Complement lectin pathway components MBL and MASP-1 promote haemostasis upon vessel injury in a microvascular bleeding model.

Background: Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model. Methods: We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry. Results: Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time. Conclusion: We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system.

Exploring the function of factor XIII free B subunit: Interactions with complement factors and a novel approach to identify potential binding partners.

Background: The factor XIII (FXIII)-B subunit has a critical function as a carrier protein to stabilize FXIII-A in plasma and supply it to its main substrate, fibrinogen. However, the function of the excess free FXIII-B circulating in plasma is still elusive. Objectives: In the present study, we explored potential interactions of free FXIII-B with complement factors and searched for novel binding partners. Methods: We tested for cofactor activity in the degradation of complement C3b and C4b and used ELISA- and surface plasmon resonance-based binding assays to investigate interactions between FXIII-B and complement components. We performed immunoprecipitation and mass spectrometry analysis to identify potential binding partners of free FXIII-B in freshly drawn plasma samples. Results: FXIII-B did not exhibit cofactor activity in the degradation of C3b and C4b similar to factor H and C4b-binding protein, nor did it bind to complement factors to a relevant extent. Identification of proteins potentially binding to free FXIII-B revealed high interindividual variation. We confirmed α2-macroglobulin (α2MG) as a candidate, although direct interactions or functional effects remain to be validated. Conclusions: Our study reveals that free FXIII-B has no direct role in regulating the complement system, despite a structural similarity to major complement regulators. Further studies are needed to validate α2MG as a binding partner and explore potential functional consequences of this binding.

Diabetes affects endothelial cell function and alters fibrin clot formation in a microvascular flow model: A pilot study.

Diabetes is a proinflammatory and prothrombotic condition that increases the risk of vascular complications. The aim of this study was to develop a diabetic microvascular flow model that allows to study the complex interactions between endothelial cells, blood cells and plasma proteins and their effects on clot formation. Primary human cardiac microvascular endothelial cells from donors without diabetes or donors with diabetes (type 1 or type 2) were grown in a microfluidic chip, perfused with non-diabetic or diabetic whole blood, and clot formation was assessed by measuring fibrin deposition in real time by confocal microscopy. Clot formation in non-diabetic whole blood was significantly increased in the presence of endothelial cells from donors with type 2 diabetes compared with cells from donors without diabetes. There was no significant difference in clot formation between non-diabetic and diabetic whole blood. We present for the first time a diabetic microvascular flow model as a new tool to study clot formation as a result of the complex interactions between endothelial cells, blood cells and plasma proteins in a diabetes setting. We show that endothelial cells affect clot formation in whole blood, attributing an important role to the endothelium in the development of atherothrombotic complications.

MASP-1 of the complement system alters fibrinolytic behaviour of blood clots.

BACKGROUND: The lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. METHODS: Rotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. RESULTS: In the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. CONCLUSION: MASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

MASP-1 of the complement system enhances clot formation in a microvascular whole blood flow model.

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.

MASP-1 Induced Clotting--The First Model of Prothrombin Activation by MASP-1.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.

MASP-1 of the complement system promotes clotting via prothrombin activation.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation.

MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBL-associated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling.