MASP-1 and MASP-2 Do Not Activate Pro–Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors

Abstract

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.

Deconstructing the Lectin Pathway in the Pathogenesis of Experimental Inflammatory Arthritis: Essential Role of the Lectin Ficolin B and Mannose-Binding Protein–Associated Serine Protease 2

“…Numerous studies have shown that MASP-1 activates MASP-2 as an essential step in the activation of the LP (57–59). Additionally, other studies have shown that MASP-1 can activate MASP-3 (60, 61). Together, these data suggest that MASP-3 is the primary activator of the AP via cleavage of pro-Df while MASP-1 is the primary activator of the LP via activation of MASP-2.…”

Mannan-Binding Lectin–Associated Serine Protease 1/3 Cleavage of Pro–Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference–Mediated Gene Silencing

“…Therefore the AP activation seen in mice MASP-1/3 −/− /fH −/− could be due to the formation of CVF-fB convertase in the presence of proDf and if so then these observations will be consistent regarding the activation of AP in fD −/− mice (39). Orozslan and colleagues recently published data in support of MASP-3 being the primary family member involved in the cleavage of proDf (40). This study has also provided evidence as to why only MASP-3 cleaves proDf as it is resistant to C1inhibitor and antithrombin, in contrast to MASP-1 or MASP-2.…”

“…However, MASP-3 can be produced by other tissues (40). Nonetheless the cleavage of proDf into Df by MASP-1/3 takes place in the extracellular space and circulation, as it is evident from our studies related to transplantation of liver under the kidney capsule.…”

“…Recently it was shown the levels of M-ficolin, MASP-1, MASP-2 and MASP-3 were significantly higher in human plasma than in the synovial fluid, except for MASP-3 indicating MASP-3 may be produced locally in the joint from patients with juvenile idiopathic arthritis (JIA)(42). There are reports that MASP-3 could cleave proDf into Df and also to fB (22, 24, 40); therefore, MASP-3 itself can form the AP convertase. MASP-3 can also by produced at different sites and then transported locally (42, 43) such as in the knee joints.…”

The emerging roles of mannose‐binding lectin‐associated serine proteases (MASPs) in the lectin pathway of complement and beyond

“…At first we determined the cleavage rates of pro‐FD by all three MASPs testing both the activated and proenzyme forms, and by thrombin. All active MASPs and thrombin were found to be efficient pro‐FD activators in vitro, on the other hand the proenzymes had no significant activity . These results imply that pro‐FD can probably be activated by various active proteases with P1 Arg specificity in vitro, and the real question is which one of these enzymes (or another enzyme) is in the active form when pro‐FD activation takes place.…”