The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by ...antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.
Tissue-on-a-chip technologies are more and more important in the investigation of cellular function and in the development of novel drugs by allowing the direct screening of substances on human ...cells. Constituting the inner lining of vessel walls, endothelial cells are the key players in various physiological processes, moreover, they are the first to be exposed to most drugs currently used. However, to date, there is still no appropriate technology for the label-free, real-time and high-throughput monitoring of endothelial function. To this end, we developed an optical biosensor-based endothelial label-free biochip (EnLaB) assay that meets all the above requirements. Using our EnLaB platform, we screened a set of plasma serine proteases as possible endothelial cell activators, and first identified the endothelial cell activating function of three important serine proteases - namely kallikrein, C1r and mannan-binding lectin-associated serine-protease 2 (MASP-2) - and verified these results in well-established functional assays. EnLaB proved to be an effective tool for revealing novel cellular mechanisms as well as for the high-throughput screening of various compounds on endothelial cells.
The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated ...serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.
The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation-fibrinolytic system. Among the three main activation routes of complement, the lectin ...pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary ("moonlighting") functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components.
Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the ...complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.
We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.
We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.
The complement and coagulation systems closely interact with each other. These interactions are believed to contribute to the proinflammatory and prothrombotic environment involved in the development ...of thrombotic complications in many diseases. Complement MASP-1 (mannan-binding lectin-associated serine protease-1) activates coagulation factors and promotes clot formation. However, this was mainly shown in purified or plasma-based static systems. Here we describe the role of MASP-1 and complement activation in fibrin clot formation in a microvascular, whole blood flow model. This microfluidic system simulates blood flow through microvessels at physiological flow and shear rates and represents the closest model system to human physiology so far. It features parallel microchannels cultured with endothelial cells in a transparent microfluidic chip allowing real-time evaluation of clot formation by confocal microscopy. To test their effects on clot formation, we added the following activators or inhibitors (individually or in combination) to whole blood and performed perfusion experiments: rMASP-1cf (recombinant active form of MASP-1), complement activator zymosan, selective MASP-1 inhibitor SGMI-1 (based on the Schistocerca gregaria protease inhibitor scaffold), classical pathway inhibitor rSALO (recombinant salivary anti-complement from Lutzomyia longipalpis). Addition of rMASP-1cf resulted in accelerated fibrin clot formation while addition of SGMI-1 delayed it. Complement activation by zymosan led to increased clot formation and this effect was partially reversed by addition of rSALO and almost abolished in combination with SGMI-1. We show for the first time a strong influence of MASP-1, complement activation and pathway-specific inhibition on coagulation in a microvascular flow system that is closest to human physiology, further underpinning the in vivo relevance of coagulation and complement interactions.
Endothelial wound-healing processes are fundamental for the maintenance and restoration of the circulatory system and are greatly affected by the factors present in the blood. We have previously ...shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) induces the proinflammatory activation of endothelial cells and is able to cooperate with other proinflammatory activators. Our aim was to investigate the combined effect of mechanical wounding and MASP-1 on endothelial cells. Transcriptomic analysis showed that MASP-1 alters the expression of wound-healing-related and angiogenesis-related genes. Both wounding and MASP-1 induced Ca
mobilization when applied individually. However, MASP-1-induced Ca
mobilization was inhibited when the treatment was preceded by wounding. Mechanical wounding promoted CREB phosphorylation, and the presence of MASP-1 enhanced this effect. Wounding induced ICAM-1 and VCAM-1 expression on endothelial cells, and MASP-1 pretreatment further increased VCAM-1 levels. MASP-1 played a role in the subsequent stages of angiogenesis, facilitating the breakdown of the endothelial capillary network on Matrigel
. Our findings extend our general understanding of endothelial wound healing and highlight the importance of complement MASP-1 activation in wound-healing processes.
Abstract Both hypoxia and the complement lectin pathway (CLP) are involved in atherosclerosis and atherosclerosis-related stroke and acute myocardial infarction (AMI). We have previously shown that ...mannose-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of CLP, induces an inflammatory phenotype of endothelial cells (ECs) by cleaving protease activated receptors (PARs). In the absence of data, we aimed to investigate whether hypoxia and MASP-1 interact at the level of ECs, to better understand their role in atherosclerosis-related diseases. Hypoxia attenuated the wound healing ability of ECs, increased ICAM-1 and decreased ICAM-2 expression and upregulated PAR2 gene expression. Hypoxia and MASP-1 increased GROα and IL-8 production, and endothelial permeability without potentiating each other’s effects, whereas they cooperatively disrupted vascular network integrity, activated the Ca 2+ , CREB and NFκB signaling pathways, and upregulated the expression of E-selectin, a crucial adhesion molecule in neutrophil homing. VCAM-1 expression was not influenced either by hypoxia, or by MASP-1. In summary, hypoxia potentiates the effect of MASP-1 on ECs, at least partially by increasing PAR expression, resulting in interaction at several levels, which may altogether exacerbate stroke and AMI progression. Our findings suggest that MASP-1 is a potential drug target in the acute phase of atherosclerosis-related diseases.
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous ...published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.
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