The synthetic LPS binding peptide 19-2.5 interferes with clotting and prevents degradation of high molecular weight kininogen in plasma.

Sepsis and septic shock are life-threatening conditions and remain an important medical problem, emphasizing the need to identify novel therapeutic approaches. Coagulation dysfunction, hypotension, disturbed microcirculation and multiorgan failure occur frequently. These severe conditions result from an overwhelming inflammatory response, induced by pathogen and damage associated molecular patterns (PAMPs and DAMPs) released into the bloodstream. In the present study, we demonstrated that the synthetic Lipopolysaccharid (LPS)-binding peptide 19-2.5 interferes with the activation of the coagulation and contact system. Moreover, binding of LPS to high molecular weight kininogen (HK), one of the major LPS carrier in blood, could be prevented by the peptide. Thus, peptide 19-2.5 might represent a promising target in the treatment of endotoxemia and sepsis, not only by its anti-inflammatory potential, but also by the anticoagulant effect, together with its ability to prevent degradation of HK.

Differential processing of high-molecular-weight kininogen during normal pregnancy.

RATIONALE: Studies identified kininogen as a potential biomarker of preeclampsia, a major cause of adverse maternal outcomes. High-molecular-weight kininogen (HK) and its activated form participate in numerous pathways associated with establishing and maintaining pregnancy. However, dynamic changes in HK and naturally occurring HK-derived peptides during the natural course of pregnancy are largely unknown. METHODS: Longitudinal serum samples during the course of normal pregnancy (trimesters T1, T2, T3) from 60 pregnant women were analyzed by western blot with an anti-HK antibody. Circulating peptides in longitudinal serum specimens derived from 50 participants were enriched using nanoporous silica thin films. Peptides were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and database searching. Relative quantification was performed using MaxQuant and in-house scripts. Normality was evaluated by either ANOVA or Friedman tests with p < 0.05 for statistical significance. RESULTS: Western blotting revealed that HK significantly decreased during normal pregnancy (T1 vs T2, p < 0.05; T1 vs T3, p < 0.0001). A 100 kDa intermediate increased during pregnancy (T1 vs T2, p < 0.005; T1 vs T3, p < 0.01). Moreover, the heavy chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.01) and light chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.05) significantly increased during pregnancy. LC/MS/MS analysis identified 180 kininogen-1 peptides, of which 167 mapped to domain 5 (D5). Seventy-three peptides with ten or more complete data sets were included for further analysis. Seventy peptides mapped to D5, and 3, 24, and 43 peptides showed significant decrease, no trend, and significant increase, respectively, during pregnancy. CONCLUSIONS: This study demonstrates dynamic changes in HK and naturally occurring HK-derived peptides during pregnancy. Our study sheds light on the gestational changes of HK and its peptides for further validation of them as potential biomarkers for pregnancy-related complications.

Polyphosphate, Zn2+ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting.

BACKGROUND: Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. OBJECTIVES AND METHODS: We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+ , to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. RESULTS: The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. CONCLUSIONS: Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

Genetic determinants of activity and antigen levels of contact system factors.

Essentials Genetic variation may provide valuable insight into the role of the contact system in thrombosis. Explored associations of genetic variants with activity, antigen, and disease in RATIO study. Two novel loci were identified: KLKB1 rs4253243 for prekallikrein; KNG1 rs5029980 for HMWK levels. Contact system variants and haplotypes were not associated with myocardial infarction or stroke. SUMMARY: Background The complex, interdependent contact activation system has been implicated in thrombotic disease, although few genetic determinants of levels of proteins from this system are known. Objectives Our primary aim was to study the influence of common F11, F12, KLKB1, and KNG1 variants on factor (F) XI activity and FXI, FXII, prekallikrein (PK) and high-molecular-weight kininogen (HMWK) antigen levels, as well as the risk of myocardial infarction and ischemic stroke. Patients/methods We analyzed samples from all 630 healthy participants, 182 ischemic stroke patients and 216 myocardial infarction patients in the RATIO case-control study of women aged < 50 years. Forty-three tagging single nucleotide variants (SNVs) were genotyped to represent common genetic variation in the contact system genes. Antigen and activity levels were measured with sandwich-ELISA-based and one-stage clotting assays. We performed single variant, age-adjusted, linear regression analyses per trait and disease phenotype, assuming additive inheritance and determined conditionally independent associations. Haplotypes based on the lead SNV and all conditionally independent SNVs were tested for association with traits and disease. Results We identified two novel associations of KLKB1 SNV rs4253243 with PK antigen (ßconditional = -12.38; 95% CI, -20.07 to -4.69) and KNG1 SNV rs5029980 with HMWK antigen (ßconditional = 5.86; 95% CI, 2.40-9.32) and replicated previously reported associations in a single study. Further analyses probed whether the observed associations were indicative of linkage, pleiotropic effects or mediation. No individual SNVs or haplotypes were associated with the disease outcomes. Conclusion This study adds to current knowledge of how genetic variation influences contact system protein levels and clarifies interdependencies.

