Final Activity Report Summary - PROLECT (A link between the complement and coagulation cascades: Identification of physiological substrates of MASP-1 using proteomics.)
Innate immunity - the non-specific and immediate response to invading microbes - is a crucial and multi-faceted first line of defence. Two processes in blood involved in innate immunity that play particularly important roles are the coagulation and complement systems. The former restricts blood loss and may immobilise microbes, thereby preventing them from gaining access to vulnerable sites in the body. In contrast, the complement system consists of a series of protein components that, when sequentially activated, result in the destruction of a microbe present in the blood. Activation is known to involve enzymes termed proteases, which recognise other specific proteins and split them into fragments known as peptides.
Preliminary work by the applicant showed that activation of particular subset of complement components, collectively known as the lectin pathway, triggers the formation of a clot in blood plasma. Furthermore, one of the enzymes involved in activating the lectin pathway, a protease known as MASP-1, had an activity that resembled that of a crucial protease termed thrombin that acts in the blood clotting pathway. The present project sought to obtain more information about these links between the coagulation and complement systems and, in particular, to define additional blood proteins that were targets for MASP-1. To achieve this aim, a new method was developed to detect proteins that are fragmented by MASP-1. When the method was tested with another well-defined protease, it readily revealed the target proteins in blood serum that were fragmented by the enzyme. In contrast, it has not yet been possible to unambiguously detect target proteins for MASP-1. However, experiments are on-going to pursue this goal. In other work, chemically synthesized peptides, that permit the sensitive measurement of the activity of MASP-1, have been produced. These have been also used to demonstrate that a potent inhibitor of the clotting enzyme thrombin also inhibits MASP-1 when it is present in a specific multi-protein complex.
The ability to selective block MASP-1 activity should greatly help efforts to define its contribution to both the activation of the complement system and the clotting process under different conditions. Further studies established that the clot produced by MASP-1 was very similar to that generated by thrombin in normal human plasma. Intriguing, MASP-1 activity during clot formation released two peptides, known as fibrinopeptides A and B, that may play a role in early activation of other parts of the innate immune system.
Preliminary work by the applicant showed that activation of particular subset of complement components, collectively known as the lectin pathway, triggers the formation of a clot in blood plasma. Furthermore, one of the enzymes involved in activating the lectin pathway, a protease known as MASP-1, had an activity that resembled that of a crucial protease termed thrombin that acts in the blood clotting pathway. The present project sought to obtain more information about these links between the coagulation and complement systems and, in particular, to define additional blood proteins that were targets for MASP-1. To achieve this aim, a new method was developed to detect proteins that are fragmented by MASP-1. When the method was tested with another well-defined protease, it readily revealed the target proteins in blood serum that were fragmented by the enzyme. In contrast, it has not yet been possible to unambiguously detect target proteins for MASP-1. However, experiments are on-going to pursue this goal. In other work, chemically synthesized peptides, that permit the sensitive measurement of the activity of MASP-1, have been produced. These have been also used to demonstrate that a potent inhibitor of the clotting enzyme thrombin also inhibits MASP-1 when it is present in a specific multi-protein complex.
The ability to selective block MASP-1 activity should greatly help efforts to define its contribution to both the activation of the complement system and the clotting process under different conditions. Further studies established that the clot produced by MASP-1 was very similar to that generated by thrombin in normal human plasma. Intriguing, MASP-1 activity during clot formation released two peptides, known as fibrinopeptides A and B, that may play a role in early activation of other parts of the innate immune system.