Activation Of Plasma Prekallikrein Research Articles

Abstract 15197: Differential Impact of Factor XII, Factor XI and Prekallikrein Deficiency on Thrombosis: A Pro-Thrombotic Vascular Action of Bradykinin

Objective: Factor XII (FXII) initiates the plasma contact system that triggers the intrinsic coagulation pathway and the kallikrein-kinin pathway via factor XI (FXI) and plasma prekallikrein (PK), respectively. The importance of the contact system for thrombosis sparing hemostasis is well established, however a comprehensive head-to-head comparison of the three proteases in arterial thrombosis is missing. Approach and Results: We produced mice deficient in FXII ( F12 -/- ), FXI ( F11 -/- ) and PK ( Klkb1 -/- ) and compared them in two distinct thrombosis models. While all (100%) of WT mice formed occlusive thrombi upon 30% ferric chloride challenge, carotid artery occlusion was reduced to 0, 4.8, 27.3 % in F12 -/- , F11 -/- and Klkb1 -/- mice (n=14-22), respectively. Upon photochemical-induced cremaster arteriolar thrombosis, occlusion time (s) prolonged to 226.1±60.4, 123.8±32.0 and 87.8±25.3 (mean±SEM, n=10-15) respectively in F12 -/- , F11 -/- and Klkb1 -/- mice, largely exceeding WT levels (38.3±12.1). A discordance was observed between in vivo thrombotic effects of the proteases and their roles for ex vivo coagulation, which ranked FXII≈FXI>PK as assayed by thromboelastography and activated partial thromboplastin time. Activation of FXII promoted thrombosis in WT and Klkb1 -/- mice, and impressively, it also accelerated thrombosis in F11 -/- mice, increasing activated PK activity and plasma bradykinin formation. Infusion of bradykinin and blockade of degradation of endogenous bradykinin increased platelet-endothelium adhesion and promoted thrombotic occlusions, which involved bradykinin mediated endothelial cell-release of platelet activating factor, rather than direct activation of platelets by bradykinin. Conclusion: FXII, FXI and PK differentially mediate experimental thrombosis in vivo and FXII/FXI appear as superior targets for thrombosis as compared to PK. PK- bradykinin axis activated by FXII contributes to thrombosis in vivo .

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

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

Predictive Value for Increased Factor XIa and Plasma Kallikrein Activity in Acute Venous Thromboembolism

Venous thromboembolism (VTE) is associated with increased coagulation activity, which in part can be attributed to the contact pathway of coagulation. Evidence from pre-clinical and epidemiological studies suggests that deficiency in factors of contact activation (e.g. coagulation factors (F) XI and FXII) protects against VTE. However, limited information exists regarding the activation of the contact system in the setting of acute VTE.In the current study, patients with confirmed VTE events (n=321) from the VTEval study and controls (n=300) from the population-based PREVENT-it pilot study were included. Plasma samples were collected from patients after confirmed VTE events or controls upon inclusion in the study. FXI as well as FXIa and plasma kallikrein (PKa) levels were assessed in plasma samples from all subjects using an activated thromboplastin time-based assay (FXI:c), a thrombin generation-based assay (CAT:FXIa) and by measuring inhibitory complexes (FXIa:antithrombin (AT), FXIa:alpha-1-antitrypsin (α1AT), FXIa:C1 esterase inhibitor (C1Inh) and PKa:C1Inh) using enzyme-linked immunoassay (ELISA). After a 2-year follow up period, a composite endpoint of recurrent VTE or death was determined.Increased FXI:c levels were determined in VTE patients compared to control individuals (124.08 ± 37.48% vs. 113.55 ± 27.99%), whereas CAT:FXIa levels were reduced in VTE patients (0 pM [IQR, 0-0.56] vs 0.56 pM [IQR, 0-0.88]). Levels of FXIa:α1AT and FXIa:AT inhibitory complexes were increased in VTE patients compared to controls (median[IQR]; 311.8 pM [238.2-424.0] vs. 202.5 pM [143.7 - 287.5] and 29.1 pM [23.4-38.3] vs 23.2 pM [19.7-29.8], respectively). Considering that 86% of the VTE patients were already on anticoagulant treatment (Table 1), investigation of their possible effect on the biomarkers revealed that only the CAT:FXIa was influenced by the presence of anticoagulants. Logistic regression models revealed a good discriminatory value for FXI:c and FXIa:α1AT (AUC=0.64 [0.6/0.69] and AUC=0.67 [0.62/0.71], respectively) to distinguish VTE from controls, whereas the other biomarkers were not able to distinguish between groups. The outcome recurrent VTE or death could be predicted by the inhibitory complexes, but not by the FXI(a) levels (Figure 1). Only the FXIa:α1AT complexes were able to both detect the presence of VTE (OR per SD [95%CI]: 1.28 [1.01-1.63], p=0.04) and predict recurrent VTE or death (HR per SD [95%CI]: 1.40 [1.2-1.62], p<0.0001).In summary, acute VTE is associated with both elevated FXI:c levels and increased activation of FXI and plasma prekallikrein, the latter specifically indicating contact activation. The generation of FXIa during acute VTE and its association with recurrent VTE suggests an important risk contribution of FXI activation. This study has added evidence favouring the utility of FXIa inhibition in the setting of acute VTE. [Display omitted] DisclosuresKnoeck: Bayer AG: Consultancy. ten Cate: Bayer AG: Other; Pfizer: Other; LEO Pharma: Other; Gideon Pharmaceuticals: Other; Alveron Pharma: Other. Wild: Bayer AG: Other, Research Funding; Boehringer Ingelheim: Other, Research Funding; Novartis Pharma: Other, Research Funding; Sanofi-Aventis: Other, Research Funding; Astra Zeneca: Other, Research Funding; Daiichi Sankyo Europe: Other, Research Funding.

Severe plasma prekallikrein deficiency: Clinical characteristics, novel KLKB1 mutations, and estimated prevalence

BackgroundSevere plasma prekallikrein (PK) deficiency is an autosomal‐recessive defect characterized by isolated activated partial thromboplastin time prolongation. To date, no comprehensive methodologically firm analysis has investigated the diagnostic, clinical, and genetic characteristics of PK deficiency, and its prevalence remains unknown. Patients/MethodsWe described new families with PK deficiency, retrieved clinical and laboratory information of cases systematically searched in the (gray) literature, and collected blood of these cases for complementary analyses. The Genome Aggregation Database (gnomAD) and the population‐based Gutenberg Health Study served to study the prevalence of mutations and relevant genetic variants. ResultsWe assembled a cohort of 111 cases from 89 families and performed new genetic analyses in eight families (three unpublished). We identified new KLKB1 mutations, excluded the pathogenicity of some of the previously described ones, and estimated a prevalence of severe PK deficiency of 1/155 668 overall and 1/4725 among Africans. One individual reported with PK deficiency had, in fact, congenital kininogen deficiency associated with decreased PK activity. One quarter of individuals had factor XII clotting activity below the reference range. Four major bleeding events were described in 96 individuals, of which 3 were provoked, for a prevalence of 4% and an annualized rate of 0.1%. The prevalence of cardiovascular events was 15% (6% <40 years; 21% 40‐65 years; 33% >65 years) for an annualized rate of 0.4%. ConclusionsWe characterized the genetic background of severe PK deficiency, critically appraised mutations, and provided prevalence estimates. Our data on laboratory characteristics and clinical course of severe PK deficiency may have clinical implications.

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.

Plasma kallikrein-kinin system contributes to peripheral inflammation in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a chronic disease, characterized by severe and refractory seizures, triggered in the hippocampus and/or amygdala, disrupting the blood-brain barrier. This disruption can sustain, or aggravate, the epileptic condition. The aim of this study was to evaluate the activation of the kallikrein-kinin system in patients with TLE, as it relates to the maintenance of blood-brain barrier. Human hippocampal sclerotic tissues removed after surgery for seizure control, plasma, and serum were used in the following assays: immunostaining for white blood cells in the TLE hippocampus, C-reactive protein in serum, quantification of plasma kallikrein (PKal) and cathepsin B (CatB) activity in serum and plasma, quantification of C1-inhibitor, analysis of high-molecular-weight kininogen (H-kininogen) fragments, and activation of plasma prekallikrein for comparison with healthy controls. Infiltration of white blood cells in the sclerotic hippocampus and a significant increase in the neutrophil/lymphocyte ratio in the blood of TLE patients were observed. High levels of C-reactive protein (TLE=1.4±0.3µg/mL), PKal (TLE=5.4±0.4U/mL), and CatB (TLE=4.9±0.4U/mL) were also evident in the serum of TLE patients comparing to controls. A strong linear correlation was observed between active CatB and PKal in the serum of TLE patients (r=0.88). High levels of cleaved H-kininogen and free PKal, and low levels of C1-inhibitor (TLE=188 ± 12µg/mL) were observed in the serum of TLE patients. Our data demonstrated that the plasma kallikrein-kinin system is activated in patients with TLE. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans.

Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells) both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

Plasma kallikrein mediates brain hemorrhage and edema caused by tissue plasminogen activator therapy in mice after stroke

AbstractThrombolytic therapy using tissue plasminogen activator (tPA) in acute stroke is associated with increased risks of cerebral hemorrhagic transformation and angioedema. Although plasma kallikrein (PKal) has been implicated in contributing to both hematoma expansion and thrombosis in stroke, its role in the complications associated with the therapeutic use of tPA in stroke is not yet available. We investigated the effects of tPA on plasma prekallikrein (PPK) activation and the role of PKal on cerebral outcomes in a murine thrombotic stroke model treated with tPA. We show that tPA increases PKal activity in vitro in both murine and human plasma, via a factor XII (FXII)–dependent mechanism. Intravenous administration of tPA increased circulating PKal activity in mice. In mice with thrombotic occlusion of the middle cerebral artery, tPA administration increased brain hemorrhage transformation, infarct volume, and edema. These adverse effects of tPA were ameliorated in PPK (Klkb1)–deficient and FXII-deficient mice and in wild-type (WT) mice pretreated with a PKal inhibitor prior to tPA. tPA-induced brain hemisphere reperfusion after photothrombolic middle cerebral artery occlusion was increased in Klkb1−/− mice compared with WT mice. In addition, PKal inhibition reduced matrix metalloproteinase-9 activity in brain following stroke and tPA therapy. These data demonstrate that tPA activates PPK in plasma and PKal inhibition reduces cerebral complications associated with tPA-mediated thrombolysis in stroke.

Plasma Prekallikrein Is Associated With Carotid Intima-Media Thickness in Type 1 Diabetes.

The hypothesis that plasma prekallikrein (PK) is a risk factor for the development of vascular complications was assessed in a study using the Diabetes Control and Complications Trial (DCCT)/Epidemiology and Diabetes Interventions and Complications (EDIC) cohort of subjects with type 1 diabetes. The circulating levels of plasma PK activity were measured in the plasma of 636 subjects with type 1 diabetes (EDIC years 3-5). Common and internal carotid intima-media thickness (IMT) were measured by B-mode ultrasonography in EDIC years 1 and 6. Plasma PK levels were positively and significantly associated with BMI, hemoglobin A1c, systolic blood pressure, total cholesterol, LDL cholesterol, and triglycerides but not with age, sex, duration of diabetes, or HDL cholesterol. Univariate and multivariable statistical models after controlling for other risk factors consistently demonstrated a positive association between plasma PK and progression of internal carotid IMT. Multivariate analysis using a general linear model showed plasma PK to be significantly associated with progression of both internal and combined IMT (Wilks Λ P value of 0.005). In addition, the mean internal carotid IMT levels were higher in subjects with plasma PK levels in the highest 10th percentile compared with subjects with plasma PK levels in the lower 10th percentile (P = 0.048). These novel findings implicate plasma PK as a risk factor for vascular disease in type 1 diabetes.

Tea Inhibits Hageman Factor

Plasma prekallikrein (PK)activation exerts both physiological and pathological effects within the cardiovascular system and CNS. Activated PK (kallikrein) controls cytokine release from human mononuclear cells, and controls both the intrinsic blood coagulation and the alternative complement cascades. The uncontrolled activation of these cascades results in the development of pathological pain and angioedema. PK is involved with the repair processes that replace injured/damaged tissue. Under pathological conditions, the activation of PK is triggered to be excessively robust. Kallikrein levels are indicated to be associated with hereditary angioedema and myocardial infarction, whereby kallikrein amplifies the generation of activated factor XII (FXIIa) and bradykinin (BK). We hypothesized that kallikrein inhibitors represent a novel class of drugs for curbing inflammation with a broad spectrum of activity. We sought to test the anti‐inflammatory effects of medicinal plants, known for their anti‐inflammatory properties in a system of BK‐forming capacity in endothelial cells and amidolytic kallikrein/FXIIa assay method. The water extracts of tea (Camellia sinensis) inhibited kallikrein and FXIIa with IC50s of 200 and 27 mg/mL. 50mg/mL crude water extracts did not influence cell viability. The extracts inhibited FXIIa ‐induced cell migration. Bioassay guided fractionation was performed on the water extracts to isolate the active compounds. Known polyphenols were ineffective in inhibiting kallikrein and FXIIa. The purified fraction displayed FXIIa inhibition of 88 ±1.5% at a concentration of 700 mg/mL. The bioactive composition comprising the aqueous extract and active fraction of tea may be used for the purpose of reducing FXIIa‐dependent inflammation and thrombosis.

Prolylcarboxypeptidase independently activates plasma prekallikrein (fletcher factor).

Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines and hormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg(9) bradykinin. It does so by cleaving a C-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identify PRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of the human PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acid sequence. Using structural and functional studies, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCP peptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning the opening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelial cells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro 637 (P(637)) and Ala 638 (A(638)). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codon is activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PK activation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is not identical to that of FXIIa.

Angiotensin II type 2 receptor-stimulated activation of plasma prekallikrein and bradykinin release: role of SHP-1

ANG II type 2 receptors (AT(2)R) elicit cardioprotective effects in part by stimulating the release of kinins; however, the mechanism(s) responsible have not been fully explored. We demonstrated previously that overexpression of AT(2)R increased expression of prolylcarboxypeptidase (PRCP; a plasma prekallikrein activator) and release of bradykinin by mouse coronary artery endothelial cells (ECs). In the present study we hypothesized that the AT(2)R-stimulated increase in PRCP is mediated by the tyrosine phosphatase SHP-1, which in turn activates the PRCP-dependent prekallikrein-kallikrein pathway and releases bradykinin. We found that activation of AT(2)R using the specific agonist CGP42112A increased SHP-1 activity in ECs, which was blocked by the AT(2)R antagonist PD123319. Activation of AT(2)R also enhanced conversion of plasma prekallikrein to kallikrein, and this effect was blunted by a small interfering RNA (siRNA) to SHP-1 and abolished by the tyrosine phosphatase inhibitor sodium orthovanadate. Treating cells with a siRNA to PRCP also blunted AT(2)R-stimulated prekallikrein activation and bradykinin release. Furthermore, blocking plasma kallikrein with soybean trypsin inhibitor (SBTI) abolished AT(2)R-stimulated bradykinin release. These findings support our hypothesis that stimulation of AT(2)R activates a PRCP-dependent plasma prekallikrein pathway, releasing bradykinin. Activation of SHP-1 may also play an important role in AT(2)R-induced PRCP activation.

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn+2, HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn2+. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Role of Prolylcarboxypeptidase in Angiotensin II Type 2 Receptor–Mediated Bradykinin Release in Mouse Coronary Artery Endothelial Cells

Activation of angiotensin II type 2 receptors (AT(2)R) causes the release of kinins, which have beneficial effects on the cardiovascular system. However, it is not clear how AT(2)R interact with the kallikrein-kinin system to generate kinins. Prolylcarboxypeptidase is an endothelial membrane-bound plasma prekallikrein activator that converts plasma prekallikrein to kallikrein, leading to generation of bradykinin from high-molecular-weight kininogen. We hypothesized that AT(2)R-induced bradykinin release is at least in part mediated by activation of prolylcarboxypeptidase. Cultures of mouse coronary artery endothelial cells were transfected with an adenoviral vector containing the AT(2)R gene (Ad-AT(2)R) or green fluorescent protein only (Ad-GFP) as control. We found that overexpression of AT(2)R increased prolylcarboxypeptidase mRNA by 1.7-fold and protein 2.5-fold compared with Ad-GFP controls. AT(2)R overexpression had no effect on angiotensin II type 1 receptor mRNA. Bradykinin release was increased 2.2-fold in AT(2)R-transfected cells. Activation of AT(2)R by CGP42112A, a specific AT(2)R agonist, increased bradykinin further in AT(2)R-transfected cells. These effects were diminished or abolished by AT(2)R blockade or a plasma kallikrein inhibitor. Furthermore, blocking prolylcarboxypeptidase with a small interfering RNA partially but significantly reduced bradykinin release by transfected AT(2)R cells either at the basal condition or when stimulated by the AT(2)R agonist CGP42112A. These findings suggest that overexpression of AT(2)R in mouse coronary artery endothelial cells increases expression of prolylcarboxypeptidase, which may contribute to kinin release.

Activation of the plasma kallikrein-kinin system on human lung epithelial cells

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.

Plasma prekallikrein levels are positively associated with circulating lipid levels and the metabolic syndrome in children

Plasma prekallikrein (PK) has been shown to be associated with cardiovascular disease (CVD) and its risk factors, but these associations have not been investigated in children. The present study examined PK activity in relation to well-established cardiovascular risk factors in a cohort of children aged 9-11 years (N=97). We found a significant and positive association between PK and fasting levels of total cholesterol (p<0.01), non-high-density lipoprotein cholesterol (p<0.01), and triglycerides (p<0.001). In addition, there was a significant association between PK activity and the metabolic syndrome, a clustering of risk factors considered to have an impact on atherosclerosis and CVD mortality. Finally, we found that children with a family history of CVD had significantly elevated PK activity. These novel findings warrant further investigations into the relationship between circulating PK levels and CVD risk factors because PK may be involved in the progression of the disease state.

