Bradykinin Generation Research Articles

Mast cell degranulation and bradykinin-induced angioedema - searching for the missing link.

Initiation of the bradykinin generation cascade is responsible for the occurrence of attacks in some types of angioedema without wheals. Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) is one such clinical entity. In this paper, we explore the existing evidence that mast cells (MCs) degranulation may contribute to the activation of the kallikrein-kinin system cascade, followed by bradykinin formation and angioedema. We present the multidirectional effects of MC-derived heparin and other polyanions on the major components of the kinin-kallikrein system, particularly on the factor XII activation. Although, bradykinin- and histamine-mediated symptoms are distinct clinical phenomena, they share some common features, such as some similar triggers and a predilection to occur at sites where mast cells reside, namely the skin and mucous membranes. In addition, recent observations indicate a high incidence of hypersensitivity reactions associated with MC degranulation in the HAE-C1-INH patient population. However, not all of these can be explained by IgE-dependent mechanisms. Mast cell-related G protein-coupled receptor-X2 (MRGPRX2), which has recently attracted scientific interest, may be involved in the activation of MCs through a different pathway. Therefore, we reviewed MRGPRX2 ligands that HAE-C1-INH patients may be exposed to in their daily lives and that may affect MCs degranulation. We also discussed the known inter- and intra-individual variability in the course of HAE-C1-INH in relation to factors responsible for possible variability in the strength of the response to MRGPRX2 receptor stimulation. The above issues raise several questions for future research. It is not known to what extent a prophylactic or therapeutic intervention targeting the pathways of one mechanism (mast cell degranulation) may affect the other (bradykinin production), or whether the number of mast cells at a specific body site and their reactivity to triggers such as pressure, allergens or MRGPRX2 agonists may influence the occurrence of HAE-C1-INH attacks at that site.

A quantitative systems pharmacology model of plasma kallikrein-kinin system dysregulation in hereditary angioedema.

Hereditary angioedema (HAE) due to C1-inhibitor deficiency is a rare, debilitating, genetic disorder characterized by recurrent, unpredictable, attacks of edema. The clinical symptoms of HAE arise from excess bradykinin generation due to dysregulation of the plasma kallikrein-kinin system (KKS). A quantitative systems pharmacology (QSP) model that mechanistically describes the KKS and its role in HAE pathophysiology was developed based on HAE attacks being triggered by autoactivation of factor XII (FXII) to activated FXII (FXIIa), resulting in kallikrein production from prekallikrein. A base pharmacodynamic model was constructed and parameterized from literature data and ex vivo assays measuring inhibition of kallikrein activity in plasma of HAE patients or healthy volunteers who received lanadelumab. HAE attacks were simulated using a virtual patient population, with attacks recorded when systemic bradykinin levels exceeded 20pM. The model was validated by comparing the simulations to observations from lanadelumab and plasma-derived C1-inhibitor clinical trials. The model was then applied to analyze the impact of nonadherence to a daily oral preventive therapy; simulations showed a correlation between the number of missed doses per month and reduced drug effectiveness. The impact of reducing lanadelumab dosing frequency from 300mg every 2weeks (Q2W) to every 4weeks (Q4W) was also examined and showed that while attack rates with Q4W dosing were substantially reduced, the extent of reduction was greater with Q2W dosing. Overall, the QSP model showed good agreement with clinical data and could be used for hypothesis testing and outcome predictions.

Abstract 155: Plasma Kallikrein Is A Key Driver Of Basal Activation Of Coagulation Under Conditions Of C1 Inhibitor Deficiency

