used as both the monomer and coordinating ligand. After imprinting polymerization and silica removal, spherical MIP (DQGHGHQ-MIP) with recognition ability was obtained successfully. The MIP could distinguish the template from one amino acid mismatched peptide. It also has surface-confined binding sites with good affinity for epitope-containing larger molecules. The MALDI-TOF analysis demonstrated that K-1944 and K-2209 could be selectively extracted from spiked human serum by the MIP. The solid phase extraction by DQGHGHQ-MIP coupled with HPLC was performed and 71–88% recoveries for K-1944 and K-2209 in spiked serum were obtained. The results demonstrated that DQGHGHQ-MIP could be used as an artificial antibody in the target peptide analysis with good extraction and sample clean-up performance.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor–dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK−/−) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK−/− mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

RATIONALE Studies identified kininogen as a potential biomarker of preeclampsia, a major cause of adverse maternal outcomes. High-molecular-weight kininogen (HK) and its activated form participate in numerous pathways associated with establishing and maintaining pregnancy. However, dynamic changes in HK and naturally occurring HK-derived peptides during the natural course of pregnancy are largely unknown. METHODS Longitudinal serum samples during the course of normal pregnancy (trimesters T1, T2, T3) from 60 pregnant women were analyzed by western blot with an anti-HK antibody. Circulating peptides in longitudinal serum specimens derived from 50 participants were enriched using nanoporous silica thin films. Peptides were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and database searching. Relative quantification was performed using MaxQuant and in-house scripts. Normality was evaluated by either ANOVA or Friedman tests with p