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The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding–associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin–associated serine protease (MASP)-1/3(−/−) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

The complement system, an essential part of the innate immune system, can be activated through three distinct routes: the classical, the alternative, and the lectin pathways. The contribution of individual activation pathways to different biological processes can be assessed by using pathway-selective inhibitors. In this paper, we report lectin pathway-specific short peptide inhibitors developed by phage display against mannose-binding lectin-associated serine proteases (MASPs), MASP-1 and MASP-2. On the basis of the selected peptide sequences, two 14-mer peptides, designated as sunflower MASP inhibitor (SFMI)-1 and SFMI-2, were produced and characterized. SFMI-1 inhibits both MASP-1 and MASP-2 with a KI of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a KI of 180 nM. Both peptides block the lectin pathway activation completely while leaving the classical and the alternative routes intact and fully functional, demonstrating that of all complement proteases only MASP-1 and/or MASP-2 are inhibited by these peptides. In a C4 deposition inhibitor assay using preactivated MASP-2, SFMI-2 is 10-fold more effective than SFMI-1 in accordance with the fact that SFMI-2 is a more potent inhibitor of MASP-2. Surprisingly, however, out of the two peptides, SFMI-1 is much more effective in preventing C3 and C4 deposition when normal human serum containing zymogen MASPs is used. This suggests that MASP-1 has a crucial role in the initiation steps of lectin pathway activation most probably by activating MASP-2. Because the lectin pathway has been implicated in several life-threatening pathological states, these inhibitors should be considered as lead compounds toward developing lectin pathway blocking therapeutics.

Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques by inducing inflammation and by functioning as an endogenous danger signal. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. In this study we hypothesized that pattern recognition molecules (PRM) from the lectin pathway bind CC and functions as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC using recombinant proteins, specific inhibitors as well as deficient and normal sera. Binding was assessed by flow cytometry and microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium dependent manner while ficolin-2 binding was calcium independent. No binding was observed for ficolin-1 or ficolin-3. Moreover, we demonstrated that IgM, but not IgG bound to CC and that C1q binding was facilitated by IgM. In conclusion our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.