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 proteaseshowever, 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.Fundacdo de Amparo a Pesquisa do Estado de Sao Paulo, FAPESPConselho Nacional de Desenvolvimento Cientifico e TecnologicoCAPESUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilFAPESP: 15/03964-6FAPESP: 16/14827-2CNPqCAPESWeb of Scienc

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 degrees 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 degrees 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilUniv Bandeirante São Paulo, Biomat & Biotechnol Res Grp, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilFAPESP: FAPESP 09/51319-1FAPESP: 09/13160-0FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6CNPq: CNPq 472403/2007-9Web of Scienc

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 anti-pain-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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biomat, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biotecnol, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilCNPq: CNPq 472403/2007-9FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6Web of Scienc

Factor XII does not initiate prekallikrein activation on endothelial cells

It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEV). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer's carrier protein, Maximal PK activation required the addition of 250 mu M or 10 mu M Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 mu M. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather. initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively. soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolystic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma. maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of contact activation and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.Univ Michigan, Dept Internal Med, Div Hematol & Oncol, Ann Arbor, MI 48109 USAUniv Copenhagen, Panum Inst, Dept Med Biochem & Genet, DK-2200 Copenhagen, DenmarkUNIFESP, Escola Paulista Med, Dept Biochem, Sao Paulo, BrazilUNIFESP, Escola Paulista Med, Dept Biochem, Sao Paulo, BrazilWeb of Scienc

Endocytosis of fluorescent Dylight 659-bradykinin free H-kininogen by CHO cells.

<p>CHO-K1 and CHO-745 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Green in Ham F-12 serum-free medium containing 50.0 μM Zn²⁺ for 20 min at 37°C and analyzed by confocal fluorescence microscopy. Dylight 659-bradykinin free H-kininogen (200 nM, red) endocytosis and intracellular localization are indicated by the colocalization with acidic vesicles labeled with LT Green. Normal endocytosis by CHO-K1: (A) LT Green; (B) Dylight 659- bradykinin free H-kininogen; (C) merged images and diphasic contrast. Normal endocytosis by CHO-745: (D) LT Green; (F) Dylight 659- bradykinin free H-kininogen; (G) merged images and diphasic contrast.</p

Hydrolysis of cell-associated H-kininogen by mature plasma kallikrein.

<p>H-kininogen (100 nM) in the presence of incubation buffer was incubated for 1 h or 30 min, respectively, with ECV304, CHO-K1 and CHO-745 cells at 37°C. After removing H-kininogen by aspiration, prekallikrein (100 nM) was added for 15 min, 30 min, 1 h or 2 h at 37°C. At the end of each time point, the incubation buffer was collected, and both the incubation buffer and the cell samples were treated with reducing sample buffer. Immunoblotting of the cells was performed with the anti-H-kininogen antibody. (A) ECV304: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (B) CHO-K1: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (C) CHO-745: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg)</p

Endocytosis of biotin-prekallikrein by CHO-745 cells.

<p>CHO-745 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. Then the cells were treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by CHO-745 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by CHO-745 cells at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).</p

Prekallikrein structure over time after interaction with CHO-K1 cells.

<p>H-kininogen (100 nM) was incubated for 30 min with CHO-K1, and after removing H-kininogen by aspiration, prekallikrein (100 nM) was added for 15 min, 30 min, 1 h or 2 h at 37°C. Alternatively, prekallikrein was added to cells at different time points in the absence of assembled H-kininogen. At the end of each time point, the incubation buffer was collected, and both the incubation buffer and the cell samples were treated with reducing sample buffer. Immunoblotting was performed with anti-plasma kallikrein U691.10 for detecting plasma kallikrein fragments containing the sequence C₃₆₄TTKTSTR₃₇₁. (A) Cells with H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (B) Cells without H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg). (C) Incubation buffer with H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (D) Incubation buffer without H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg)</p