Kallikrein-kinin Cascade Research Articles

Factor XII and prekallikrein promote microvascular inflammation and psoriasis in mice.

Psoriasis is an autoimmune inflammatory skin disease, featuring microvascular abnormalities and elevated levels of bradykinin. Contact activation of Factor XII can initiate the plasma kallikrein-kinin cascade, producing inflammation and angioedema. The role of Factor XII in psoriasis is unknown. The effects of deficiency of Factor XII or its enzymatic substrate, prekallikrein, were examined in the imiquimod-induced mouse model of psoriasis. Skin microcirculation was assessed using intravital confocal microscopy and laser Doppler flowmeter. A novel antibody blocking Factor XII activation was evaluated for psoriasis prevention. Expression of Factor XII was markedly up-regulated in human and mouse psoriatic skin. Genetic deletion of Factor XII or prekallikrein, attenuated imiquimod-induced psoriatic lesions in mice. Psoriatic induction increased skin microvascular blood perfusion, causing vasodilation, hyperpermeability and angiogenesis. It also promoted neutrophil-vascular interaction, inflammatory cytokine release and enhanced Factor XII / prekallikrein enzymatic activity with elevated bradykinin. Factor XII or prekallikrein deficiency ameliorated these microvascular abnormalities and abolished bradykinin increase. Antagonism of bradykinin B2 receptors reproduced the microvascular protection of Factor XII / prekallikrein deficiency, attenuated psoriatic lesions, and prevented protection by Factor XII / prekallikrein deficiency against psoriasis. Furthermore, treatment of mice with Factor XII antibody alleviated experimentally induced psoriasis and suppressed microvascular inflammation. Activation of Factor XII promoted psoriasis via prekallikrein-dependent formation of bradykinin, which critically mediated psoriatic microvascular inflammation. Inhibition of contact activation represents a novel therapeutic strategy for psoriasis.

Cerebral edema in acute stroke: Effect of thrombolytic treatment

Cerebral edema (CED) is a common complication of ischemic stroke in Intensive Care Unit. Although frequently observed in patients undergoing intravenous thrombolytic (IVT) treatment, the pathogenic role of recombinant tissue plasminogen activator (rtPA) in CED induction has not yet been definitively clarified. The aim of our study is to verify the relationship between CED and rtPA in patients affected by acute ischemic stroke, without reperfusion signs, evaluating the CED growth rate in the first week after stroke onset. In this single-center, retrospective observational study, we included all consecutive patients with acute stroke undergone multi-parameter monitoring of vital signs in the sub-intensive care Unit. We included both patients undergoing systemic IVT and standard medical treatment (n-IVT) with the following time of CT scan: within 4.5 h onset, 24 ± 12 h, 72 ± 24 h and 120 ± 24 h from the stroke onset. Of 1753 with acute ischemic stroke patients screened, 810 patients were included in the study (218 IVT and 592 n-IVT). No significant difference was observed at baseline in age, gender, NIHSS score or infarcted area, while hemorrhagic transformation rate was significantly higher in IVT than in n-IVT group. We observed a significant increase of CED growth rate in IVT patients compared to n-IVT patients only between 24 and 72 h from the ischemic event (respectively 1.85cm3/h and 0.89cm3/h; p = 0.031) regardless of the presence of HT. No significant difference was observed in growth rate between 3 and 5 days following rtPA administration or in overall growth rate. Although the pathogenetic mechanism of rtPA determining CED remains uncertain, our data suggests rtPA can act as a “trigger” of edema onset and progression. Therefore, drugs interfering with specific molecular pathways, such as kallikrein-kinin cascade, could constitute an effective strategy to reduce the risk of development and progression of rtPA-related cerebral edema in patients with acute stroke. Further studies are needed to define the molecular pathways involved in the genesis of CE in humans and to verify the efficacy of specific drugs.

