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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.

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.

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.

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.

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.

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.

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.

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

Angiotensin-converting enzyme (ACE) inhibitors are extensively prescribed to treat patients with hypertension, congestive heart failure, and diabetic nephropathy. A small fraction of these patients (approximately 0.7%) develop angioedema, manifested by swelling of the lips and oropharynx. Angioedema of oropharynx is a medical emergency that can lead to asphyxiation and death. The angioedema is due to bradykinin generated from high molecular weight kininogen by kallikrein, which is derived from plasma prekallikrein by action of the factor XIIa, factor Xia, or prolylcarboxypeptidase. Bradykinin induces vasodilation and increased vascular permeability. ACE is the major degrading enzyme of bradykinin in the intravascular department. ACE inhibitors inhibit the proteolytic inactivation of bradykinin. We report a patient with oropharyngeal angioedema associated with an ACE inhibitor with complete absence of plasma prekallikrein due to homozygous mutation (Ser97PhefsTer173).

BACKGROUND The lack of specific biomarkers makes the diagnosis of hereditary angioedema (HAE) with normal levels of C1-inhibitor (C1INH) protein (HAE-nl-C1INH) and idiopathic non-histaminergic angioedema (INHA) difficult. Confirming or excluding these diagnoses is a significant challenge for clinicians evaluating patients with angioedema. OBJECTIVE To develop a reliable biomarker that would aid the diagnosis of HAE-nl-C1INH and INHA. METHODS A total of 154 consecutive patients referred for angioedema at a single centre were enrolled and evaluated. Subjects were clinically phenotyped based on clinical history and response to treatment by clinicians blinded to laboratory assay results. Plasma kallikrein activity was measured by the cleavage of the fluorometric substrate Z-Phe-Arg-AMC-HCL in plasma samples stimulated ex vivo with submaximal doses of dextran sulphate. RESULTS Stimulated plasma kallikrein activity (mean relative fluorescence units/min ± SD) was significantly increased in both HAE-nl-C1INH (1804 ± 600) and INHA (1579 ± 371) subjects compared to non-swelling controls (171 ± 46) and histaminergic angioedema (133 ± 30) subjects. Using a threshold cut-off based on the normal controls, HAE-nl-C1INH and INHA subjects could be differentiated from histaminergic angioedema subjects with high sensitivity (negative predictive value 86%-89%) and specificity (positive predictive value 80%-100%). CONCLUSION AND CLINICAL RELEVANCE The stimulated kallikrein activity assay allows differentiation of bradykinin- from histamine-mediated angioedema. The assay could feasibly be considered as a potential clinical tool for the diagnosis of bradykinin-mediated angioedema.

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer’s disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent downstream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide β-amyloid (Aβ)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

Background: Hypotensive transfusion reactions are adverse events typified by a sudden decrease in blood pressure that usually occurs within the first minutes after the initiation of a transfusion and resolves shortly after the transfusion is stopped. Due to current passive reporting practices, the incidence is likely underreported, but recent studies estimate an incidence of 1.3 cases per 10000 RBC units. The pathophysiology of these reactions are not fully understood. One hypothesis proposed is that increased bradykinin (BK), a nonapeptide released from cleavage of high molecular weight kininogen (HK), as seen with the use of negatively charged leukoreduction filters and the use of angiotensin-converting enzyme inhibitors, is a major contributor to the pathophysiology. We have recently demonstrated that red blood cell derived microvesicles (RBCMVs) from aging red blood cell (RBC) units are able to trigger thrombin generation via kallikrein activation - a predominant enzyme to cleave high molecular weight kininogen (Noubouossie, Blood, 2020). Thus, we hypothesize that the same RBCMVs would lead to bradykinin generation and might explain these hypotensive events. Objectives: To determine if RBC storage lesion-derived microvesicles are able to facilitate HK cleavage and BK release. Methods: RBCMVs were prepared from 4 recently expired RBC units (42 or 43 day old, AS-3 preserved, prestorage leukoreduced, all A+) via a series of centrifugations and washes. RBCMVs were quantified and characterized using nanoparticle tracking analysis. Obtained RBCMVs were first assessed for the capacity to initiate thrombin generation in microvesicle free human plasma via a substrate cleavage assay. Next, RBCMVs were added to a buffer reaction containing prekallikrein and HK, and kininogen cleavage was assessed via western blot. RBCMVs were also mixed with microvesicle-free human plasma and analyzed for evidence of kallikrein activation, cleavage of high molecular weight kininogen, and bradykinin production by ELISA. Cohn fractionation of plasma was used to enrich for BK. Results: RBCMVs were enumerated and concentrated to 7.5 ± 1.4 x 10 12 per mL (mean±SD size 160 ± 29µm). RBCMVs were able to initiate thrombin generation principally via contact pathway activation, independently of tissue factor. In a buffer system RBCMVs demonstrated activity to generate kallikrein with a sequential high molecular weight kininogen cleavage in a dose-dependent manner. Exclusion of kallikrein from the buffer system or addition of the small molecule inhibitor of kallikrein - ecallantide - halted cleavage of kininogen. A dose-dependent cleavage of high molecular weight kininogen indicated that RBCMVs could cause BK release in plasma; this was confirmed via an independent assay of Cohn -fractionated samples. Conclusions: Results of this current study demonstrate that RBCMVs are leading to high HK cleavage via kallikrein activation in vitro. We suspect that the same mechanisms could lead to BK generation in patients receiving older RBC units, possibly increasing the risk for hypotensive events from transfusion. Disclosures Karafin: Westat, Inc.: Consultancy. Key: Sanofi: Consultancy; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

