Cleavage Of HK Research Articles

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.

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.

Protective Effects of Kininogen-1 Gene Deficiency in Mouse Models of Venous Thrombosis

Background- High molecular weight Kininogen (HK) is a nonenzymatic co-factor of the contact activation system. HK binds prekallikrein (PK) and FXI to surfaces in proximity to FXII, amplifying PK activation by FXIIa and the reciprocal activation of FXII by activated PK (PKa), as well as FXI activation of FXIIa. PKa cleavage of HK also liberates bradykinin-a proinflammatory and vasoactive nanopeptide. The aim of this study was to define the pro-thrombotic role of kininogen in venous thrombosis (VT) and to use in vivo serial analysis of thrombus development to understand the recruitment and retention of platelets in the growing thrombus in the absence and presence of kininogen.Methods- The development of VT in mice deficient in kininogen (mKng1-/-) was compared to that in their wild-type littermates. A femoral-saphenous stasis VT model was prepared by ligating both saphenous and femoral veins. Next VT formation, growth, and dissolution (n=3 for each group) was monitored using intravital microscopy (IVM) via a multichannel epifluorescence microscope (Nikon Eclipse 90i). To induce stasis VT, FITC-dextran (10 mg/kg, ex/em 488/520 nm) was injected retro-orbitally, and then continuous light irradiation (20x objective, 475nm/35nm) of the saphenous vein was applied for 5 minutes. FITC-dextran fluorescence angiography monitored thrombus formation and dissolution. Immediately after VT formation, platelet accumulation at the thrombus site was monitored in the Cy5 channel (630/38 nm) via injection of a GPIbβ antibody conjugated with Dylight-649 (150nmol/kg), over time. All images were identically windowed in each channel, and thrombus area was measured using NIH ImageJ software. To corroborate IVM studies, we also evaluated a complete stasis model of inferior vena cava (IVC) ligation (n=7-8 per group). Thrombi were harvested after 48 hours and thrombus weight and length were measured to estimate thrombus mass. FXI circulates in blood as a homodimer along with HK. We determined the effect of kininogen deficiency on FXI activity. FXI activity assay used a combination of inhibitors, serially, to monitor the cleavage of substrate specific to activated FXI and release of chromogen, as a function of FXI activity. Finally, to determine the effects of Kng1 deficiency on bleeding, tail vein bleeding times were also determined (n=8 per group).Results- In femoral-saphenous stasis VT, thrombus developed in both groups immediately following FITC-channel light irradiation. However, thrombus size was smaller in Kng1-/- as compared to WT (Figure 1). Results from serial IVM of VT indicated faster thrombus dissolution in the Kng1-/- group. Lower platelet signals, as shown at 2 and 6 hours in the Kng1-/- mice may be consistent with this hypothesis. Thrombus area analysis suggested decreased thrombus formation in the Kng1-/- animals, and temporal analysis indicated faster dissolution by 6 hours (Figure 2). IVC ligation results corroborated the findings of femoral-saphenous DVT model, demonstrating that thrombus weight was significantly lower in Kng1-/- mice as compared to WT (p<0.001, Figure 3). FXI activity was also decreased in the Kng1-/- group (p<0.10). Tail vein bleeding times, however, showed no increased bleeding in Kng1-/- mice.Conclusion- These initial results suggest a pro-thrombotic role of kininogen and a protective role of kininogen deficiency in two murine venous thrombosis models, without incurring a bleeding penalty. Thrombus dissolution was faster and platelet accumulation was inhibited in Kng1-/- mice. These findings suggest that targeting kininogen may provide a new approach to prevent and treat venous thrombosis. [Display omitted] DisclosuresMcCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria. Jaffer: Mercator, Inc.: Other: Sponsred research.