C1 inhibitor (C1INH) functions as a key endogenous inhibitor of contact pathway coagulation factors XII, XI and plasma kallikrein (PKa). Patients with a congenital deficiency in C1INH suffer from a rare swelling disorder called hereditary angioedema (HAE) caused by excess bradykinin generation. Patients with HAE also have elevated circulating levels of markers of activation of coagulation and an increased risk of venous thromboembolism, phenotypes recapitulated in C1INH-deficient mice. We sought to evaluate the molecular mechanism by which C1INH deficiency leads to increased basal activation of coagulation and venous thrombosis. We hypothesized that PKa serves as a key driver of activation of coagulation in humans and mice with congenital C1INH deficiency. Patients with C1INH deficiency-associated HAE were treated with the PKa inhibitor lanadelumab with plasma samples collected before and after treatment. Plasma levels of coagulation markers and contact pathway-initiated thrombin generation were determined. C1INH-deficient mice were treated with the PKa inhibitor avoralstat, the FXIIa inhibitor garadacimab, FXII small interfering RNAs, FXI antisense oligonucleotides, the anti-tissue factor antibody 1H1 or appropriate controls and plasma coagulation markers were measured. Treatment of HAE patients with the PKa inhibitor lanadelumab significantly reduced plasma levels of prothrombin fragment 1.2, thrombin antithrombin (TAT) complexes and D-dimer at 6 months after treatment initiation (P<0.05). Interestingly, landalumab had no significant effect on contact pathway-initiated thrombin generation. In experiments with C1INH deficient mice, TAT complexes were significantly reduced by PKa inhibition (P<0.01) but not by inhibition of FXIIa or FXIa. Interestingly, tissue factor inhibition also significantly reduced plasma TAT complexes (P<0.01). This data suggests that under conditions of C1INH deficiency PKa potentiates activation of coagulation through a non-canonical pathway involving tissue factor. These findings demonstrate that C1INH is a physiologically relevant anticoagulant with C1INH deficiency resulting in enhanced kallikrein-mediated activation of basal coagulation.

Hereditary Angioedema: Novel Molecules for Treatment of Acute Attacks and Long-Term Prophylaxis

Hereditary angioedema (HAE) is a rare disease caused by a genetic alteration of the SERPING1 gene and characterized by recurrent attacks of angioedema that involve the skin, and the mucosae of the gastrointestinal tract and upper airways, which significantly affect the quality of life of patients. Nowadays there are effective drugs for both 1. treating acute attacks and 2. preventing attacks with a long-term prophylaxis. However, there are some unmet needs for HAE treatment, and therefore several novel molecules are under active testing for this clinical condition. Novel drugs will simplify the mode of administration (oral versus parenteral for both on demand treatment or long-term prophylaxis), prolong the interval between administrations (up to 3–6 months of efficacy with a single administration), target more specifically the central enzymes involved in the generation of bradykinin, the ultimate mediator of angioedema (prekallikrein, activated plasma kallikrein or activated factor XII), and potentially determine a definitive cure for the disease by genetic manipulation of the altered gene (SERPING1) or other downstream genes (KLKB1). In this review we provide a panoramic view of all new medications that are under active experimentation and will probably transform and enrich all of the therapeutic armamentarium for treating this disease.

Forms of Hereditary Angioedema That Are Independent of Kallikrein and Factor XIIa System

