Protease activity in single-chain prekallikrein.

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

Hereditary angioedema: Investigational therapies and future research.

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 ß2 receptor with sustained action properties, testing of products to determine whether the ß1 receptor 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%.

The complement and contact activation systems: partnership in pathogenesis beyond angioedema.

The blood plasma contains four biologically important proteolytic cascades, which probably evolved from the same ancestral gene. This in part may explain why each cascade has very similar "initiating trigger" followed by sequential and cascade-like downstream enzymatic activation pattern. The four cascades are: the complement system, the blood clotting cascade, the fibrinolytic system, and the kallikrein-kinin system. Although much has been written about the interplay between all these enzymatic cascades, the cross-talk between the complement and the kinin generating systems has become particularly relevant as this interaction results in the generation of nascent molecules that have significant impact in various inflammatory diseases including angioedema and cancer. In this review, we will focus on the consequences of the interplay between the two systems by highlighting the role of a novel molecular link called gC1qR. Although this protein was first identified as a receptor for C1q, it is now recognized as a multiligand binding cellular protein, which serves not only as C1q receptor, but also as high affinity (KD  ≤ 0.8 nM) binding site for both high molecular weight kininogen (HK) and factor XII (FXII). At inflammatory sites, where atherogenic factors such as immune complexes and/or pathogens can activate the endothelial cell into a procoagulant and proinflammatory surface, the two pathways are activated to generate vasoactive peptides that contribute in various ways to the inflammatory processes associated with numerous diseases. More importantly, since recent observations strongly suggest an important role for both pathways in cancer, we will focus on how a growing tumor cluster can employ the byproducts derived from the two activation systems to ensure not only its survival and growth, but also its escape into distal sites of colonization.

Diagnosis, pathogenesis, and treatment of chronic spontaneous urticaria.

BACKGROUND: Chronic Spontaneous Urticaria (CSU) is an endogenous disorder that is strongly associated with autoimmunity, particularly with immunoglobulin G (IgG) antibody to the alpha subunit of the IgE receptor seen in 35-40% of patients. Basophils and cutaneous mast cells can be activated and lead to a late-phase-like perivascular infiltration about small venules and hive formation. METHODS: Review of current literature. RESULTS: Antibody to thyroid antigens are seen in 25% of patients; a small fraction of these may be clinically hypothyroid (Hashimoto's Thyroiditis). Forty percent of patients have angioedema, but not laryngeal edema. Therapy typically begins with second-generation antihistamines (H1 receptor blockers) up to four times a day. The failure rate is substantial, and estimates vary from 25% to 50%. The drug of choice for antihistamine resistant cases is omalizumab, at 300 mg/month, which is effective in 70% of patients. H-2-antagonists and leucotriene antagonists are no longer recommended because the literature does not support additional efficacy beyond blockage of H-1 receptors. For patients unresponsive to antihistamines and omalizumab, cyclosporine is recommended next. This is similarly effective in 65-70% of patients; however, assessment of blood pressure and renal function need to be followed every 4-6 weeks. Corticosteroid should not be employed chronically; however, a brief course of 3-10 days can be used acutely for severe exacerbations. Other agents, such as dapsone, sulfasalazine, or hydroxychloroquin, can be tried when the aforementioned medications fail, but the results are unpredictable because they have not been shown to have efficacy beyond the placebo effect (25-30%), and have not been studied in patients for whom the aforementioned approach i.e. antihistamines, omalizumab, and cyclosporine has failed. CONCLUSION: High dose antihistamines, omalizumab and cyclosporine (in that order) are effective and recommended for therapy of CUS, an inflammatory skin disorder associated with autoimmunity in 45% of patients.

Cytokine and estrogen stimulation of endothelial cells augments activation of the prekallikrein-high molecular weight kininogen complex: Implications for hereditary angioedema.

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.

Deficiency of plasminogen activator inhibitor 2 in plasma of patients with hereditary angioedema with normal C1 inhibitor levels.