高齢になってから診断された先天性プレカリクレイン欠乏症の1症例

The proband is a 69-year-old woman with purpura and subcutaneous hematoma.We investigated this patient with prekallikrein (PK) deficiency, using both standard coagulation study and molecular genetic analysis of the PK gene. In a coagulation study, the prothrombin time (PT) was normal but the activated partial thromboplastin time (APTT) was prolonged. Preincubation of normal plasma with APTT reagent caused shortening of abnormal clotting time. Plasma PK activity was <1%. Her parents were cousins. Molecular genetic analysis showed a homozygous Gly401Glu substitution in exon 11 in the PK gene. This mutation has already been reported in a Japanese patient as PK Tokushima. Gly401 is positioned in PK light chain, which encodes the serine protease domain. The disulfide binding is formed between Cys400 and Cys416, thus Gly401 is located next to His415, which is one of the activation peptides and is important in supporting the correct conformation of proteins. Therefore, we suggest that this mutation may prevent formation of disulfide binding and reduce enzyme activity. In conclusion, in the elderly case with prolonged APTT, we should consider the contact factor deficiency and determine PK activity apart from the abnormality of coagulation factor VIII, IX, XI and XII activities.

The elusive physiologic role of Factor XII

Physiologic hemostasis upon injury involves many plasma proteins in a well-regulated cascade of proteolytic reactions to form a clot. Deficiency of blood coagulation Factors VIII, IX, or XI is associated with hemophilia. Factor XII (FXII) autoactivates by contact with a variety of artificial or biologic negatively charged surfaces (contact activation), resulting in blood coagulation and activation of the inflammatory kallikrein-kinin and complement systems. However, surprisingly, individuals deficient in FXII rarely suffer from bleeding disorders. Most biologic surfaces that activate FXII become expressed in disease states. Investigators have long searched for physiologic activators of FXII and its role in vivo. In this issue of the JCI, Maas et al. show that misfolded protein aggregates produced during systemic amyloidosis allow for plasma FXIIa and prekallikrein activation and increased formation of kallikrein-C1 inhibitor complexes, without Factor XIa activation and coagulation (see the related article beginning on page 3208). This study describes a novel biologic surface for FXII activation and activity, which initiates inflammatory events independent of hemostasis.

Changes in plasma prekallikrein activity, blood pressure and left ventricular thickness in hypertensive and normotensive diabetic rats

The present study examined plasma prekallikrein (PK) activity, left ventricular wall thickness (LVWT) and mean arterial blood pressure (BP) in diabetic and nondiabetic spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The mean arterial BP was significantly raised (p < 0.01) in diabetic WKY rats and diabetic SHR when compared with their respective controls. The LVWT was significantly (p < 0.01) increased in diabetic WKY rats when compared with that in the control WKY rats. There was also an increase (p < 0.01) in the LVWT in the SHR and diabetic SHR when compared with that in the control WKY rats and diabetic WKY rats, respectively. The plasma PK activities were higher (p < 0.01) in diabetic WKY rats, SHR, and diabetic SHR against the mean value of control WKY rats. In this animal study, these findings suggest for the first time that higher plasma PK activity may serve as an indicator for predicting high BP and left ventricular hypertrophy in diabetic and hypertensive conditions. The possible significance of these observations is discussed.

Over-Expression of Prolylcarboxypeptidase Enhances Plasma Prekallikrein Activation on Chinese Hamster Ovary Cell Membranes.

Recent investigations indicate that there is a physiologic plasma prekallikrein (PK) activator on the membrane of endothelial cells (HUVEC). PK complexes with high molecular weight kininogen (HK) and HK is PK's receptor on HUVEC. When assembled on HUVEC, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP has been shown to be a PK activator when isolated from HUVEC (JBC 277:17962, 2002) and produced as a recombinant protein (Blood 103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary cells (CHO). CHO were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (EGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation are 2–3-fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells parallels the increase in PRCP antigen expression on the transfected cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells is blocked by siRNA to the translation initiation site of PRCP, anti-PRCP antibody, and Z-Pro-Pro-aldehyde dimethyl acetate, with IC50 of the last 2 inhibitors of 0.01 and 7.0 mM respectively. Cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry indicate that its over-expression results in its placement on the CHO cell plasma membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation. The endothelial cell membrane expression of PRCP resulting in PK activation contributes to the constitutive level of bradykinin in the intravascular compartment to regulate blood pressure homeostasis.