The role of the kallikrein-kinin system, matrix metalloproteinases, and tissue inhibitors of metalloproteinases in the early restenosis of covered stents in the femoropopliteal arterial segment.

OBJECTIVE: The purpose of this study was to investigate the roles of the kallikrein-kinin system and matrix metalloproteinases (MMPs) in the development of arterial restenosis attributable to intimal hyperplasia in the femoropopliteal arteries. METHODS: This report describes a single-center prospective study of 27 patients with peripheral artery disease who required percutaneous transluminal angioplasty and stenting of the femoropopliteal segment using covered stent grafts. The blood concentrations of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, and tissue inhibitors of metalloproteinases were measured by enzyme-linked immunosorbent assay. RESULTS: Four (15%) of the treated patients developed restenosis at the 6-month follow-up evaluation. These patients had significantly lower levels of high-molecular-weight kininogens (24 hours; P < .05) and low-molecular-weight kininogens (before, P < .05; 24 hours, P < .01; 6 months, P < .05) and lower levels of tissue inhibitor of metalloproteinases-2 (6 months; P < .05) than the patients without restenosis. The activity levels of plasma and tissue kallikrein, kininase II, and MMPs did not differ significantly between the patients with and without restenosis. CONCLUSIONS: This study demonstrates an involvement of the kallikrein-kinin system in in-stent restenosis, although we could not confirm the participation of metalloproteinases in the restenosis process.

High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.

BACKGROUND: The inherited deficiency of C1-inhibitor (C1-INH), which can be quantitative (type I) or qualitative (type II), is characterized by recurrent attacks of oedema, and it is known as hereditary angioedema due to C1-INH deficiency (HAE-C1-INH). The frequency of symptoms varies widely among patients and in the same patient during life. OBJECTIVE: To identify laboratory markers of disease severity in HAE-C1-INH patients. METHODS: We studied 162 patients with differently severe HAE-C1-INH during remission, 31 HAE-C1-INH patients during attacks, and 81 normal controls, evaluating complement parameters, spontaneous plasma kallikrein activity, the capacity of plasma to inhibit exogenous kallikrein activity, and cleavage of high-molecular-weight kininogen (HK). Sixty-five HAE-C1-INH patients were screened for mutations in the C1-INH gene. RESULTS: As expected, plasma C1-INH levels and activity and C4 levels were low in the HAE-C1-INH patients. Spontaneous plasma kallikrein activity in patients in remission was higher than in controls (P = 0.001) and increased during acute attacks (P = 0.01), whereas the capacity of inhibiting kallikrein activity was lower in patients in remission than in controls (P = 0.001) and further reduced during attacks (P = 0.001). HAE-C1-INH patients in remission had higher levels of cleaved HK than controls (P = 0.001), and these further increased during acute attacks (P = 0.001). Cleaved HK levels were higher in highly symptomatic HAE-C1-INH patients than in those with less frequent attacks (P = 0.001). Thirty-five different mutations in the C1-INH gene were equally distributed in patients with different attack frequencies. CONCLUSIONS: Measuring plasma levels of cleaved HK may be a sensitive mean of assessing disease severity in HAE-C1-INH patients.

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.

Identification of a high molecular weight kininogen fragment as a marker for early gastric cancer by serum proteome analysis.

BACKGROUND: Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific. METHODS: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n = 81) and age- and sex-matched healthy controls (n = 66). RESULTS: Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209 Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209 Da peptide (AUC = 0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC = 0.593, carbohydrate antigen 19-9 AUC = 0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209 Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients. CONCLUSIONS: Serum levels of the 2209 Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.

Mechanisms by which cleaved kininogen inhibits endothelial cell differentiation and signalling.

We have shown that cleaved high-molecular-weight kininogen inhibits endothelial cell tube and vacuole formation in a concentration-dependent manner and this correlates with its recognised anti-angiogenic activity. The antibody against the urokinase plasminogen activator receptor (uPAR) mimicked the inhibitory effect of cleaved kininogen (HKa) on apoptosis (HKa: 30% and uPAR antibody: 26%) and tube formation. In tumour angiogenesis, cancer cells release angiogenic stimulators, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), thus stimulating the transformation of endogenous pro-uPA to uPA. The proteolytic enzyme urokinase plasminogen activator (uPA) then binds to its receptor in a complex with its inhibitor PAI-1, which results in the internalisation of this complex, and activates extracellular signal-regulated kinase (ERK). Recycling of the uPAR regulates the migration of endothelial cells (ECs). ERK activation stimulates migration and proliferation and suppresses apoptosis of ECs. HKa disrupted the uPA-uPAR complex, inhibited ERK activation, and blocked the internalization of uPAR, eventually resulting in cell death and cell motility arrest. Both are critical steps in angiogenesis.

Kininogen in autoimmune uveitis: decrease in peripheral blood stream versus increase in target tissue.

PURPOSE: Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. METHODS: In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify differentially expressed proteins. The expression pattern of a candidate protein in retina and vitreous was validated by Western blots and immunohistochemistry. RESULTS: Ten differentially expressed proteins could be identified by mass spectrometry (MALDI-TOF/TOF). Five proteins--IgM, IgG4 hc, serotransferrin, alpha-2HS-glycoprotein, and complement factor B--were upregulated in the uveitic state, whereas the five proteins albumin, apolipoprotein A-IV and H, IgG5 hc, and high-molecular-weight kininogen (HK) showed a significantly lower expression in sera of uveitis cases. Of interest, kininogen was significantly upregulated in the target tissues vitreous and retina. HK is a plasma protein with multiple physiological functions, with an important role in inflammation and promoting neovascularization. Most interesting is the as of yet unaddressed association of HK with uveitis. Immunohistochemistry showed coexpression of kininogen and VEGF in inflamed eyes. CONCLUSIONS: Since neovascularization plays a major role in the pathogenesis of uveitis, the identification of a proangiogenic factor in the retina presents an important finding and may contribute to elucidating the pathogenesis of uveitis.

The plasma kallikrein-kinin system: its evolution from contact activation.

The plasma kallikrein-kinin system consists of the proteins factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen. It was first recognized as a surface-activated coagulation system that is activated when blood or plasma interacts with artificial surfaces. Although surface-activated contact activation occurs in vivo in the case of tissue destruction or a developing thrombus, the physiologic basis for the activation and function of this system has not been delineated. New investigations indicate that there is a proteolytic pathway on cells for PK activation independent of FXII. This pathway for PK with subsequent FXII activation indicates physiologic activities. These activities include blood pressure regulation and modulation of thrombosis risk independently of hemostasis. Furthermore, they include regulation of endothelial cell proliferation, angiogenesis and apoptosis through a cellular-based, outside-in signaling system. The present characterizations of this system, which incorrectly had been thought to initiate coagulation, represent an evolution of understanding in this field.

Activation of the contact system and inflammation after thrombolytic therapy in patients with acute myocardial infarction.

Thrombolytic therapy activates the contact system, and factor XII activation may activate the coagulation cascade and inflammation. It is not known whether an early inflammatory response is induced by thrombolytic therapy in patients with acute myocardial infarction (AMI). We prospectively measured the plasma levels of activated factor XII, cleaved kininogen, prothrombin fragment 1 + 2 (as indexes of the contact phase and coagulation activation), and interleukin-6 and C-reactive protein (CRP) (as indexes of inflammation) in 39 patients hospitalized for AMI within 12 hours of symptom onset: 26 receiving thrombolytic therapy and 13 heparin alone. Blood samples were collected at baseline and after 90 minutes and 24 hours. Patients undergoing thrombolysis had a significant early increase in activated factor XII (from 2.2 ng/ml at baseline to 4.7 ng/ml after 90 minutes; p = 0.0001), cleaved kininogen (from 26% to 37%; p = 0.001), and fragment 1 + 2 (from 1.4 to 2.1 nmol/L; p = 0.0001), whereas the 24-hour levels were similar to baseline levels. The levels of interleukin-6 significantly increased during the first 90 minutes (from 3.9 to 6.3 microg/ml; p = 0.001), and were even higher after 24 hours (11.9 ng/ml, p = 0.0001). CRP levels increased only after 24 hours (p = 0.0001). There were no changes in these parameters in patients receiving heparin alone, except for a 24-hour increase in interleukin-6 and CRP levels. Thus, in patients with AMI receiving thrombolytic therapy, early activation of inflammation parallels the activation of the contact system and the coagulation cascade, which might contribute to microvascular obstruction and reperfusion injury.

Kinins and cytokines in plasma and cerebrospinal fluid of patients with neuropsychiatric lupus.

OBJECTIVE: To evaluate the kinin system components and selected cytokines in plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPL). METHODS: We studied 29 women with active NPL and 29 healthy women matched to patients for age. Low (LKg) and high molecular weight kininogen (HKg) and cytokine concentrations [interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, and tumor necrosis factor-a (TNF-a)] were determined by ELISA. The activities of tissue kallikrein, plasma prekallikrein, and kininase II were assayed by their action on selective substrates. RESULTS: Compared to controls, patients with NPL presented increased plasma and CSF levels of LKg, HKg, and prekallikrein, increased activity of tissue kallikrein and kininase II, and increased levels of IL-6, IL-10, and TNF-a (p < 0.001 each comparison). IL-1beta levels were increased in patient plasma (p < 0.001), whereas plasma IL-8 levels did not differ from controls. IL-1beta and IL-8 were not detected in CSF of patients or controls. CONCLUSION: The increased levels of kininogen fractions, kallikreins, and kininase II in patient plasma and CSF indicate overactivity of the kinin system, suggesting intense kinin production. Since kinins may induce the production of proinflammatory cytokines including IL-1beta, IL-6, and TNF-a, these findings support the participation of kinins and cytokines in the acute manifestations of NPL. Most of the variables evaluated in patients' CSF increased proportionally in relation to plasma levels. In contrast, the activity of tissue kallikrein in patient CSF increased out of proportion to plasma levels, appearing to be locally synthesized in response to brain involvement.

Chronic intestinal inflammation and angiogenesis in genetically susceptible rats is modulated by kininogen deficiency.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis associated with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK), we backcrossed Brown Norway rats having an HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome. The HK values of F5WT rat plasma and F5HKd rat plasma were 0.62 +/- 0.20 and 0.08 +/- 0.03 U/ml, respectively. Among the inflammatory changes, the mean gross gut, total intestinal histologic and liver granuloma score and the white blood count were significantly lower in the F5HKd than the F5WT rats. Plasma T-kininogen was significantly less in F5HKd. Angiogenesis (mean vascular density) in the cecum was decreased significantly in F5HKd compared to F5WT. These results indicate the importance of the kallikrein-kinin system in this model of chronic enterocolitis and systemic inflammation.

Changes in high molecular weight kininogen levels during and after cardiopulmonary bypass surgery measured using a chromogenic peptide substrate assay.

High molecular weight kininogen (HK) is a co-factor in the blood-contact activation system. A chromogenic peptide substrate assay for HK (HKcs) has been developed in which test plasmas are mixed with diluted HK-deficient plasma and incubated with a soluble contact system activator that activates prekallikrein and factor XII. Calcium chloride, a synthetic thrombin inhibitor and a chromogenic peptide substrate for activated factor X (FXa) are then added. The FXa generated cleaves the FXa substrate releasing p-nitroanaline, which is measured photometrically. Test plasma HK values were calculated from a standard curve generated using a pooled normal plasma. Acceptable intra-assay and inter-assay precision values were obtained and levels of HK up to 200% were measurable. The assay measured HK in plasmas deficient in factor XII, prekallikrein and factor XI, was not affected by antiphospholipid antibodies and gave an acceptable correlation (r = 0.95) when normal plasmas and mixtures of HK-deficient and normal pooled plasma, calculated to give HK levels of 25 and 50%, were compared using HKcs and a HK one-stage clotting assay. The HKcs was used to measure HK levels in seven patients undergoing cardiopulmonary bypass (CPB). HK levels fell significantly during CPB (P = 0.0014) and were significantly higher (P = 0.016) 6 days after CPB, suggesting that HK may be a positive acute-phase reacting protein.