The original version of this article unfortunately contained a mistake. The image of Fig. 1 should be corrected. In the previous figure, we had shown that the plasma kallikrein cleaves both high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) to produce bradykinin. However, the plasma kallikrein activates only high molecular weight kininogen (HMWK) while low molecular weight kininogen (LMWK) is activated by tissue kallikrein. Hence we have removed low molecular weight kininogen (LMWK) in the revised figure. We wish to acknowledge Dr. Allen P. Kaplan, Medical University of South Carolina, Charleston, SC, USA for pointing out the error in Fig. 1. The corrected Fig. 1 is given below.

Aim: C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. Materials & methods: A novel ion-pair 2D LC–MS/MS assay was developed to measure the 46 kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction. Results: The LLOQ was 200 ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%. Conclusion: A reagent-free LC–MS assay has been developed for the quantitation of 46 kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.

In this study, we show that mice lacking high-molecular-weight kininogen (HK) were resistant to lipopolysaccharide (LPS)-induced mortality and had significantly reduced circulating LPS levels. Replenishment of HK-deficient mice with human HK recovered the LPS levels and rendered the mice susceptible to LPS-induced mortality. Binding of HK to LPS occurred through the O-polysaccharide/core oligosaccharide, consistent with the ability to bind LPS from K. pneumoniae, P. aeruginosa, S. minnesota, and different E. coli strains. Binding of LPS induced plasma HK cleavage to the two-chain form (HKa, containing a heavy chain [HC] and a light chain [LC]) and bradykinin. Both HKa and the LC, but not the HC, could disaggregate LPS. The light chain bound LPS with high affinity (Kd = 1.52 × 10−9 M) through a binding site in domain 5 (DHG15). A monoclonal antibody against D5 significantly reduced LPS-induced mortality and circulating LPS levels in wild-type mice. Thus, HK, as a major LPS carrier in circulation, plays an essential role in endotoxemia.

The kinin system is activated during inflammation. Plasma prekallikrein is cleaved on the extracellular surface of endothelial cells or neutrophils generating plasma kallikrein. This, in turn, cleaves high molecular weight kininogen (HK) releasing bradykinin and Lys-bradykinin, with subsequent

Background Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU. Objective To study biomarkers of bradykinin release in CSU. Methods Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE). Results One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels. Conclusions cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema. Clinical relevance If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

Plasma of patients with types I and II hereditary angioedema is unstable if incubated in a plastic (i.e., inert) vessel at 37 °C manifested by progressively increasing formation of bradykinin. There is also a persistent low level of C4 in 95 % of patients even when they are symptomatic. These phenomena are due to the properties of the C1r subcomponent of C1, factor XII, and the bimolecular complex of prekallikrein with high molecular weight kininogen (HK). Purified C1r auto-activates in physiologic buffers, activates C1s, which in turn depletes C4. This occurs when C1 inhibitor is deficient. The complex of prekallikrein-HK acquires an inducible active site not present in prekallikrein which in Tris-type buffers cleaves HK stoichiometrically to release bradykinin, or in phosphate buffer auto-activates to generate kallikrein and bradykinin. Thus immunologic depletion of C1 inhibitor from factor XII-deficient plasma (phosphate is the natural buffer) auto-activates on incubation to release bradykinin. Normal C1 inhibitor prevents this from occurring. During attacks of angioedema, if factor XII auto-activates on surfaces, the initial factor XIIa formed converts prekallikrein to kallikrein, and kallikrein cleaves HK to release bradykinin. Kallikrein also rapidly activates most remaining factor XII to factor XIIa. Additional cleavages convert factor XIIa to factor XIIf and factor XIIf activates C1r enzymatically so that C4 levels approach zero, and C2 is depleted. There is also a possibility that kallikrein is generated first as a result of activation of the prekallikrein-HK complex by heat shock protein 90 released from endothelial cells, followed by kallikrein activation of factor XII.