BACKGROUND: Hereditary angioedema with normal C1 inhibitor levels (HAE-N) is associated with a Factor XII mutation in 30% of subjects; however, the role of this mutation in the pathogenesis of angioedema is unclear. OBJECTIVE: We sought evidence of abnormalities in the pathways of bradykinin formation and bradykinin degradation in the plasma of patients with HAE-N both with and without the mutation. METHODS: Bradykinin was added to plasma, and its rate of degradation was measured by using ELISA. Plasma autoactivation was assessed by using a chromogenic assay of kallikrein formation. Plasminogen activator inhibitors (PAIs) 1 and 2 were also measured by means of ELISA. RESULTS: PAI-1 levels varied from 0.1 to 4.5 ng/mL (mean, 2.4 ng/mL) in 23 control subjects, from 0.0 to 2 ng/mL (mean, 0.54 ng/mL) in patients with HAE-N with a Factor XII mutation (12 samples), and from 0.0 to 3.7 ng/mL (mean, 1.03 ng/mL) in patients with HAE-N without a Factor XII mutation (11 samples). PAI-2 levels varied from 25 to 87 ng/mL (mean, 53.8 ng/mL) in control subjects and were 0 to 25 ng/mL (mean, 4.3 ng/mL) in patients with HAE-N with or without the Factor XII mutation. Autoactivation at a 1:2 dilution was abnormally high in 8 of 17 patients with HAE-N (4 in each subcategory) and could be corrected by supplemental C1 inhibitor in 4 of them. Bradykinin degradation was markedly abnormal in 1 of 23 patients with HAE-N and normal in the remaining 22 patients. CONCLUSIONS: Bradykinin degradation was normal in all but 1 of 23 patients with HAE-N studied. By contrast, there was a marked abnormality in PAI-2 levels in patients with HAE-N that is not seen in patients with C1 inhibitor deficiency. PAI-1 levels varied considerably, but a statistically significant difference was not seen. A link between excessive fibrinolysis and bradykinin generation that is estrogen dependent is suggested.

Therapy of chronic urticaria: a simple, modern approach.

OBJECTIVE: To examine the available treatment choices for chronic spontaneous urticaria (CSU) and discuss a new paradigm for treating such patients. DATA SOURCES: The literature regarding treatment is reviewed, including considerations of published guidelines. Attention is focused on the most recent evidence indicating particular efficacy of omalizumab. RESULTS: Omalizumab has been found to have considerable efficacy in phase 2 and phase 3 trials in which more than 900 patients have been studied. A response rate of 65% is seen in patients resistant to antihistamines as well as to histamine2 blockers and leukotriene antagonists, and 40% of patients are completely free of hives as long as therapy is continued. In addition, serious adverse events have not been seen. Only cyclosporine can match this response rate (excluding steroids), but the adverse effect profile (blood pressure and renal function) is substantial by comparison. Double-blind, placebo-controlled studies of other agents often listed as alternatives are lacking (ie, whether their success rate exceeds the 25%-30% placebo response is uncertain). The mechanism by which omalizumab works in CSU is not clear because the response rate is unrelated to the autoimmune profile and can occur rapidly (ie, within a few days). CONCLUSION: Omalizumab has exceptional efficacy for antihistamine-resistant CSU with an excellent adverse effect profile.

The thrombogenicity of C1 esterase inhibitor (human): review of the evidence.

Thromboembolic events associated with human plasma-derived C1 esterase inhibitor (C1-INH) use in patients with hereditary angioedema (HAE) have been reported in the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System database. The purpose of this article is to review and assess the strength of available evidence regarding the thrombogenicity of human plasma-derived C1-INH. A PubMed search was conducted of English language articles from January 1990 to December 2013 reporting the thrombogenicity of C1-INH. Original research articles were selected if the following criteria were met: (1) C1-INH was the focus of the study and (2) the authors addressed the pro- or antithrombotic potential of C1-INH. Additional articles on the clinical use of C1-INH in disease states other than HAE were obtained using reference lists of selected articles. Pivotal studies and prescribing information for C1-INH products were also reviewed. Limited animal and clinical data suggest that C1-INH, particularly at high doses of up to 500 U/kg (compared with the U.S. FDA-approved 20-U/kg dose), may be prothrombotic. In contrast, C1-INH has been used in some patients with myocardial infarction, ischemic stroke, sepsis, and capillary leak syndrome at off-label supratherapeutic doses (up to 100 U/kg) without evidence of a thrombogenic effect. Based on our review, thromboembolic events reported with C1-INH use are rare and patients with HAE who experienced such events often have underlying thromboembolic risk factors.

Factor XII-independent activation of the bradykinin-forming cascade: Implications for the pathogenesis of hereditary angioedema types I and II.

