The coagulation and complement pathways simultaneously promote homeostasis in response to injury but cause tissue damage when unregulated. Mechanisms by which they cooperate are poorly understood. To delineate their interactions, we studied the effects of thrombin and C5 convertase on C5 in purified and plasmabased systems, measuring release of the anaphylatoxin C5a, and generation of C5b, the initial component of the lytic membrane attack complex. Thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b. However, thrombin efficiently cleaved C5 at a newly identified, highly conserved R947 site, generating previously undescribed intermediates C5 T and C5b T . Tissue factor-induced clotting of plasma led to proteolysis of C5 at a thrombin-sensitive site corresponding to R947 and not R751. Combined treatment of C5 with thrombin and C5 convertase yielded C5a and C5b T , the latter forming a C5b T -9 membrane attack complex with significantly more lytic activity than with C5b-9. Our findings provide a new paradigm for complement activation, in which thrombin and C5 convertase are invariant partners, enhancing the terminal pathway via the generation of newly uncovered C5 intermediates. Delineating the molecular links between coagulation and complement will provide new therapeutic targets for diseases associated with excess fibrin deposition and complement activation. (Blood. 2012;120(8):1717-1725)

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12(-/-) mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.

The complement system plays an integral part of a host's innate immunity, and its activation is highly dependent on the chemistry and structure of a "foreign" target surface. We determined that the conformational state of glycopolymer chains, defined by the grafting density (chains/nm(2)), on the nanoparticle (NP) surface acts as a "molecular switch" for complement activation and amplification, and the protein corona on the NP surface dictates this process. A grafting density threshold was determined, below which minimal complement activation was observed and above which substantial complement activation was detected. The glycopolymer-grafted NPs activated complement via the alternative pathway. The chemical structure of pendent sugar units on the grafted polymer was also an important determinant for complement activation. NPs grafted with glucose-containing polymer activated complement at a lower grafting density compared to NPs grafted with galactose-containing polymer. Analysis of complement activation products C3a and SC5b-9 followed a similar pattern. Complement activation on the NP surface was independent of particle size or concentration for a given conformational state of grafted polymer. To gain insight into a putative surface-dependent mechanism of complement activation, we determined the nature of adsorbed protein corona on various NPs through quantitative mass spectrometry. Elevated levels of two pro-complement proteins, factors B and C3, present on the NP surface grafted with glycopolymer chains at high grafting density compared to low grafting density surface, may be responsible for its complement activity. Galactose polymer modified NPs adsorbed more of the negative regulator of complement, factor H, than the glucose surface, providing an explanation for its lower level of complement activation.

Blood coagulation involves a series of enzymatic protein complexes that assemble on the surface of anionic phospholipid. To investigate whether apolipoproteins affect coagulation reactions, they were included during the preparation of anionic phospholipid vesicles using a detergent solubilization-dialysis method. Apolipoprotein components of high density lipoproteins, especially apolipoprotein A-I, had a pronounced anticoagulant effect. The anionic phospholipids lost their procoagulant effect when the vesicle preparation method was performed in the presence of apolipoprotein A-I. The anionic phospholipid-apolipoprotein A-I particles were 8 -10 nm in diameter and contained around 60 -80 phospholipid molecules, depending on the phospholipid composition. The phospholipids of these particles were unable to support the activation of prothrombin by factor Xa in the presence of factor Va and unable to support binding of factor Va, whereas binding of prothrombin and factor Xa were efficient. Phospholipid transfer protein was shown to mediate transfer of phospholipids from liposomes to apolipoprotein A-I-containing reconstituted high density lipoprotein. In addition, serum was also shown to neutralize the procoagulant effect of anionic liposomes and to efficiently mediate transfer of phospholipids from liposomes to either apolipoprotein A-I-or apolipoprotein B-containing particles. In conclusion, apolipoprotein A-I was found to neutralize the procoagulant properties of anionic phospholipids by arranging the phospholipids in surface areas that are too small to accommodate the prothrombinase complex. This anionic phospholipid scavenger function may be an important mechanism to control the exposure of such phospholipids to circulating blood and thereby prevent inappropriate stimulation of blood coagulation.

Key Points Polyphosphate suppresses complement via the terminal pathway by destabilizing C5b,6, thereby reducing the lytic capacity of the membrane attack complex. Polyphosphate, a novel negative regulator of complement, bridges coagulation and complement and is a potential therapeutic target.