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

Lorenz Jenny,József Dobó,Verena Schroeder,Péter Gál

doi:10.1371/journal.pone.0144633

Abstract

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.

Highlights

The complement system is a central part of the innate immune system and has a crucial function in the clearance of pathogens from the circulation
We have shown that mannan-binding lectin-associated serine protease (MASP)-1 is able to induce clot formation by an alternative prothrombin activation and identified R155, R271, and R393 as the putative cleavage sites [8]
In order to test the functionality of the three prothrombin mutants in terms of their clot formation ability, they were incubated with fibrinogen (2 mg/ml) and activated factor X (FXa) (5 μg/ml), measured on a thrombelastograph (Table 1)

Summary

Introduction

The complement system is a central part of the innate immune system and has a crucial function in the clearance of pathogens from the circulation. The lectin pathway is one of three possible ways of the complement system to encounter threats due to infection. It recognises its targets by binding of mannan-binding lectin (MBL) or ficolins to specific patterns on foreign and/or altered surfaces [1]. This leads to the activation of the MBL-associated serine proteases (MASPs) MASP-1, MASP-2, and MASP-3 which produce C3 convertase via C2 and C4 cleavage (reviewed in [2]). Beside its central role in the lectin pathway, MASP-1 has been shown to interact with the coagulation system [4]. The serine protease domain of MASP-1 is more closely related to PLOS ONE | DOI:10.1371/journal.pone.0144633 December 8, 2015

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