Hereditary angioedema (HAE) is an inherited disorder characterized by recurrent episodes of severe soft tissue swelling involving skin and mucous membranes. In most cases, edema is caused by excessive formation of the vasoactive nanopeptide bradykinin (BK) due to dysregulation of the plasma kallikrein-kinin system (KKS). The KKS consists of the zymogens prekallikrein (PK) and factor XII (FXII) and the cofactor/substrate high-molecular-weight kininogen (HK). PK and FXII reciprocally convert each other to the proteases plasma kallikrein (PKa) and FXIIa. PKa cleaves HK after Lys362 and Arg371 to release bradykinin. In most cases of HAE, increased KKS activation is due to low plasma activity of C1-Inhibitor (C1-INH), the main regulator of PKa and FXIIa. However, ~10% of HAE patients have normal C1-INH activity (HAEnC1). A relationship between fibrinolysis and kinin formation was first described in 1971. The fibrinolytic protease plasmin activates FXII and cleaves HK, although it is unclear that this results in significant bradykinin generation. Recently mutations in the PLG gene encoding plasminogen (Lys311Glu) and the KNG1 gene encoding HK and the related protein low-molecular-weight kininogen (LK, Met361Lys) were identified in patients with HAEnC1. Here we describe how the fibrinolytic system contributes to angioedema in patients with these mutations. Human Glu-plasminogen (Plg), HK and LK were expressed in HEK293 cells. Lys311 in plasminogen was changed to glutamic acid, and Met361 in HK and LK were changed to lysine, by site directed mutagenesis. Wild type (Plg-Lys311) and variant (Plg-Glu311) plasminogen were converted to plasmin (Plm-Lys311 and Plm-Glu311) with urokinase. The capacity of plasmin to cleave HK and LK were studied with SDS-PAGE, western blots and kinin ELISA. Cleavage of wild type HK-Met361 and LK-Met361 and variant HK-Lys361 and LK-Lys361 by PKa and plasmin were assessed in a similar manner. The types of kinin released during reactions were determined by mass spectroscopy. Normal plasma was supplemented with Plg-Lys311 or Plg-Glu311, and tPA was added to generate plasmin. Bradykinin release was substantially greater with Plm-Glu311 than Plm-Lys311. This held when reactions were run in plasma lacking FXII or PK, indicating activation of the KKS was not responsible for kinin production. Using plasma-derived proteins, PKa released bradykinin 100-fold faster from HK than LK, and released bradykinin from HK ~50-fold faster than did plasmin. PKa and plasmin released bradykinin at comparable relatively slow rates from LK. Plm-Glu311 released bradykinin from HK 10-fold faster, and from LK 3-fold faster, than in reactions with Plm-Lys311. In experiments in which kininogen deficient plasma was supplemented with either HK or LK to a physiologic concentration, the majority of bradykinin released by Plm-Glu311 was from LK. PKa-catalyzed release of bradykinin from wild type HK-Met361 and variant HK-Lys361 comparably. The same held for LK-Met361 and LK-Lys361. However, plasmin released kinin from HK-Lys361 and LK-Lys361 3.3-fold and 13-fold faster than from HK-Met361 and LK-Met361. While the kinin released from HK-Met361 and LK-Met361 was bradykinin, plasmin released the decapeptide Lys-bradykinin (kallidin) from HK-Lys361 and LK-Lys36. Our studies indicate that angioedema in some patients with HAEnC1 may be due to fibrinolytic activity, and not to KKS activity. Both HK and LK may be substrates in such patients. As the plasma LK concentration is ~4-fold higher than HK, it may be the main pathologic kinin source. The plasmin Lys311Glu substitution converts the protease into a more efficient kininogenase. Fibrinolytic activity is normally brisk in the mouth, perhaps explaining why patients with the Lys311Glu mutation primarily suffer from oral-lingual angioedema. The findings raise the possibility that oral-lingual edema after tPA infusion or ACE inhibitor administration may also be due to plasmin-mediated cleavage of LK and HK. The Met361Lys substitution in HK and LK introduces a novel protease cleavage site immediately upstream from a normal PKa cleavage site. The novel site is preferred over the normal site by plasmin, but not PKa. Several treatments for HAE directed at neutralizing PKa or FXIIa activity have been developed. Our results suggest that such drugs may be less effective in patients with HAEnC1 in which disease mechanisms operate independently of the KKS.

Hypersensitivity reactions amongst Hungarian Patients with Hereditary Angioedema due to C1-Inhibitor Deficiency

BackgroundIn hereditary angioedema (HAE) due to C1-inhibitor deficiency (C1-INH-HAE), bradykinin-mediated submucosal and/or subcutaneous angioedema dominates the clinical picture. The deficiency of C1-inhibitor can lead to the over-activation of the complement system. Complement plays an important role in all types of hypersensitivity reactions. On the other hand, during the degranulation of mast cells, heparin is also released amongst other substances. Heparin can activate the plasma kinin-kallikrein system, leading to bradykinin generation. These observations suggest a possible connection between C1-INH-HAE and mast cell-mediated hypersensitivity reactions. ObjectiveTo assess the occurrence of hypersensitivity reactions in the Hungarian C1-INH-HAE population. MethodsPatients filled out a questionnaire of 112 questions, either online or on paper. The questions were about hypersensitivity and C1-INH-HAE symptoms, the relation between these 2, general health, and demographic data. The study protocol was approved by the institutional review board of Semmelweis University, Budapest, and informed consent was obtained from the participants. ResultsOne hundred and six patients (64 female, 42 male, median age 46 years) responded, with 63.2% having hypersensitivity. Hypersensitivity was provoked by pollen in 25.5% of patients, by contact sensitivity in 22.6%, by food in 21.7%, by insect sting in 19.8%, by pet in 15.1%, by drug in 14.2%, by dust mite in 5.7%, and by mold in 1.9%. In 11 patients, hypersensitivity symptoms appeared after the diagnosis of C1-INH-HAE. Six hypersensitive patients experienced improvement in their symptoms; 42 remained the same, but none experienced worsening after the diagnosis of C1-INH-HAE. In 7.8% of the hypersensitive patients, a C1-INH-HAE attack worsened the hypersensitivity symptoms, while 15.7% of the hypersensitive patients experienced a C1-INH-HAE attack provoked by contact with the provoking factor. ConclusionWhile 63.2% of our C1-INH-HAE patients have reported hypersensitivity symptoms, Eurostat's latest data puts the prevalence of self-reported allergies in Hungary at 19.3%. Since in our experience most Hungarian patients report hypersensitivity reactions as allergies, this may support a possible connection between the 2 diseases, but further molecular studies are needed.

STAR-0215 is a Novel, Long-Acting Monoclonal Antibody Inhibitor of Plasma Kallikrein for the Potential Treatment of Hereditary Angioedema.

Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a deficiency in functional C1 esterase inhibitor (C1-INH), a serpin family protein that blocks the activity of plasma kallikrein. Insufficient inhibition of plasma kallikrein results in the overproduction of bradykinin, a vasoactive inflammatory mediator that produces both pain and unpredictable swelling during HAE attacks, with potentially life-threatening consequences. We describe the generation of STAR-0215, a humanized immunoglobulin G1 (IgG1) antibody with a long circulating half-life (t1/2) that potently inhibits plasma kallikrein activity, with a >1000-fold lower affinity for prekallikrein and no measurable inhibitory activity against other serine proteases. The high specificity and inhibitory effect of STAR-0215 is demonstrated through a unique allosteric mechanism involving N-terminal catalytic domain binding, destabilization of the activation domain, and reversion of the active site to the inactive zymogen state. The YTE (M252Y/S254T/T256E) modified fragment crystallizable (Fc) domain of STAR-0215 enhances pH-dependent neonatal Fc receptor (FcRn) binding, resulting in a prolonged t1/2 in vivo (⁓34 days in cynomolgus monkeys) compared to antibodies without this modification. A single subcutaneous dose of STAR-0215 ({greater than or equal to} 100 mg) was predicted to be active in patients for 3 months or longer, based on simulations using a minimal physiologically based pharmacokinetic (mPBPK) model. These data indicate that STAR-0215, a highly potent and specific antibody against plasma kallikrein with extended t1/2, is a potential agent for long-term preventative HAE therapy administered every 3 months or less frequently. Significance Statement STAR-0215 is a YTE-modified immunoglobulin G1 monoclonal antibody with a novel binding mechanism that specifically and potently inhibits the enzymatic activity of plasma kallikrein and prevents the generation of bradykinin. It has been designed to be a long-lasting prophylactic treatment to prevent attacks of HAE and to decrease the burden of disease and the burden of treatment for people with HAE.

Genetic Variants Leading to Urticaria and Angioedema and Associated Biomarkers.