Plasma kallikrein modulates immune cell trafficking during neuroinflammation via PAR2 and bradykinin release

Blood-brain barrier (BBB) disruption and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of neuroinflammatory disorders like multiple sclerosis (MS). Recent evidence suggests that the kallikrein-kinin and coagulation system might participate in this process. Here, we identify plasma kallikrein (KK) as a specific direct modulator of BBB integrity. Levels of plasma prekallikrein (PK), the precursor of KK, were markedly enhanced in active CNS lesions of MS patients. Deficiency or pharmacologic blockade of PK renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by a remarkable reduction of BBB disruption and CNS inflammation. In vitro analysis revealed that KK modulates endothelial cell function in a protease-activated receptor-2-dependent manner, leading to an up-regulation of the cellular adhesion molecules Intercellular Adhesion Molecule 1 and Vascular Cell Adhesion Molecule 1, thereby amplifying leukocyte trafficking. Our study demonstrates that PK is an important direct regulator of BBB integrity as a result of its protease function. Therefore, KK inhibition can decrease BBB damage and cell invasion during neuroinflammation and may offer a strategy for the treatment of MS.

C5a and Bradykinin Receptor Cross-Talk Regulates Innate and Adaptive Immunity in Trypanosoma cruzi Infection

Complement and the kallikrein-kinin cascade system are both activated in injured tissues. Little is known about their partnership in the immunopathogenesis of Chagas disease, the chronic infection caused by the intracellular protozoan Trypanosoma cruzi. In this study, we show that pharmacological targeting of the C5a receptor (C5aR) or the bradykinin B2 receptor (B2R) inhibited plasma leakage in hamster cheek pouch topically exposed to tissue culture trypomastigotes (TCTs). Further, angiotensin-converting enzyme inhibitors potentiated TCT-evoked paw edema in BALB/c, C57BL/6, and C5-deficient A/J mice through activation of joint pathways between C5aR/B2R or C3aR/B2R. In addition to generation of C5a and kinins via parasite-derived cruzipain, we demonstrate that macrophages internalize TCTs more efficiently through joint activation of C5aR/B2R. Furthermore, we found that C5aR targeting markedly reduces NO production and intracellular parasitism in macrophages. We then studied the impact of C5aR/B2R cross-talk in TCT infection on the development of adaptive immunity. We found that IL-12p40/70 expression was blunted in splenic dendritic cells by blocking either C5aR or B2R, suggesting that codominant signaling via C5aR and B2R fuels production of the Th1-polarizing cytokine. Finally, we assessed the impact of kinins and C5a liberated in parasite-laden tissues on Th cell differentiation. As predicted, BALB/c mice pretreated with angiotensin-converting enzyme inhibitors potentiated IFN-γ production by Ag-specific T cells via C5aR/B2R cross-talk. Interestingly, we found that B2R targeting upregulated IL-10 secretion, whereas C5aR blockade vigorously stimulated IL-4 production. In summary, we describe a novel pathway by which C5aR/B2R cross-talk couples transendothelial leakage of plasma proteins to the cytokine circuitry that coordinates antiparasite immunity.

Current treatment of hereditary angioedema: An update on clinical studies

Hereditary angioedema (HAE) is a rare, potentially life-threatening disease that manifests as recurrent episodes of nonpruritic swelling that may affect the extremities, face, genitalia, gastrointestinal tract, and/or larynx. HAE is the result of a deficiency of functional C1-esterase inhibitor (C1-INH), a key regulator of the complement, coagulation, and kallikrein-kinin cascades. In HAE patients, overactivation of the kallikrein-kinin cascade results in excessive release of bradykinin, the mediator of the pain and swelling that is characteristic of HAE. Historically, treatment options for HAE have been limited, but newly approved and emerging therapies, such as C1-INH replacement products, a plasma kallikrein inhibitor, and a bradykinin B₂-receptor antagonist, appear to provide safe and effective relief for a significant proportion of patients with HAE. Because they may have therapeutic and practical advantages over existing HAE therapies, the new agents have the potential to improve the overall management of patients with HAE. This article reviews the results from recent clinical trials of these drugs and considers their role in clinical practice.