Domain 5 of kinin-free high molecular weight kininogen inhibits the adhesion of many tumor cell lines, and it has been reported that the histidine-glycine-lysine (HGK)-rich region might be responsible for inhibition of cell adhesion. The authors developed HGK-containing hexapeptide, glutamic acid-cysteine-glycine (ECG)-HGK, and evaluated the utility of Tc-99m ECG-HGK for tumor imaging. Hexapeptide, ECG-HGK was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling efficiency was evaluated. The uptake of Tc-99m ECG-HGK within HT-1080 cells was evaluated in vitro. In HT-1080 tumor-bearing mice, gamma imaging and biodistribution studies were performed. The complexes Tc-99m ECG-HGK was prepared in high yield. The uptake of Tc-99m ECG-HGK within the HT-1080 tumor cells had been demonstrated by in vitro studies. The gamma camera imaging in the murine model showed that Tc-99m ECG-HGK was accumulated substantially in the HT-1080 tumor (tumor-to-muscle ratio = 5.7 ± 1.4 at 4 h), and the tumoral uptake was blocked by the co-injection of excess HGK (tumor-to-muscle ratio = 2.8 ± 0.6 at 4 h). In the present study, Tc-99m ECG-HGK was developed as a new tumor imaging agents. Our in vitro and in vivo studies revealed specific function of Tc-99m ECG-HGK for tumor imaging.

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe-/- mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.

Background Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK). Methods Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit. Results The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage. Conclusions Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts. Electronic supplementary material The online version of this article (10.1186/s13071-018-2704-0) contains supplementary material, which is available to authorized users.

Capsule Summary Bradykinin concentrations and the ratio of bradykinin to its stable metabolite BK1-5 (RPPGF) were significantly increased in patients presenting with angiotensin-converting enzyme (ACE) inhibitor-associated angioedema compared to ACE inhibitor-treated controls. Cleavage of high molecular weight kininogen was not increased in patients with ACE inhibitor-associated angioedema, indicating that increased bradykinin concentrations result from decreased degradation.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr(309) in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Apoptotic cells, by externalizing phosphatidylserine (PS) as a hallmark feature, are procoagulant. However, the mechanism by which apoptotic cells activate coagulation system remains unknown. Intrinsic coagulation pathway is initiated by coagulation factor XII (FXII) of contact activation system. The purpose of this study was to determine whether FXII is involved in procoagulant activity of apoptotic cells. Using Western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells, but not with viable cells, resulted in rapid cleavage and activation of FXII in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of contact activation system. As detected by flow cytometry, FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. Direct association of FXII with PS was confirmed in a surface plasmon resonance assay. Clotting time of FXII-deficient plasma induced by apoptotic cells was significantly prolonged, which was fully reversed by replenishment with FXII. Corn trypsin inhibitor, a FXII inhibitor, completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which was not affected by an inhibitory anti-tissue factor antibody. However, blocking of PS by annexin V, inhibition of FXII or the deficiency of FXII suppressed apoptotic cells-induced thrombin generation. Addition of purified FXII to XII-deficient plasma recovered thrombin generation to the normal plasma level. In conclusion, FXII binds to apoptotic cells via PS and becomes activated, thereby constituting a novel mechanism mediating the procoagulant activity of apoptotic cells.

