Abstract
IntroductionThe 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.MethodsWe 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.ResultsAt 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.ConclusionThe 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.
Highlights
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
The plasma kallikrein-kinin system proteins have been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, coagulopathy, angiogenesis, epithelial cell apoptosis, adipocyte differentiation, and stromal remodeling and the interaction with cell surface could be a mechanism for controlling their activities [7]
We showed that human plasma prekallikrein, the zymogen form of plasma kallikrein, and reversibly binds to human umbilical vein endothelial cells (HUVECs) in the presence or absence of exogenously applied Hkininogen
Summary
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. The plasma kallikrein/kinin system, which comprises the contact system proteins plasma prekallikrein, high molecular weight kininogen (H-kininogen) and Factor (F)XII is a physiologic mediator of vascular biology and inflammatory reactions. Human plasma kallikrein is a protease that was first found to affect hemostasis by amplifying FXII activation and inflammation by Hkininogen hydrolysis and bradykinin release. The plasma kallikrein-kinin system proteins have been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, coagulopathy, angiogenesis, epithelial cell apoptosis, adipocyte differentiation, and stromal remodeling and the interaction with cell surface could be a mechanism for controlling their activities [7]. Besides regulating VEGF-VEGFR signaling system [15], [16], [17] cell surface proteoglycans can regulate angiogenesis by modulating plasma kallikrein-kinin system activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