Bradykinin Is a Proximal Event in Experimental Cerebral Malaria

Human malaria is a complex disease and a leading cause of mortality in children under 5 years of age. Plasmodium falciparum (Pf) is the agent responsible for cerebral malaria. Parasite infected erythrocytes are sequestered in the brain vasculature, disrupting the blood-brain-barrier, and with systemic inflammation leading to progressive brain edema. The precise pathophysiologic mechanism(s) underlying brain swelling in CM is not known. Recent work from our laboratories indicates that there is a role for bradykinin (BK) in fluid transport in human brain microvascular endothelial cells (Front Med 6:75, 2019).We examined the role of bradykinin (BK) in pediatric CM. Initial studies showed recombinant falcipain-2, a cysteine protease contained in the parasite digestive vacuole, was inhibited by high molecular weight kininogen (HK), with an IC 50=36 nM. Further, falcipain-2, but not the related protease falcipain 3, hydrolyzed the chromogenic substrate S2302 (Pro-Phe-Arg-pNA) at pH 7.4 with an 88 nM K m. These results suggest that falcipain-2 has plasma kallikrein-like activity. HK is both an inhibitor and substrate of falcipain-2. Molar excess HK to falcipain-2 (ratio 8:1 to 2:1) blocked the proteolytic activity of the cysteine protease at pH 7.4. Equal molar falcipain-2 to HK (1:1) resulted in kallikrein-like cleavage of HK with stable BK liberation over 1 h. Molar excess falcipain-2 to HK (1:2 and greater) led to progressive HK cleavage into smaller proteins and peptides. The falcipain-2 major cleavages observed by N-terminal sequencing were in Domain 3 of the heavy chain of HK, the cysteine protease inhibitory region (I 292ASFSQNCDIYPGKDF 303, D 320IPTNSPELEETLT 334, and E 412KKIYPTVNCQPLG 425). P. falciparum trophozoite lysates completely hydrolyzed purified and plasma HK into a ~64 kDa heavy chain and ~46 kDa light chain in buffer containing EDTA, pepstatin, and PMSF. The cysteine proteinase inhibitor E64 blocked this cleavage, suggesting that the relevant activity was that of a cysteine protease.Plasma from Kenyan children presenting with CM (fever, parasitemia, coma) had evidence of circulating cHK, indicative of BK released from HK. Forty percent (8 of 20) of CM patients had no intact 120 kDa HK at hospital entry. In contrast, only 16% (3 of 8) of children with uncomplicated malaria had detectable cHK. In CM patients, the HK level before antimalarial treatment (58 ± 3.9 µg/ml) was significantly lower than the value after clinical recovery (69 ± 3.6 µg/ml; p<0.04) as measured by competitive ELISA. We also examined the roles of BK and HK in experimental cerebral malaria. 10 6 infected red blood cells with P. berghei ANKA were injected intraperitoneally into wild-type (C57BL/6) and total kininogen deficient (kgn1 -/-) C57BL/6 mice. The level of parasitemia on day 5 post-infection was ≥ 8% for both groups of mice (Figure 1). The kgn1 -/- mice had protected neuronal function measured by SHIRPA score relative to wild-type mice. Cerebral edema detected in wild- type mice by Evans Blue dye extravasation test was nearly completely attenuated in kgn1 -/- mice. Corroborative studies were performed in BK B2 receptor deleted (bdkrb2 -/-) mice. In mice with 15% parasitemia for both genotypes, there was significantly less neurologic function deterioration and a 30% reduction in cerebral Evans blue extravasation into brain parenchyma in the bdkrb2 -/- mice.These data strongly suggest that falcipain-2 liberates BK from HK by acting like plasma kallikrein and in high concentrations destroys HK's cysteine protease inhibitory region. Some children with CM have in vivo evidence of prior HK proteolysis. Total kininogen deficiency protects mice from lethal experimental CM. Taken together, these data suggest that bradykinin is a proximal mediator of cerebral malaria. [Display omitted] DisclosuresMcCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure

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

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

High Molecular Weight Kininogen but Not Factor XII Deficiency Attenuates Acetaminophen-Induced Liver Injury in Mice

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&amp;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&amp;lt;0.05) and 24 hours (1546 ± 302 vs. 3857 ± 493 U/L; p&amp;lt;0.01) after injection of APAP. The attenuation of liver injury was observed despite the lack of a significant effect on APAP-induced coagulation activation (plasma TAT levels [mean±SEM]: 72.9 ± 8.2 μg/L in HK+/+ vs. 70.8 ± 4.3 μg/L in HK-/-). HK deficiency also reduced plasma levels of IL-6 (75.8%; p&amp;lt;0.001), number of infiltrating neutrophils in liver (59.8%; p&amp;lt;0.001) and liver necrotic area (28.1%; p&amp;lt;0.001) 24 hours after APAP challenge. Importantly, the protective effects of HK deficiency were completely reversed by the injection of HK protein into APAP-treated HK-/- mice. 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&amp;lt;0.01) and PAP complexes (64.4 fold; p&amp;lt;0.01). Using Western blotting analysis, BK ELISA, and mass spectrometry analysis we demonstrated that plasmin efficiently cleaves HK in a buffer system as well as in mouse and human plasma. Importantly, plasmin mediated cleavage of HK resulted in the release of BK and generation of cleaved HK fragments. 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.