Elevated levels of plasma prekallikrein, high molecular weight kininogen and factor XI in coronary heart disease.

Increased levels of hemostatic factors may play a role in the pathogenesis of myocardial infarction by triggering thrombin formation. We measured factor XII (FXII), factor XI (FXI), plasma prekallikrein (PK) and high-molecular-weight kininogen (HK) in 200 patients having survived myocardial infarction for at least 2 months, and in 100 healthy controls. We found significantly elevated levels of FXI clotting activity (FXI:C), HK:C and of the amidolytic activity of PK (PK:Am) among the patients as compared to the controls. Plasma levels of FXI:C, HK:C and PK:Am in the highest quartile were associated with an odds ratio of 1.9 (95% CI: 1.0-3.8), 2.0 (95% CI: 1.0-4.0) and 5.4 (95% CI: 2.6-11.2), respectively, compared to the respective plasma levels in the lowest quartile. After correction for established clinical and laboratory risk factors, the association between PK:Am plasma levels and myocardial infarction remained significant (P=0.0007). Combination of high PK:Am plasma levels and smoking or arterial hypertension, respectively, resulted in a more than additive relative risk for myocardial infarction.

High molecular weight kininogen deficiency: a patient who underwent cardiac surgery.

A 66 year old male, referred for cardiac surgery, was found to have high molecular weight kininogen deficiency (activity <1%). Apart from activated partial thromboplastin time (APTT) >300 s, tests of haemostasis were otherwise normal (factors VIII, IX, XI, XII and prekallikrein). No inhibitor of coagulation was found. The activated coagulation time (ACT) was 800 s pre-operatively and >1000 s after heparin. Heparin levels were measured directly by an anti-Xa chromogenic assay, with values of between 2.9 and 3.2 u/ml during cardiopulmonary bypass. Thrombin-antithrombin levels rose from 2.3*g/l before surgery to a peak of 83.5*g/l at the end of cardiopulmonary bypass. Cross linked fibrin d-dimers (XDP) levels rose from 100 ng/ml before operation to 600 ng/ml after protamine administration. The patient had no excess bleeding and no thrombotic complications from surgery. This patient shows that high molecular weight kininogen is not required for thrombin formation or fibrinolysis during cardiac surgery and illustrates the need to measure heparin directly in patients with such contact factor deficiencies.

Whale high-molecular-weight and low-molecular-weight kininogens.

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.

Kallikrein-kininogen system activation and bradykinin (B2) receptors in indomethacin induced enterocolitis in genetically susceptible Lewis rats.

BACKGROUND: The plasma kallikrein-kinin (K-K) system is activated in acute and chronic relapsing intestinal inflammation induced in Lewis rats by intramural injection of exogenous bacterial components. AIMS: To determine whether this effect is model specific, K-K system activation was investigated in a modified indomethacin induced enterocolitis model, as well as bradykinin 2 (B2) receptor distribution in the normal and acutely inflamed intestine. METHODS: Lewis rats injected with daily sublethal doses of indomethacin for two days developed acute (two days) and chronic (14 days) intestinal inflammation. Plasma prekallikrein (amidolytic), high molecular weight kininogen (HK, coagulant) and cleavage of HK (western blot) were assayed to detect K-K activation. RESULTS: Liver and spleen weights were significantly higher, and body weights and haematocrit values were significantly lower in the indomethacin group than in the control group. During both acute and chronic phases, rats displayed K-K system activation manifested by a significant decrease in plasma prekallikrein and HK functional levels, and by HK cleavage. Plasma T kininogen (a major acute phase protein) was significantly elevated. B2 receptors were identified in both normal and inflammatory intestine with more prominent specific immunohistochemical staining in the acutely inflamed tissue. CONCLUSIONS: K-K system activation occurs in association with both acute and chronic phases of intestinal injury, regardless of the triggering agent, suggesting that activation of this system is integrally involved in intestinal inflammation in genetically susceptible hosts. Localisation of B2 receptors across intestinal layers provides a structural basis for the kinin function in the intestine.