Advances in next generation sequencing technologies, as well as their expanded accessibility and clinical use over the past 2 decades, have led to an exponential increase in the number of identified single gene disorders. Among these are primary atopic disorders-inborn errors of immunity resulting in severe allergic phenotypes as a primary presenting feature. Two cardinal aspects of type I immediate hypersensitivity allergic reactions are hives and angioedema. Mast cells (MCs) are frequent primary drivers of these symptoms, but other cells have also been implicated. Even where MC degranulation is believed to be the cause, mediator-induced symptoms may greatly vary among individuals. Angioedema-particularly in the absence of hives-may also be caused by hereditary angioedema conditions resulting from aberrant regulation of contact system activation and excessive bradykinin generation or impairment of vascular integrity. In these patients, swelling can affect unpredictable locations and fail to respond to MC-directed therapies. Genetic variants have helped delineate key pathways in the etiology of urticaria and nonatopic angioedema and led to the development of targeted therapies. Herein, we describe the currently known inherited and acquired genetic causes for these conditions, highlight specific features in their clinical presentations, and discuss the benefits and limitations of biomarkers that can help distinguish them.

Anti-HK antibody inhibits the plasma contact system by blocking prekallikrein and factor XI activation invivo.

A dysregulated plasma contact system is involved in various pathological conditions, such as hereditary angioedema, Alzheimer disease, and sepsis. We previously showed that the 3E8 anti-high molecular weight kininogen (anti-HK) antibody blocks HK cleavage and bradykinin generation in human plasma exvivo. Here, we show that 3E8 prevented not only HK cleavage but also factor XI (FXI) and prekallikrein (PK) activation by blocking their binding to HK in mouse plasma invivo. 3E8 also inhibited contact system-induced bradykinin generation invivo. Interestingly, FXII activation was also inhibited, likely because of the ability of 3E8 to block the positive feedback activation of FXII by kallikrein (PKa). In human plasma, 3E8 also blocked PK and FXI binding to HK and inhibited both thrombotic (FXI activation) and inflammatory pathways (PK activation and HK cleavage) of the plasma contact system activation exvivo. Moreover, 3E8 blocked PKa binding to HK and dose-dependently inhibited PKa cleavage of HK. Our results reveal a novel strategy to inhibit contact system activation invivo, which may provide an effective method to treat human diseases involving contact system dysregulation.

Treatment of hereditary angioedema-single or multiple pathways to the rescue.

Hereditary angioedema (HAE) is a rare disease caused by mutations in the SERPING1 gene. This results in deficient or dysfunctional C1 esterase inhibitor (C1-INH) and affects multiple proteases involved in the complement, contact-system, coagulation, and fibrinolytic pathways. Current options for the treatment and prevention of HAE attacks include treating all affected pathways via direct C1-INH replacement therapy; or specifically targeting components of the contact activation system, in particular by blocking the bradykinin B2 receptor (B2R) or inhibiting plasma kallikrein, to prevent bradykinin generation. Intravenously administered plasma-derived C1-INH (pdC1-INH) and recombinant human C1-INH have demonstrated efficacy and safety for treatment of HAE attacks, although time to onset of symptom relief varied among trials, specific agents, and dosing regimens. Data from retrospective and observational analyses support that short-term prophylaxis with intravenous C1-INH products can help prevent HAE attacks in patients undergoing medical or dental procedures. Long-term prophylaxis with intravenous or subcutaneous pdC1-INH significantly decreased the HAE attack rate vs. placebo, although breakthrough attacks were observed. Pathway-specific therapies for the management of HAE include the B2R antagonist icatibant and plasma kallikrein inhibitors ecallantide, lanadelumab, and berotralstat. Icatibant, administered for treatment of angioedema attacks, reduced B2R-mediated vascular permeability and, compared with placebo, reduced the time to initial symptom improvement. Plasma kallikrein inhibitors, such as ecallantide, block the binding site of kallikrein to prevent cleavage of high molecular weight kininogen and subsequent bradykinin generation. Ecallantide was shown to be efficacious for HAE attacks and is licensed for this indication in the United States, but the labeling recommends that only health care providers administer treatment because of the risk of anaphylaxis. In addition to C1-INH replacement therapy, the plasma kallikrein inhibitors lanadelumab and berotralstat are recommended as first-line options for long-term prophylaxis and have demonstrated marked reductions in HAE attack rates. Investigational therapies, including the activated factor XII inhibitor garadacimab and an antisense oligonucleotide targeting plasma prekallikrein messenger RNA (donidalorsen), have shown promise as long-term prophylaxis. Given the requirement of lifelong management for HAE, further research is needed to determine how best to individualize optimal treatments for each patient.