Abstract P267: Aging &amp; Endothelial Cell Senescence: Does Prolylcarboxypeptidase Predict Endothelial Dysfunction?

Changes due to cellular senescence may lead to the formation of a unique cellular morphology such as the transformation of endothelium from an anticoagulant to a potentially prothrombotic surface. Investigations were performed to determine whether the ratio of prolylcarboxypeptidase (PRCP) gene expression to the rate of telomere shortening in vascular endothelial cells would predict endothelial dysfunction and subsequent endothelial surface alteration from anticoagulant to potentially prothrombotic surface. Here, we present evidence for a novel mechanism of senescence-induced cell-surface expression of PRCP involving activation of the plasma kallikrein-kinin system (KKS). We found that the abilities of human pulmonary vein endothelial cells (HPVECs) to express PRCP first incrementally increased at early passages and then decreased after passage 18, in contrast to changes in average human telomerase reverse transcriptase (hTERT) mRNA expression levels. In agreement with the changes in average PRCP expression, incubation of prekallikrein (PK) and high molecular weight kininogen (HK) plasma proteins resulted in increased cleavage of HK, higher enzymatic activities of kallikrein, and increased liberation of bradykinin (BK). Furthermore, the alterations in PK and BK generation was prevented using PRCP-siRNA or PRCP inhibitor (z-Pro- prolinal) that blocked PK activation in passage 18 by 60% and 80% respectively. SDD31 that interferes with the binding of PK on HK reduced kallikrein generation by 90%. There was a direct correlation between PRCP-dependent BK generation and cell permeability. Our results were supported when we found that nitric oxide (NO), a downstream product of the PRCP-catalyzed HK-PK system, also shared the same peak in formation as PRCP. In contrast, increased eNOS expression remained unchanged after passage 12. While β-Gal rose after passage 18, calnexin expression like PRCP dropped significantly in passage 30. These data are the first to demonstrate the inverse relationship between hTERT and PRCP expression prior to an apparent onset of EC replicative senescence, which could have profound implications as a prognostic tool for predicting disease progression and cardiovascular event rates.

The blood fluke Schistosoma mansoni cleaves the coagulation protein high molecular weight kininogen (HK) but does not generate the vasodilator bradykinin

BackgroundSchistosomes 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).MethodsFollowing 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.ResultsThe 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.ConclusionsSince 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.

High-molecular-weight kininogen and its cleavage product bradykinin are critical in the pathogenesis of autoantibody-induced arthritis

Abstract High-molecular-weight kininogen (HK) is a major component of plasma kallikrein-kinin system (KKS), the activation of which mediates HK cleavage to release bradykinin (BK). BK is a nonapeptide with potent proinflammatory and vasoactive activities. Although patients with rheumatoid arthritis (RA) exhibits the activation of the KKS and the cleavage of HK in plasma, the role of HK in arthritis pathogenesis has never been characterized. In this study we evaluated the phenotype of our newly-generated HK-deficient (Kng1−/−) mice in arthritis. To induce autoantibody-induced arthritis, two groups of Kng1+/+ and Kng1−/− mice were injected intraperitoneally with anti-type II collagen antibody cocktail (4 mg) on day 0 and lipopolysaccharide (LPS, 50 μg) on day 3. Although wild-type (WT) mice developed severe arthritis, the Kng1−/− mice exhibited significantly attenuated disease severity as evaluated by joint diameter change and clinical scores (p&amp;lt;0.0001). Compared with WT mice, Kng1−/− mice displayed reduced synovial hyperplasia and cartilage erosions, and decreased protein and mRNA levels of IL-1b and IL-6 in joint tissue (p&amp;lt;0.001). BK level and kallikrein activity in Kng1−/− plasma were significantly lower than that of WT mice. Incubation with collagen-anti-collagen antibody complex mediated BK production in human plasma. Consistent with the phenotype of Kng1−/− mice, the mice lacking BK receptors also displayed a reduced arthritis severity, as well as a decrease in IL-1b and IL-6 production in joint tissue. Our study provides the first genetic evidence indicating an important role of HK in the pathogenesis of arthritis, and the blockade of HK cleavage to prevent BK production might be a novel therapeutic strategy for RA treatment.

Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.

1Factor XII-Independent Activation of the Complex of Prekallikrein with High Molecular Weight Kininogen: Implications for Hereditary Angioedema

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 (C1INH) or corn trypsin inhibitor (CTI). CTI does not inhibit kallikrein. Addition of heat shock protein 90 (Hsp90) leads to conversion of prekallikrein to kallikrein in a zinc-dependent reaction. Our goal is to determine whether these reactions occur in plasma. C1INH was removed from factor XII deficient plasma by immunoadsorption. Plasma was incubated in polystyrene plates and assayed for kallikrein formation. We now demonstrate that prekallikrein-HK will activate to kallikrein in phosphate-containing buffers and the rate is further accelerated upon addition of Hsp90. In 1983, Fields et al (J Allergy Clin Immunol 72: 54-60, 1983) reported that incubation of HAE plasma at 37°C leads to progressive formation of bradykinin, presumably by activation of factor XII and formation of kallikrein when C1INH is absent. Activation of the prekallikrein-HK complex is an alternative possibility. Prolonged incubation of plasma deficient in both factor XII and C1INH led to conversion of prekallikrein to kallikrein and cleavage of HK as was seen with HAE plasma but not normal plasma. These results indicate that C1INH stabilizes the prekallikrein-HK complex to prevent HK cleavage either by prekallikrein and/or prekallikrein-HK autoactivation. In HAE, 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.

Angio-Oedema Induced by Oestrogen Contraceptives Is Mediated by Bradykinin and Is Frequently Associated with Urticaria

Background: Hereditary C1-inhibitor (C1-Inh) deficiency is associated with ‘bradykinin-mediated angio-oedema’ (BK-AO) and is believed not to be associated with urticaria. Acquired AO has been related to oestrogen contraceptives. Objective: To demonstrate that AO precipitated by oestrogens and characterized by nonfunctional C1-Inh is mediated by BK and to evaluate the occurrence of urticaria in these patients. Methods: A retrospective evaluation of patients referred for AO related to oestrogen was undertaken. Circulating C1-Inh, high molecular weight kininogen (HK) and enzymes involved in the metabolism of bradykinin were investigated. Results: Fifteen patients were included. HK cleavage concurrent to oestrogen intake was demonstrated in 10 patients with available plasma. Eight patients reported recurrent or chronic urticaria. Discontinuation of the contraceptive resulted in a return to native C1-Inh and HK in all cases studied and to normal kininogenase activity in all but one. The clinical manifestations completely disappeared in 6 patients and improved in 7 after the withdrawal of oestrogen. Conclusion: Patients display extensive cleavage of HK in the plasma, which supports that AO precipitated by oestrogen contraception is BK-mediated. Recurrent urticaria may have been underestimated in this context. The presence of recurrent urticaria should not systematically rule out the diagnosis of BK-AO when the history is suggestive.

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen.

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10(6) cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.

High molecular weight kininogen binds to laminin - characterization and kinetic analysis

High molecular weight kininogen (HK) is an abundant plasma protein that plays a central role for the function of the kallikrein/kinin/kininogen system. Thus, cleavage of HK by kallikrein liberates bradykinin, which stimulates vascular repair and a two-chain protein, activated HK (HKa), which induces apoptosis in proliferating endothelial cells. The localization of these events remains obscure, although the basement membrane may be of importance. Analyzing the interaction between HK and HKa and selected basement membrane proteins, we observed that they bound to the major noncollageneous proteins laminin, but not to vitronectin or fibronectin coated on microtiter plates. The binding to laminin was Zn2+ independent. However, at low but not at high concentrations of albumin, Zn2+ increased the affinity for the binding by abolishing an inhibitory effect of Ca2+. Recombinant human kininostatin encompassing the amino acid sequence, Arg439-Ser532 but not the endothelial cell binding peptide sequence (His479-His498; HKH20) within kininostatin inhibited the binding of HKa to laminin. This established that the amino acid sequence Arg439-Lys478 in domain 5 of HK is of importance for its binding to laminin. Extensive proteolytic cleavage of HK and HKa with kallikrein abolished the binding to laminin, releasing a 12 kDa anti-kininostatin reacting peptide. On the basis of these results, we propose that the binding of HK to laminin is a primary event, which secures proper localization of the cleavage products for subsequent interaction with the endothelium to promote inflammatory and pro- and anti-angiogenic activities.