For decades, immunologists have considered the complement system as a paradigm of a proteolytic cascade that, acting cooperatively with the immune system, enhances host defense against infectious organisms. In recent years, advances made in thrombosis research disclosed a functional link between activated neutrophils, monocytes, and platelet-driven thrombogenesis. Forging a physical barrier, the fibrin scaffolds generated by synergism between the extrinsic and intrinsic (contact) pathways of coagulation entrap microbes within microvessels, limiting the systemic spread of infection while enhancing the clearance of pathogens by activated leukocytes. Insight from mice models of thrombosis linked fibrin formation via the intrinsic pathway to the autoactivation of factor XII (FXII) by negatively charged "contact" substances, such as platelet-derived polyphosphates and DNA from neutrophil extracellular traps. Following cleavage by FXIIa, activated plasma kallikrein (PK) initiates inflammation by liberating the nonapeptide bradykinin (BK) from an internal domain of high molecular weight kininogen (HK). Acting as a paracrine mediator, BK induces vasodilation and increases microvascular permeability via activation of endothelial B2R, a constitutively expressed subtype of kinin receptor. During infection, neutrophil-driven extravasation of plasma fuels inflammation via extravascular activation of the kallikrein-kinin system (KKS). Whether liberated by plasma-borne PK, tissue kallikrein, and/or microbial-derived proteases, the short-lived kinins activate immature dendritic cells via B2R, thus linking the infection-associated innate immunity/inflammation to the adaptive arm of immunity. As inflammation persists, a GPI-linked carboxypeptidase M removes the C-terminal arginine from the primary kinin, converting the B2R agonist into a high-affinity ligand for B1R, a GPCR subtype that is transcriptionally upregulated in injured/inflamed tissues. As reviewed here, lessons taken from studies of kinin receptor function in experimental infections have shed light on the complex proteolytic circuits that, acting at the endothelial interface, reciprocally couple immunity to the proinflammatory KKS.

Background: Unregulated plasma kallikrein proteolytic activity can result from C1-inhibitor deficiency, causing excessive and potentially fatal edema. Results: The antibody DX-2930 potently and specifically inhibits plasma kallikrein and exhibits a long plasma half-life. Conclusion: An antibody protease inhibitor can lead to potent and specific bioactivity. Significance: DX-2930 could be an effective therapeutic for the prophylactic inhibition of plasma kallikrein-mediated diseases., Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.

Acetaminophen (APAP) toxicity is a common cause of acute liver failure (ALF). Dysregulation of coagulation is well documented in ALF patients. Recent reports indicate that small subset of ALF patients (~10%) exhibit spontaneous bleeding, while thrombotic complications are more frequent. Animal studies demonstrated that the inhibition of tissue factor-initiated thrombin generation attenuates APAP-induced liver injury in mice via both fibrin- and protease activated receptor 1-dependent mechanisms. Activation of FXII leads to two events: propagation of coagulation by activation of FXI (intrinsic pathway) and activation of plasma prekallikrein to kallikrein, with subsequent cleavage of high molecular weight kininogen (HK) into bradykinin (BK) and cleaved HK fragments (contact pathway). There is a growing interest in therapeutic targeting of FXII or FXI to prevent thrombosis, primarily because this goal may be attainable without incurring a significant bleeding risk. In this study, we evaluated if the intrinsic coagulation pathway contributes to the activation of coagulation and liver injury in a mouse model of APAP-induced ALF. We postulated that targeting FXII would attenuate coagulation and reduce liver injury without affecting hemostasis. In addition, FXII deficiency could provide a further protection by preventing activation of contact pathway and subsequent reduction of inflammatory response. We used 12 weeks old FXII, FXI, prekallkrein and HK deficient male mice and their respective WT controls. Mice were fasted for 16 hours followed by a single intraperitoneal injection of APAP (400 mg/kg) or sterile saline. Livers and plasma samples were collected 6 and 24 hours after injections. Hepatocellular necrosis was determined on liver sections stained with H&E. Plasma levels of alanine transaminase (ALT- marker of liver injury), thrombin-antithrombin (TAT) complexes, plasmin-α2 antiplasmin (PAP) complexes and interleukin-6 were analyzed using commercially available assays. HK cleavage was determined using BK ELISA, Western blot, and mass spectrometry analysis. Consistently with previously published data, plasma levels of ALT, TAT and IL-6, as well as liver injury scores were significantly elevated in APAP-challenged mice compared to saline-injected WT mice at both 6 and 24 hours. Surprisingly, neither FXII, FXI, nor prekallikrein deficiency had statistically significant effects on any of these parameters in APAP-challenged mice at either time point. Interestingly, however, plasma levels of ALT were significantly attenuated in HK-/- mice (n=23-34) compared to HK+/+ (n=19-31) controls at both 6 (1278 ± 184 vs. 1850 ± 235 U/L; p It has been previously shown that plasminogen deficiency protects against APAP-induced liver injury. Indeed, APAP administration activated the fibrinolytic system in WT mice as demonstrated by increased plasma levels of active tissue plasminogen activator (19.5 fold; p In summary, our data indicate that the extrinsic but not the intrinsic coagulation pathway drives the coagulation-mediated pathologies associated with APAP-induced liver injury in mice. HK contributes to the liver injury independently of thrombin generation. We propose that in APAP-challenged mice, HK cleavage and downstream hepatotoxicity, are mediated by plasmin rather than FXIIa-dependent generation of kallikrein. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Key:Uniqure BV: Research Funding.