Pharmacological suppression of the kallikrein kinin system with KVD900: An orally available plasma kallikrein inhibitor for the on-demand treatment of hereditary angioedema.

BackgroundHereditary angioedema (HAE) is a rare genetic disease that leads to recurrent episodes of swelling and pain caused by uncontrolled plasma kallikrein (PKa) activity. Current guidelines recommend ready availability of on‐demand HAE treatments that can be administered early upon attack onset. This report describes the pharmacological and pharmacodynamic properties of the novel oral small‐molecule PKa inhibitor KVD900 as a potential on‐demand treatment for HAE.MethodsPharmacological properties of KVD900 on PKa and closely related serine proteases were characterized using kinetic fluorogenic substrate activity assays. Effects of KVD900 on PKa activity and kallikrein kinin system activation in whole plasma were measured in the presence of dextran sulphate (DXS)‐stimulation using a fluorogenic substrate and capillary immunoassays to quantify high molecular weight kininogen (HK), plasma prekallikrein and Factor XII cleavage. Pharmacodynamic effects of orally administered KVD900 were characterized in plasma samples from six healthy controls in a first in human phase 1 clinical trial and from 12 participants with HAE in a phase 2 clinical trial.ResultsKVD900 is a selective, competitive and reversible inhibitor of human PKa enzyme with a Ki of 3.02 nM. The association constant (Kon) of KVD900 for PKa is >10 × 106 M−1 s−1. Oral administration of KVD900 in a first‐in‐human clinical trial achieved rapid and near complete inhibition of DXS‐stimulated PKa enzyme activity and HK cleavage and reduced plasma prekallikrein and Factor XII activation in plasma. In individuals with HAE, orally administered KVD900 inhibited DXS‐stimulated PKa activity in plasma by ≥95% from 45 min to at least 4 h post‐dose and provided rapid protection of HK from cleavage.ConclusionKVD900 is a fast‐acting oral PKa inhibitor that rapidly inhibits PKa activity, kallikrein kinin system activation and HK cleavage in plasma. On‐demand administration of KVD900 may provide an opportunity to halt the generation of bradykinin and reverse HAE attacks.

A mechanism for hereditary angioedema caused by a lysine 311–to–glutamic acid substitution in plasminogen

AbstractPatients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains

AbstractFactor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro–hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid–sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

The versatile role of the contact system in cardiovascular disease, inflammation, sepsis and cancer.

The human contact system consists of plasma proteins, which - after contact to foreign surfaces - are bound to them, thereby activating the zymogens of the system into enzymes. This activation mechanism gave the system its name - contact system. It is considered as a procoagulant and proinflammatory response mechanism, as activation finally leads to the generation of fibrin and bradykinin. To date, no physiological processes have been described that are mediated by contact activation. However, contact system factors play a pathophysiological role in numerous diseases, such as cardiovascular diseases, arthritis, colitis, sepsis, and cancer. Contact system factors are therefore an interesting target for new therapeutic options in different clinical conditions.

Factor VII activating protease (FSAP) is not essential in the pathophysiology of angioedema in patients with C1 inhibitor deficiency

BackgroundExcessive bradykinin (BK) generation from high molecular weight kininogen (HK) by plasma kallikrein (PK) due to lack of protease inhibition is central to the pathophysiology of hereditary angioedema (HAE). Inadequate protease inhibition may contribute to HAE through a number of plasma proteases including factor VII activating protease (FSAP) that can also cleave HK. ObjectiveTo investigate the interaction between FSAP and C1 inhibitor (C1Inh) and evaluate the potential role of FSAP in HAE with C1Inh deficiency. Materials and methodsPlasma samples from 20 persons with HAE types 1 or 2 in remission were studied and compared to healthy controls. We measured and compared antigenic FSAP levels, spontaneous FSAP activity, FSAP generation potential, activation of plasma pre-kallikrein (PPK) by FSAP, and the formation of FSAP-C1Inh and FSAP-alpha2-antiplasmin (FSAP-α2AP) complexes. Furthermore, we measured HK cleavage and PK activation after activation of endogenous pro-FSAP and after addition of exogenous FSAP. ResultsIn plasma from HAE patients, there is increased basal FSAP activity compared to healthy volunteers. HAE plasma exhibits decreased formation of FSAP-C1Inh complexes and increased formation of FSAP-α2AP complexes in histone-activated plasma. Although exogenous FSAP can cleave HK in plasma, this was not seen when endogenous plasma pro-FSAP was activated with histones in either group. PK was also not activated by FSAP in plasma. ConclusionIn this study, we established that FSAP activity is increased and the pattern of FSAP-inhibitor complexes is altered in HAE patients. However, we did not find evidence suggesting that FSAP contributes directly to HAE attacks.