Critical role of kallikrein in hereditary angioedema pathogenesis: A clinical trial of ecallantide, a novel kallikrein inhibitor

Hereditary angioedema (HAE) is a rare, autosomal-dominant disorder caused by C1 inhibitor gene mutation. Patients with HAE experience intermittent attacks of edema affecting the oropharynx, abdomen, gastrointestinal tract, and limbs. C1 inhibitor is the primary endogenous inhibitor of the kallikrein-kinin (contact) cascade. Unregulated kallikrein activation generates bradykinin, the likely mediator of the swelling and pain characterizing HAE attacks. Ecallantide, a novel, recombinant protein, potently inhibits kallikrein. This is the first placebo-controlled assessment in human beings of a therapeutic intervention to improve symptoms of HAE attacks under the hypothesis that the contact cascade is the putative pathway responsible for HAE pathology. To determine the safety and efficacy of ecallantide in patients with HAE. This double-blind, placebo-controlled, ascending-dose study assessed efficacy and tolerability of ecallantide (5, 10, 20, or 40 mg/m(2) intravenously) in individuals experiencing acute HAE attacks (N = 49). Twelve patients were assigned to each dose level: 10 to ecallantide and 2 to placebo, per cohort. Ecallantide treatment ameliorated the symptoms of HAE attacks: 72.5% (29/40) of patients treated with ecallantide versus 25.0% (2/8) of placebo patients reported significant improvement in symptoms within 4 hours (P = .0169). Ecallantide was well tolerated at all doses. Ecallantide, a potent, specific inhibitor of plasma kallikrein, significantly improved HAE symptoms over placebo. The trial provides strong support for the role of the kallikrein-kinin cascade and its end product, bradykinin, in the pathophysiology of HAE. Further clinical trials are underway. Ecallantide is a promising new therapy for HAE attacks.

Interaction of Hemoglobin Vesicles, a Cellular-Type Artificial Oxygen Carrier, with Human Plasma: Effects on Coagulation, Kallikrein-Kinin, and Complement Systems

Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro. Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

Pathogenic Factors of Vibrios with Special Emphasis on Vibrio vulnificus

AbstractFor Abstract see ChemInform Abstract in Full Text.

Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma

Background: The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells...

Pathogenic Factors of Vibrios with Special Emphasis on <i>Vibrio vulnificus</i>

Bacteria of the genus Vibrio are normal habitants of the aquatic environment and play roles for biocontrole of aquatic ecosystem, but some species are believed to be human pathogens. These species can be classified into two groups according to the types of diseases they cause: the gastrointestinal infections and the extraintestinal infections. The pathogenic species produce various pathogenic factors including enterotoxin, hemolysin, cytotoxin, protease, siderophore, adhesive factor, and hemagglutinin. We studied various pathogenic factors of vibrios with special emphasis on protease and hemolysin of V. vulnificus. V. vulnificus is now recognized as being among the most rapidly fatal of human pathogens, although the infection is appeared in patients having underlying disease(s) such as liver dysfunction, alcoholic cirrhosis or haemochromatosis. V. vulnificus protease (VVP) is thought to be a major toxic factor causing skin damage in the patients having septicemia. VVP is a metalloprotease and degrades a number of biologically important proteins including elastin, fibrinogen, and plasma proteinase inhibitors of complement components. VVP causes skin damages through activation of the Factor XII-plasma kallikrein-kinin cascade and/or exocytotic histamine release from mast cells, and a haemorrhagic lesion through digestion of the vascular basement membrane. Thus, the protease is the most probable candidate for tissue damage and bacterial invasion during an infection. Pathogenic roles and functional mechanism of other factors including hemolysins of V. vulnificus and V. mimicus are also shown in this review article.

Generation of active fragments from human zymogens in the brady kinin-generating cascade by extracellular proteases from Vibrio vulnificus and V. parahaemolyticus

Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous skin lesions on limbs. This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant. The metalloprotease was confirmed to activate human factor XII–plasma kallikrein–kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen. Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII). In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V. parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens. These results in part may explain why only V. vulnificus often causes serious edematous skin damages in humans.

Visualization of the sequential changes in immunolabelled tissue kininogenase which accompany follicular development and luteinization of angiogenic granulosa cells of the ovary

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns, rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin-treated and nontreated control rats. Preliminary experiments suggest that when granulosa and endothelial cells are co-cultured, granulosa cells participate in the formation of vascular capillary tubes. This inherent capacity of granulosa cells to behave and respond like endothelial cells may be of importance in the aetiology of ovarian angiogenesis, which drives new blood vessel formation in the ovary. Recently, we demonstrated that tissue kininogenase showed intense immunolabelling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells possess the same capacity to express the kallikrein–kinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro-forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stage and segments of the ovarian follicle by immunoperoxidase, fluorescent microscopy (confocal) and in situ hybridization.