N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.

Diabetic retinopathy (DR) occurs, to some extent, in most people with at least 20 years’ duration of diabetes mellitus. The progression of DR to its sight-threatening stages is usually associated with the worsening of underlying retinal vascular dysfunction and disease. The plasma kallikrein-kinin system (KKS) is activated during vascular injury, where it mediates important functions in innate inflammation, blood flow, and coagulation. Recent findings from human vitreous proteomics and experimental studies on diabetic animal models have implicated the KKS in contributing to DR. Vitreous fluid from people with advanced stages of DR contains increased levels of plasma KKS components, including plasma kallikrein (PK), coagulation factor XII, and high-molecular-weight kininogen. Both bradykinin B1 and B2 receptor isoforms (B1R and B2R, respectively) are expressed in human retina, and retinal B1R levels are increased in diabetic rodents. The activation of the intraocular KKS induces retinal vascular permeability, vasodilation, and retinal thickening, and these responses are exacerbated in diabetic rats. Preclinical studies have shown that the administration of PK inhibitors and B1R antagonists to diabetic rats ameliorates retinal vascular hyperpermeability and inflammation. These findings suggest that components of plasma KKS are potential therapeutic targets for diabetic macular edema.

Background When the prekallikrein-high molecular weight kininogen complex is bound to endothelial cells, prekallikrein is stoichiometrically converted to kallikrein because of release of heat shock protein-90 (Hsp90). Although bradykinin formation is typically initiated by factor XII autoactivation, it is also possible to activate factor XII either by kallikrein, thus formed, or by plasmin. Objective Because attacks of hereditary angioedema can be related to infection and/or exposure to estrogen, we questioned whether estrogen or cytokine stimulation of endothelial cells could augment release of Hsp90 and prekallikrein activation. We also tested release of profibrinolytic enzymes, urokinase, and tissue plasminogen activator (TPA) as a source for plasmin formation. Methods Cells were stimulated with agonists, and secretion of Hsp90, urokinase, and TPA was measured in the culture supernatants by ELISA. Activation of the prekallikrein-HK complex was measured by using pro-phe-arg-p-nitroanilide reflecting kallikrein formation. Results Hsp90 release was stimulated with optimal doses of estradiol, IL-1, and TNF-α (10 ng/mL) from 15 minutes to 120 minutes. TPA release was not augmented by any of the agonists tested but urokinase was released by IL-1, TNF-α, and thrombin (positive control), but not estrogen. Augmented activation of the prekallikrein-HK complex to generate kallikrein was seen with each agonist that releases Hsp90. Addition of 0.1% factor XII relative to prekallikrein-HK leads to rapid formation of kallikrein; factor XII alone does not autoactivate. Conclusions IL-1, TNF-α, and estrogen stimulate release of Hsp90 and augment activation of the prekallikrein-HK complex to generate kallikrein and bradykinin. IL-1 and TNF-α stimulate release of urokinase, which can convert plasminogen to plasmin and represents a possible source for plasmin generation in all types of hereditary angioedema, but particularly hereditary angioedema with normal C1 inhibitor with a factor XII mutation. Both kallikrein and plasmin activate factor XII; kallikrein is 20 times more potent on a molar basis.

Two major kallikreins are known in mammals. Plasma kallikrein (KLKB1) possesses a unique structure of four apple-domains and a trypsin domain. Tissue (glandular) kallikrein (KLK) possesses only a trypsin domain. KLKB1 binds to high molecular weight kininogen (KNG) with high affinity and preferentially releases bradykinin (BK). KLKB1 also digests plasminogen to plasmin and participates in surface-dependent activation of blood coagulation, fibrinolysis, and inflammation. Kallikrein converts prorenin to renin to activate the renin-angiotensin system. No KLKB1 was found in teleosts but KLKB1-like lectin that could be involved in immune function was found. KLK is highly selective to release [Lys0]-BK from both high and low molecular weight KNGs. In addition, KLK is involved in proteolytic cascades for semen liquefaction through hydrolysis of seminogelin and desquamation of the skin by cleavage of cellular adhesion proteins. High KLK3 expression is a marker for prostate cancer.