Neutrophil‐derived heparin binding protein (HBP) is an endogenous activator of the kallikrein‐kinin system

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to release of heparin binding protein (HBP) causing increased vascular permeability. We have previously reported data suggesting that the kallikrein‐kinin system (KKS) is involved in PMN‐evoked plasma extravasation. Plasma prekallikrein (PK), factor XII (FXII) and high molecular weight kininogen (HK) are principal components of the KKS. Activated PK cleaves HK, liberating bradykinin (BK) which triggers EC contraction. Endogenous activators of this system are poorly defined. Here, we investigated the role of HBP in the activation of the KKS. Stimulation of EC monolayers with HBP caused an increase in paracellular permeability that was abolished with BK B1 and B2 receptor antagonists or a peptide displacing HK from the EC surface. Western blot analysis further revealed that HBP is able to induce cleavage of HK on EC. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐evoked plasma changes in vascular permeability in vivo. Plasma extravasation was markedly increased 4 h after thioglycollate injection. Treatment with BK receptor antagonists or an antibody to HBP significantly reduced the plasma exudation. These results demonstrate a critical role of the KKS in PMN‐evoked increase in vascular permeability. The data suggest that PMN‐derived HBP may constitute an endogenous activator of this system.

Endothelial-cell apoptosis induced by cleaved high-molecular-weight kininogen (HKa) is matrix dependent and requires the generation of reactive oxygen species

AbstractHigh–molecular-weight kininogen (HK) is an abundant plasma protein that plays a central role in activation of the kallikrein-kinin system. Cleavage of HK by plasma kallikrein results in release of the nonapeptide bradykinin (BK), leaving behind cleaved high–molecular-weight kininogen (HKa). Previous studies have demonstrated that HKa induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in vivo, activities mediated primarily through its domain 5. However, the mechanisms by which these effects occur are not well understood. Here, we demonstrate that HKa induces apoptosis of endothelial cells cultured on gelatin, vitronectin, fibronectin, or laminin but not collagen type I or IV. The ability of HKa to induce endothelial-cell apoptosis is dependent on the generation of intracellular reactive oxygen species and associated with depletion of glutathione and peroxidation of endothelial-cell lipids, effects that occur only in cells cultured on matrix proteins permissive for HKa-induced apoptosis. Finally, the ability of HKa to induce endothelial-cell apoptosis is blocked by the addition of reduced glutathione or N-acetylcysteine. These studies demonstrate a unique role for oxidant stress in mediating the activity of an antiangiogenic polypeptide and highlight the importance of the extracellular matrix in regulating endothelial-cell survival.

Inhibition of Angiogenesis by Cleaved High Molecular Weight Kininogen (HKa) and HKa Domain 5

High molecular weight kininogen (HK) is an abundant, multi-domain plasma protein that circulates in plasma primarily in its single chain form. Proteolytic cleavage of HK by plasma kallikrein releases the vasoactive nanopeptide bradykinin (BK), and converts HK into two-chain HK (HKa). BK appears to have pro-angiogenic activity, most likely mediated through binding to B1 and B2 receptors on endothelial cells. Conversely, HKa and its domain 5, but not (single chain) HK, have potent anti-angiogenic activity comparable to other endogenous angiogenesis inhibitors. The mechanism by which HKa exerts its anti-angiogenic activity remains controversial, but appears to involve binding to cell surface tropomyosin and induction of apoptosis of proliferating endothelial cells. A role for tropomyosin in mediating the anti-angiogenic signals of other anti-angiogenic proteins such as endostatin and histidine-proline-rich glycoprotein (HPRG) has also been reported. Here we review the physiological importance of high molecular weight kininogen in angiogenesis, with emphasis on the mechanism(s) by which this activity is mediated.

High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β through the Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Mononuclear Cells.

High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation

AbstractCrohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.