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 ± 0.3 µg/mL), PKal (TLE = 5.4 ± 0.4 U/mL), and CatB (TLE = 4.9 ± 0.4 U/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 ± 12 µg/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.

Bradykinin release avoids high molecular weight kininogen endocytosis.

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.

The involvement of proteoglycans in the human plasma prekallikrein interaction with the cell surface.

INTRODUCTION: The aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen (H-kininogen), influence this interaction. METHODS: We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type) and CHO-745 (deficient in proteoglycans). Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule. RESULTS: At 37°C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4±0.010 and 1,070.3±0.001 pixels/cell, respectively, for ECV304 and 1,319.1±0.003 and 631.3±0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein colocalized with LysoTracker in the absence and presence of exogenous H-kininogen at levels of 76.0% and 88.5%, respectively, for ECV304 and at levels of 40.7% and 57.0%, respectively, for CHO-K1. After assembly on the cell surface, a plasma kallikrein fragment of 53 kDa was predominant in the incubation buffer of all the cell lines studied, indicating specific proteolysis; plasma kallikrein fragments of 48-44 kDa and 34-32 kDa were also detected in the incubation buffer, indicating non-specific cleavage. Bradykinin free H-kininogen internalization was not detected in CHO-K1 or CHO-745 cells at 37°C. CONCLUSION: The prekallikrein interaction with the cell surface is temperature-dependent and independent of exogenously applied H-kininogen, which results in prekallikrein endocytosis promoted by proteoglycans. Prekallikrein proteolysis/activation is influenced by H-kininogen/glycosaminoglycans assembly and controls plasma kallikrein activity.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

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 Zn(2+). 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.

Involvement of heparan sulfate proteoglycans in cellular uptake of high molecular weight kininogen.

In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5- related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488- HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-beta-cyclodextrin, FCCP and 2-deoxy-D-glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.

Plasma prekallikrein/kallikrein processing by lysosomal cysteine proteases.

Plasma kallikrein plays a role in coagulation, fibrinolysis and inflammation. Cathepsins B and L participate in (patho)physiological processes such as peptide antigen processing, tissue remodeling events, protein turnover in cells, hormone processing and tumor invasion. The present work analyzes the processing of prekallikrein/kallikrein by lysosomal cathepsins. Prekallikrein is not hydrolyzed by catB, and catL generates an inactive fragment of prekallikrein. Both kallikrein chains are hydrolyzed by catL and the light chain is mainly hydrolyzed by catB; kallikrein activity is lower after incubation with catL compared to catB. Our data suggest that the plasma kallikrein/ kinin system can be controlled by cathepsins.

High molecular weight kininogen as substrate for cathepsin B.

We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.