Two major kallikreins are known in mammals. Plasma kallikrein (KLKB1) possesses a unique structure of four apple-domains and a trypsin domain. Tissue (glandular) kallikrein (KLK) possesses only a trypsin domain. KLKB1 binds to high molecular weight kininogen (KNG) with high affinity and preferentially releases bradykinin (BK). KLKB1 also digests plasminogen to plasmin and participates in surface-dependent activation of blood coagulation, fibrinolysis, and inflammation. Kallikrein converts prorenin to renin to activate the renin-angiotensin system. No KLKB1 was found in teleosts but a KLKB1-like lectin that could be involved in immune function was found. KLK is highly selective to release [Lys0]-BK from both high and low molecular weight KNGs. In addition, KLK is also involved in proteolytic cascades for semen liquefaction through hydrolysis of seminogelin and desquamation of the skin by cleavage of cellular adhesion proteins. High KLK3 expression is a marker for prostate cancer.

Polyphosphate (polyP) was recently discovered to be stored in a subset of the secretory granules of human platelets (the blood cell that supports formation of clots) and to be secreted upon activation of these cells. It is also present in other human cell types and is present in infectious microorganisms. Work from our laboratory and others has now shown that polyphosphate is a novel, potent modulator of blood clotting that likely plays roles in hemostasis, thrombosis, inflammation, and the host response to pathogens. Polyphosphate acts at multiple points in the coagulation cascade, providing a template for initiation of the contact pathway of clotting, enhancing the activation of factor V (a critical cofactor in clotting whose accelerated activation results in an earlier thrombin burst), and markedly enhancing the rate of activation of factor XI by thrombin (resulting in marked amplification of thrombin generation). Polyphosphate also acts on the formation and degradation of fibrin by becoming incorporated into polymerizing fibrin fibrils (rendering them thicker and obscuring the binding sites for fibrinolytic proteins, which in turn delays clot degradation). Therapeutic agents targeting polyphosphate may have the potential to limit thrombosis with fewer hemorrhagic complications than conventional anticoagulant drugs that target essential proteases of the blood-clotting cascade.

Kininogen (KNG) is the precursor protein of the kallikrein-kinin system (KKS). High molecular weight (HMW) and low molecular weight (LMW) KNGs are formed by alternative splicing of kng1 in mammals. HMW KNG interacts with plasma kallikrein to produce bradykinin (BK) and is involved in the contact activation of the blood coagulation process. LMW KNG interacts with tissue kallikreins to produce [Lys0]-BK. Amphibians have lost functional KNG but they produce skin kinins from other secretory proteins as poisons for self-protection. The histidine-rich domain of HMW KNG can release anti-microbial and anti-fungal peptides for immune functions. HMW KNG also downregulates endothelial cell proliferation and migration, inhibits angiogenesis, and reduces apoptosis. LMW KNG inhibits the aggregation of thrombocytes. The cystatin domains on KNG are identical to those of alpha-cysteine proteinase inhibitors and act as natural inhibitors for cysteine cathepsins.

Bradykinin (BK) is a vasoactive nonapeptide cleaved from circulating kininogens and that is degraded by angiotensin converting enzyme (ACE). It has been reported that the PR3 protease from human neutrophil releases an alternate peptide of 13 amino acids, Met-Lys-BK-Ser-Ser, from high molecular weight kininogen. We have studied vascular actions of this kinin. Its affinity for recombinant B 1 and B 2 receptors is very low, as assessed by the binding competition of [ 3 H]Lys-des-Arg 9 -BK and [ 3 H]BK, respectively, but Met-Lys-BK-Ser-Ser effectively displaced a fraction of [ 3 H]enalaprilat binding to recombinant ACE. Mutant recombinant ACE constructions revealed that affinity gap between BK and Met-Lys-BK-Ser-Ser is larger for the N-terminal catalytic site than for the C-terminal one, based on competition for the substrate Abz-Phe-Arg-Lys(Dnp)-Pro-OH in an enzymatic assay. Met-Lys-BK-Ser-Ser is a low potency stimulant of the rabbit aorta (bioassay for B 1 receptors), but the human isolated umbilical vein, a contractile bioassay for the B 2 receptors, responded to Met-Lys-BK-Ser-Ser more than expected from the radioligand binding assay, this agonist being ∼30-fold less potent than BK in the vein. Venous tissue treatment with the ACE inhibitor enalaprilat reduced the apparent potency of Met-Lys-BK-Ser-Ser by 15-fold, while not affecting that of BK. In the rabbit isolated jugular vein, Met-Lys-BK-Ser-Ser is nearly as potent as BK as a contractile stimulant of endogenous B 2 receptors (EC 50 values of 16.3 and 10.5 nM, respectively), but enalaprilat reduced the potency of Met-Lys-BK-Ser-Ser 13-fold while increasing that of BK 5.3-fold. In vascular tissue, ACE assumes a paradoxical activating role for Met-Lys-BK-Ser-Ser.