BACKGROUND: We have previously reported that prekallikrein expresses an active site when it is bound to high-molecular-weight kininogen (HK) and can digest HK to produce bradykinin. The reaction is stoichiometric and inhibited by C1 inhibitor (C1-INH) or corn trypsin inhibitor. Addition of heat shock protein 90 leads to conversion of prekallikrein to kallikrein in a zinc-dependent reaction. OBJECTIVE: Our goal was to determine whether these reactions are demonstrable in plasma and distinguish them from activation through factor XII. METHODS: Plasma was incubated in polystyrene plates and assayed for kallikrein formation. C1-INH was removed from factor XII-deficient plasma by means of immunoadsorption. RESULTS: We demonstrate that prekallikrein-HK will activate to kallikrein in phosphate-containing buffers and that the rate is further accelerated on addition of heat shock protein 90. Prolonged incubation of plasma deficient in both factor XII and C1-INH led to conversion of prekallikrein to kallikrein and cleavage of HK, as was seen in plasma from patients with hereditary angioedema but not plasma from healthy subjects. CONCLUSIONS: These results indicate that C1-INH stabilizes the prekallikrein-HK complex to prevent HK cleavage either by prekallikrein or by prekallikrein-HK autoactivation to generate kallikrein. In patients with hereditary angioedema, kallikrein and bradykinin formation can occur without invoking factor XII activation, although the kallikrein formed can rapidly activate factor XII if it is surface bound.

The bradykinin-forming cascade in anaphylaxis and ACE-inhibitor induced angioedema/airway obstruction.

Anaphylaxis is a potentially life-threatening multi-system allergic reaction to a biological trigger resulting in the release of potent inflammatory mediators from mast cells and basophils and causing symptoms in at least two organ systems that generally include skin, lungs, heart, or gastrointestinal tract in any combination. One exception is profound hypotension as an isolated symptom. There are two types of triggers of anaphylaxis: immunologic and non-Immunologic. Immunologic anaphylaxis is initiated when a foreign antigen directly binds to IgE expressed on mast cells or basophils and induces the release of histamine and other inflammatory substances resulting in vasodilation, vascular leakage, decreased peripheral vascular resistance, and heart muscle depression. If left untreated, death by shock (profound hypotension) or asphyxiation (airway obstruction) can occur. The non-immunologic pathway, on the other hand, can be initiated in many ways. A foreign substance can directly bind to receptors of mast cells and basophils leading to degranulation. There can be immune complex activation of the classical complement cascade with the release of anaphylatoxins C3a and C5a with subsequent recruitment of mast cells and basophils. Finally, hyperosmolar contrast agents can cause blood cell lysis, enzyme release, and complement activation, resulting in anaphylactoid (anaphylactic-like) symptoms. In this report we emphasize the recruitment of the bradykinin-forming cascade in mast cell dependent anaphylactic reactions as a potential mediator of severe hypotension, or airway compromise (asthma, laryngeal edema). We also consider airway obstruction due to inhibition of angiotensin converting enzyme with a diminished rate of endogenous bradykinin metabolism, leading not only to laryngeal edema, but massive tongue swelling with aspiration of secretions.

A Comprehensive Management Approach in Pediatric and Adolescent Patients With Hereditary Angioedema.

Hereditary angioedema (HAE) is a rare autosomal-dominant disorder; most cases are characterized by low plasma levels of C1 esterase inhibitor (C1-INH). Clinical manifestations of HAE due to C1-INH deficiency include unpredictable, acute, recurrent episodes of nonpruritic swelling that can affect the face, trunk, limbs, and the respiratory, gastrointestinal, and genitourinary tracts. Attacks can be disfiguring, disabling, painful, and even life-threatening if laryngeal swelling occurs. Symptoms of HAE generally manifest in childhood. Effective medications are available and approved to treat HAE in children. However, evidence informing use of these medications in pediatric clinical practice is limited. Hereditary angioedema management plans are critical to optimize outcomes and should address on-demand treatment for acute attacks and plans to prevent potentially fatal laryngeal attacks. The plan should also comprise a holistic approach to address nonclinical aspects of HAE, including quality of life (QoL) and psychological issues. This article provides an overview of HAE management principles that health care providers can apply to treat pediatric patients to improve their QoL.