Cleavage of High Molecular Weight Kininogen and Bradykinin Release By Red Blood Cell Microvesicles As a Putative Mechanism for Hypotensive Transfusion Reactions

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. DisclosuresKarafin: 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.

Elevated salivary activity of mast cell chymase of periodontitis patients, and a new bradykinin generation cascade, mediating the cross-talks between mast cell and gingival fibroblast

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.

Berotralstat (BCX7353): Structure-Guided Design of a Potent, Selective, and Oral Plasma Kallikrein Inhibitor to Prevent Attacks of Hereditary Angioedema (HAE).

Hereditary angioedema (HAE) is a rare and potentially life-threatening disease that affects an estimated 1 in 50 000 individuals worldwide. Until recently, prophylactic HAE treatment options were limited to injectables, a burdensome administration route that has driven the need for an oral treatment. A substantial body of evidence has shown that potent and selective plasma kallikrein inhibitors that block the generation of bradykinin represent a promising approach for the treatment of HAE. Berotralstat (BCX7353, discovered by BioCryst Pharmaceuticals using a structure-guided drug design strategy) is a synthetic plasma kallikrein inhibitor that is potent and highly selective over other structurally related serine proteases. This once-daily, small-molecule drug is the first orally bioavailable prophylactic treatment for HAE attacks, having successfully completed a Phase III clinical trial (meeting its primary end point) and recently receiving the U.S. Food and Drug Administration's approval for the prophylactic treatment of HAE attacks in patients 12 years and older.

English

BACKGROUND Surgical removal of impacted third molar is widely carried out in general dental practice and is usually associated with postoperative inflammatory sequelae like pain, swelling, and trismus. The objective of the study was to assess and compare the efficacy of oral bromelain and Serratiopeptidase for the control of post-operative sequelae following surgical removal of impacted third molar. METHODS A randomized, comparative clinical study was conducted on sixty patients who were undergoing impacted third molar surgery, and were allocated into two groups each comprising 30 patients. Conventional drugs included Cap amoxicillin 500 mg bid, Tab. metronidazole 200 mg tid, and Tab. diclofenac sodium 50 mg bid that were given to both the groups. Group I was treated with Tab. bromelain 200 mg tid along with conventional treatment for 5 days. Group II received a combination of conventional treatment and Tab. Serratiopeptidase 10 mg bid for 5 days. The parameters such as measurement of pain, facial width and trismus were analysed on postoperative days 1, 2 and 7. RESULTS The study comprised of totally 60 otherwise healthy patients. The group of patients who received bromelain along with diclofenac sodium seemed to be effective in controlling post-operative sequelae like pain (P < 0.05), swelling (P < 0.05) but not trismus (P < 0.05) when compared to the group who received Serratiopeptidase with diclofenac sodium. CONCLUSIONS The intensified anti-inflammatory action of bromelain, was possibly by inhibiting the generation of bradykinin and negative action on the prostaglandin pathway. The present study assessed the clinical effect of bromelain and serratiopeptidase on pain, facial swelling and trismus. KEY WORDS Bromelain, Oral Bromelain, Serratiopeptidase, Proteolytic Enzyme, Third Molar Surgery

Analysis of cold activation of the contact system in hereditary angioedema with normal C1 inhibitor

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.