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.

BACKGROUND AND PURPOSE Kallikrein acts on high molecular weight kininogen (HK) to generate HKa (cleaved HK) and bradykinin (BK). BK exerts its effects by binding to B2 receptors. The activation of B2 receptors leads to the formation of tissue plasminogen activator, nitric oxide (NO) and prostacyclin (PGI2). An elevated kallikrein-dependent pathway has been linked to cardiovascular disease risk. The aim of this study was to investigate whether our novel plasma kallikrein inhibitor abolishes kallikrein-mediated generation of BK from HK and subsequent BK-induced NO and PGI2 formation, thereby influencing endothelial pathophysiology during chronic inflammatory diseases. EXPERIMENTAL APPROACH Kinetic analysis was initially used to determine the potency of PF-04886847. Biochemical ligand binding assays, immunological methods and calcium flux studies were used to determine the selectivity of the kallikrein inhibitor. In addition, the effect of PF-04886847 on BK-induced relaxation of the rat aortic ring was determined in a model of lipopolysaccharide-induced tissue inflammation. KEY RESULTS Evidence was obtained in vitro and in situ, indicating that PF-04886847 is a potent and specific inhibitor of plasma kallikrein. PF-04886847 efficiently blocked calcium influx as well as NO and PGI2 formation mediated through the BK-stimulated B2 receptor signalling pathway. PF-04886847 blocked kallikrein-induced endothelial-dependent relaxation of isolated rat aortic rings pre-contracted with phenylephrine. CONCLUSIONS AND IMPLICATIONS PF-04886847 was shown to be the most potent small molecule inhibitor of plasma kallikrein yet described; it inhibited kallikrein in isolated aortic rings and cultured endothelial cells. Overall, our results indicate that PF-04886847 would be useful for the treatment of kallikrein-mediated inflammatory disorders.

In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) of B. forficata leaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharose. The molecular weight 30 kDa was estimated by SDS-PAGE and zymography, and the N-terminal sequence and CD spectra indicated a relationship with the papain family of cysteine proteinases. Denominated baupain, the enzyme was activated by dithiotreitol and inhibited by E-64 and iodoacetamide, but not by benzamidine, TLCK, TPCK and EDTA. The S2 and S1 substrate specificity of baupain, assayed with two series of fluorescence resonance energy transfer (FRET) peptide substrates derived from Abz-KLRSSK-Q-EDDnp, indicates a preference for Phe and Tyr at P2 position over Leu found in papain. Baupain releases bradykinin from HMWK (human high molecular weight kininogen) though its proteolytic activity is blocked by the sequence motif QVVA of kininogen ( K iapp = 1.9 × 10 −8 M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain ( K iapp = 0.18 × 10 −9 M).

Apoptosis can be induced in a variety of pathological disorders, including inflammation, autoimmune diseases, and chemotherapy. When cells undergo apoptosis, they express phosphatidylserine (PS) on cell membrane surface and thus become procoagulant. Although it has been known that the procoagulant activity of apoptotic cells are tightly associated with thrombotic disorders, such as atherothrombosis and Trousseau syndrome, the mechanisms by which apoptotic cells activate the coagulation system and enhance blood clotting are largely unknown. In this study we investigated which coagulation factor(s) is involved in this process. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells induced by Dexamethasone (DXMS), but not with viable cells, resulted in rapid cleavage and activation of FXII. Moreover, apoptotic cells-mediated FXII activation was significantly increased in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of plasma contact system. However, incubation of apoptotic cells did not cause dramatic changes of other coagulation factors, suggesting a selective association of FXII activation with apoptotic cells. Activation of FXII by apoptotic cells was markedly inhibited by a specific anti-kallikrein antibody, indicating the activation of the contact system by apoprotic cells. Flow cytometric measurement showed that FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. A surface plasmon resonance assay showed a direct binding of FXII to PS (KD=3.9E-9 M). When challenged by apoptotic cells, clotting time of plasma from FXII-knockout mice was significantly prolonged, which was reversed by replenishment with human FXII. Moreover, an inhibitory anti-FXII antibody completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which were attenuated by PS blocker annexin V, an inhibitory anti-FXII antibody, and the deficiency of FXII, respectively. Addition of human FXII to XII-deficient plasma recovered thrombin generation. As evaluated by ELISA, the levels of thrombin-antithrombin complex in circulation were significantly increased when apoptotic cells were intravenously injected into wild-type mice, but not in FXII-knockout mice. In conclusion, FXII plays an important role in apoptotic cells-mediated procoagulant activity. Disclosures No relevant conflicts of interest to declare.