Contact-system activation in children with vasculitis

The contact system triggers the kallikrein-kinin cascade, liberating bradykinin from high-molecular-weight kininogen. Effectors of the contact system have proinflammatory and vasoactive properties. Vasculitis is a condition characterised by inflammation around vessel walls, leading to secondary tissue damage for which the underlying molecular mechanisms are poorly understood. Our aim was to investigate contact-system activation in children with vasculitis. We compared 17 children, aged 4-19 years, with vasculitis, engaging the skin, joints, intestines, or kidneys, with 21 controls, aged 2-18 years. We analysed proteolysis of high-molecular-weight kininogen by immunoblotting. Plasma bradykinin concentrations were quantified by ELISA. Kidney and skin biopsies were stained in situ for kinins. Concentrations of heparin binding protein (HBP) were quantified by ELISA. We noted extensive proteolysis of high-molecular-weight kininogen in the plasma of 13 of 17 patients, but in only one of 21 controls (p<0.0001). Bradykinin concentrations were higher in the patients' plasma (median 320 ng/L, range <1-19680) than in plasma from controls (11 ng/L, <1-304; p=0.0004). Patients had local release of kinins at sites of inflammation in kidney and skin biopsies. HBP values were raised in patients (17.4 microg/L, 5.4-237.6) compared with controls (6 microg/L, 2.5-43.4; p=0.008). Activation of the contact system could play a part in the pathogenesis of vasculitis, and explain the inflammation, pain, vasodilatation, and oedema seen in patients.

Expression of the tissue kallikrein-kinin cascade in granulosa cells of the ovary.

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin-treated and non-treated control rats. Recently, we demonstrated that tissue kininogenase showed intense immunolabeling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells associated with ovarian vascularization possess the same capacity to express the kallikrein-kinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stages and segments of the ovarian follicle by immunoperoxidase assay, confocal fluorescent microscopy and in situ hybridization.

Comparison of tissue kallikrein and kinin receptor expression in gastric ulcers and neoplasms

Recent studies have suggested the possible involvement of the kallikrein–kinin cascade in gastric inflammatory diseases and malignant transformation in peptic ulcers. Germ-line mutations of genes responsible for repairing DNA mismatch may cause the transformation of chronic peptic ulceration to malignancy. The aim of the study was to compare the expression of the serine protease, tissue kallikrein (TK) and kinin peptide receptors, B1 and B2, in human gastric carcinoma and ulcers. Furthermore, experiments were designed to ask the question whether holistic changes known to occur in carcinoma would be reflected in the expression of the neutrophil kallikrein–kinin cascade. Expression of tissue kallikrein and kinin receptors in both tumour and ulcer tissue and circulating neutrophils of cancer and ulcer patients was determined by immunolabelling techniques, using specific antibodies. The immunolabelled images were visualised by light, confocal and electron microscopy. This is a first study that provides strong evidence for enhanced expression of TK in peptic ulceration and gastric malignancy, suggestive of a crucial role for serine proteases in cancer.

Kallikrein and kinin receptor expression in inflammation and cancer.

The kallikrein family of serine proteases has been investigated in many inflammatory disorders as molecular mapping, gene characterisation and cloning of kinin receptor genes have unfolded experimentally. In the molecular events of the inflammatory response the kallikrein cascade plays a significant role, since it is considered to initiate and maintain systemic inflammatory responses and immune-modulated disorders. A primary event is the chemotactic attraction of neutrophils which deliver the kallikrein-kinin cascade to sites of cellular injury and carcinogenic transformation of cells. The present study establishes the casual involvement of the kallikrein cascade in infection, inflammatory joint disease, acute transplant rejection, renal glomerular diseases, angiogenesis and carcinoma. We provide strong evidence for new or enhanced expression of kinin B1 receptors in inflammation, and additionally the induction of kallikrein genes in angiogenesis and carcinoma. The results provide insights into possible roles of kallikrein inhibitors and kinin receptor antagonists.

Effects of Vibrio vulnificus metalloprotease on the capillaries: pathological actions and inactivation by alpha-macroglobulin

Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia, characterized by hemorrhagic and edematous damage to the skin of limbs. When injected into the dorsal skin, an extracellular metalloprotease from this vibrio (V. vulnificus protease: VVP) enhanced the vascular permeability through activation of the Hageman factor-plasma kallikrein-kinin cascade and/or stimulation of exocytotic histamine release. Additionally, VVP caused the hemorrhagic skin lesion through disorganization of the vascular basement membrane layer due to specific degradation of type IV collagen, which is known to form the backbone structure of the basement membrane. However, injected VVP was quickly inactivated by a plasma glycoprotein, alpha-macroglobulin, at a molar ratio of 1:1. This glycoprotein was leaked from the capillaries by the actions of VVP, which resulted in in situ inactivation by physical entrapment. When VVP (45,000 Da) was incubated at 37 degrees C, a 35,000 Da fragment was generated by the autocatalytic removal of a 10,000 Da C-terminal polypeptide. This N-terminal fragment showed significant proteolytic activity, however, because of a markedly decreased affinity to the protein substrates, its permeability-enhancing and hemorrhagic activity was reduced to less than 50%. These findings indicate that the C-terminal polypeptide is not essential for but promotes skin reactions caused by VVP.

Different susceptibility of human tissue prokallikrein to cleavage by neutrophil proteinases

The recent discovery of tissue prokallikrein (TproK) on the cell surface of human neutrophils prompted this study to test the sensitivity of the kallikrein precursor to proteolysis by neutrophil-derived elastase and cathepsin G. Purified recombinant human TproK was readily degraded by cathepsin G via distinct cleavage sites into two major fragments of 5 and 8 kDa which are devoid of enzymatic activity. Leukocyte elastase caused a rapid conversion of TproK into [des-Ile1]-TK, a differentially processed tissue kallikrein (TK) isoform with truncated N-terminus that appeared to be resistant to further cleavage by elastase. Kinetic data demonstrated the cleavage of amidolytic kallikrein substrates and the release of kinin from HMW kininogen, but the single amino acid deletion results in a severe reduction of catalytic activity for [des-Ile1]-TK compared to the mature enzyme. These in vitro observations support the possibility that the inter-relationships among TproK and neutrophil proteinases may provide a sensitive regulatory system to balance the kallikrein–kinin cascade during inflammatory events.

Kallikrein–kinin in infection and cancer

This review article describes the mechanism of enhancement of vascular permeability in infectious disease and cancer. This phenomenon is primarily mediated by bradykinin, nitric oxide and other unique vascular mediators. They are highly intermingled with each other in these disease states. Furthermore, these mediators are elicited in various in vivo settings most frequently induced by bacterial proteases, and indirect or direct activation of kallikrein–kinin cascade at one or more steps. The key steps involve bacterial proteases or cellular components including lipopolysaccharides. Thus, the use of appropriate protease inhibitors or antagonists, or scavengers in the case of nitric oxide, superoxide or peroxynitrite, are anticipated to attenuate the clinical manifestation induced by such mediators. It also explained that fluid accumulation in ascitic or pleural compartments in the case of carcinomatosis in terminal cancer patients can be largely attributed to bradykinin or related mechanism. Systemic bacterial dissemination is also facilitated by bradykinin, or suppressed by kinin antagonists as well as by the inhibition of kinin production, respectively. Thus, control of the level of such vascular mediators appears important both in infectious disease and in cancer. α1-Protease inhibitor, which inhibits neutrophil elastase, is inactivated by oxidative metabolites such as superoxide and peroxynitrite, and this effect activates matrix metalloproteinases. This indicates that oxidative stress activates proteolytic potential, and thus accelerates the degenerative process upon infection.

176 Coagulation activation markers in the diagnosis of deep vein thrombosis following total hip replacement

Bradykinin (BK), an important mediator of allergic reactions and pain induction, is released by the activation of the plasma kallikrein-kinin (K-K) cascade. Neurotropin® is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus and is widely used clinically in Japan as an effective agent for these disorders. Since its mechanism of action is not clearly known, we have investigated the effects of Neurotropin on the human plasma K-K system. In dextran sulfate-activated plasma, Neurotropin inhibited the formation of BK, the cleavage of high molecular weight kininogen (HK) and the formation of kallikrein-C1 inhibitor and activated coagulation factor XII (FXIIa)-C1 inhibitor complexes. Experiments using purified enzyme of the K-K cascade indicated that Neurotropin inhibited surface-mediated activation of coagulation factor XII (FXII) and the activation of prekallikrein by FXIIa. Neurotropin also inhibited the binding of FXII and HK to the activating surface. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and consequently the formation of BK.