The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.

The plasma bradykinin-forming cascade and the complement pathways share many elements, including cross-activation, common control mechanisms, and shared binding proteins. The C1 inhibitor (C1 INH) is not only the inhibitor of activated C1r and C1s, but it is the key control protein of the plasma bradykinin-forming cascade. It inhibits the autoactivation of Factor XII, the ability of Factor XIIa to activate prekallikrein and Factor XI, the activation of high molecular weight kininogen (HK) by kallikrein, and the feedback activation of Factor XII by kallikrein. Thus in the absence of C1 INH (hereditary angioedema or acquired C1 INH deficiency) there is unimpeded formation of bradykinin leading to angioedema. Activated Factor XII (Factor XIIa, 80,000 kDa) is further cleaved by kallikrein or plasmin to yield Factor XII fragment (Factor XIIf, 30,000 kDa) and Factor XIIf can activate the C1r subcomponent of C1, particularly when C1 INH (which inhibits Factor XIIf) is absent. Once bradykinin is formed, it causes vasodilatation and increased vascular permeability by interaction with constitutively expressed B-2 receptors. However degradation of bradykinin by carboxypeptidase N (in plasma) or carboxypeptidase M (on endothelial cells) yields des-arg-9 ( Kerbiriou and Griffin, 1979 ) bradykinin which interacts with B-1 receptors. B-1 receptors are induced in inflammatory states by cytokines such as Interleukin 1 and its interaction with bradykinin may prolong or perpetuate the vascular response until bradykinin is completely inactivated by angiotensin converting enzyme or aminopeptidase P, or neutral endopeptidase. The entire bradykinin-forming cascade is assembled and can be activated along the surface of endothelial cells in zinc dependent reactions involving gC1qR, cytokeratin 1, and the urokinase plasminogen activated receptor (u-PAR). Although Factors XII and HK can be shown to bind to each one of these proteins, they exist in endothelial cells as two bimolecular complexes; gC1qR-cytokeratin 1, which preferentially binds HK, and cytokeratin 1–u-PAR which preferentially binds Factor XII. The gC1qR, which binds the globular heads of C1q is present in excess and can bind either Factor XII or HK however the binding sites for HK and C1q have been shown to reside at opposite ends of gC1qR. Activation of the bradykinin-forming pathway can be initiated at the cell surface by gC1qR-induced autoactivation of Factor XII or direct activation of the prekallikrein–HK complex by endothelial cell-derived heat-shock protein 90 (HSP 90) or prolylcarboxypeptidase with recruitment or Factor XII by the kallikrein produced.

Inflammatory bowel disease and arthritis are associated with contact activation that results in cleavage of kininogen to form high molecular weight kininogen (HKa) and bradykinin. We have previously demonstrated that HKa can stimulate inflammatory cytokine and chemokine secretion from human monocytes. We now show that HKa can upregulate tissue factor antigen and procoagulant activity on human monocytes as a function of time (1-4 h) and HKa concentration (75-900 nM). The amino acid sequence responsible to block HKa effects is G440-H455. The HKa receptor macrophage-1 (Mac-1; CD11b18) is the binding site as shown by inhibition by a monoclonal antibody to CD11b/18. Chemical inhibitors of JNK, ERK, and p38 signaling pathways block cell signaling, as does an inhibitor to the transcription factor NF-kappaB. A combination of monoclonal antibodies to TNF-alpha and IL-1beta but neither alone inhibited the HKa induction of tissue factor. These results suggest that HKa mimics LPS by triggering a paracrine pathway in monocytes that depends on TNF-alpha and IL-1beta. Antibodies to kininogen or peptidomimetics might be a useful and safe therapy in inflammatory diseases or sepsis involving cytokines.