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Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin Foley, Jonathan H.; Walton, Bethany L.; Aleman, Maria M.; O'Byrne, Alice M.; Lei, Victor; Harrasser, Micaela; Foley, Kimberley A.; Wolberg, Alisa Sue; Conway, Edward M. Feb 6, 2016

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Research Papernolemersh Cod Kinn, Uill, 8on, CReceived 3 November 2015Received in revised form 4 February 2016Accepted 5 February 20161. Introduction and nucleosomes, which provide a scaffold for aggregating plateletsand ultimately throm-ruited early in throm-sion or resolution, areEBioMedicine 5 (2016) 175–182Contents lists available at ScienceDirectEBioMej ourna l homepage: wwwpoorly understood.However, C5a, themost potent chemotactic comple-ment activation fragment, is released following proteolytic cleavage ofAbbreviations:MAC, membrane attack complex; VWF, von Willebrand factor; R751,arginine 751; TAT, thrombin antithrombin; IVC, inferior vena cava; VFKck, Val-Phe-Lys-C5 and is considered a critical determinant of neutrophil recruitmentchloromethylketone; PPACK, Phe-Pro-Arg-chloromethylketone; FeCl3, ferric chloride;and C5a. These pathways cooperate to trigger platelet, neutrophil, andmonocyte recruitment and activation (von Bruhl et al., 2012). Thelocally accumulated cells release proteases, reactive oxygen species,other immunemediators, vessel wall remodeling,bus resolution (Wakefield et al., 2008).The mechanisms by which leukocytes are recbus formation and later during thrombus extenThe coagulation system and innate immunity are coordinatelyactivated and highly integrated during venous and arterial thrombusformation and progression (von Bruhl et al., 2012; Engelmann andMassberg, 2013; Fuchs et al., 2012). Vascular endothelial activation ordamage causes release of ultralarge von Willebrand factor (VWF) andP-selectin from Weibel-Palade bodies, and local activation of comple-ment with liberation of anaphylatoxic and chemotactic factors C3aand red blood cells and further promote coagulation and fibrin formation(Fuchs et al., 2012). Several complement factors, including C3, C4, C3a,C5a and factor H are incorporated into the thrombus, where theymodu-late thrombus stability and the inflammatory process (Distelmaier et al.,2009; Howes et al., 2012). The fibrinolytic system and plasmin-mediatedproteolysis are also intimately coupled to the axis of thrombus develop-ment and inflammation by controlling fibrin degradation, activation ofmatrix metalloproteinases, infiltration of monocytes/macrophages andtPA, tissue-type plasminogen activator; NETs, neutropprotease activated receptor 1; MCP1-1, monocyte chinterleukin-8; FDP, fibrin degradation product.⁎ Corresponding author at: Centre for Blood Research, 4University of British Columbia, Vancouver, BC V6T 1Z3, CaE-mail address: ed.conway@ubc.ca (E.M. Conway).http://dx.doi.org/10.1016/j.ebiom.2016.02.0112352-3964/© 2016 The Authors. Published by Elsevier B.VFibrinolysisThrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interac-tions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step,mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediatingC3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels ofthrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombinin C3a/C5a production. However, clot weight strongly correlatedwith C5a, suggesting processes triggered duringthrombosis promote C5a generation. Since thrombosis elicitsfibrinolysis,we hypothesized that plasmin activatesC5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded thatof thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activatoradministration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immuneresponse by liberating C5a and inducing MAC assembly. These new insights may lead to the development ofstrategies to limit thrombus formation and/or enhance resolution.© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).Available online 6 February 2016Keywords:ThrombosisComplementLeukocytesThrombinArticle history:a b s t r a c ta r t i c l e i n f oComplement Activation in Arterial and Veby PlasminJonathan H. Foley a,b,c, Bethany L. Walton d, Maria M. AMicaela Harrasser b, Kimberley A. Foley e, Alisa S. Wolba Centre for Blood Research, Department of Medicine, Life Sciences Institute, University of Britib Department of Haematology, UCL Cancer Institute, University College London, London, Unitec Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free NHS Trust, Londod Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel He Cancer Care and Epidemiology, Queen's Cancer Research Institute, Queen's University, Kingsthil extracellular traps; PAR1,emoattracant protein-1; IL-8,306-2350 Health SciencesMall,nada.. This is an open access article underus Thrombosis is Mediatedan d, Alice M. O'Byrne a, Victor Lei a,g d, Edward M. Conway a,⁎lumbia, 2350 Health Sciences Mall, LSC4306, Vancouver V6T 1Z3, Canadagdomnited Kingdom19 Brinkhous-Bullitt Building, CB# 7525, Chapel Hill, NC 27599-7525, USAanadadicine.eb iomed ic ine.comand activation in thrombosis (Distelmaier et al., 2009; Salmon et al.,2002; Pierangeli et al., 2005). Moreover, terminal complement pathwaycomplexes formed as C5 is activated, have multiple procoagulant prop-erties (Langer et al., 2013; Hamilton et al., 1990). Thus, there is interestin understanding how C5a and the other major complement-derivedthe CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).176 J.H. Foley et al. / EBioMedicine 5 (2016) 175–182chemotactic factor, C3a, are generated, so that novel therapeutic strate-gies may be designed to prevent thrombosis.Complement activation typically proceeds via three pathways —classical, lectin and alternative—which converge to form C3 convertasesthat proteolyse C3 into C3b with release of C3a (Ricklin et al., 2010). Ascomplement activation exceeds a threshold, and the density of C3bincreases, the specificity of the convertase shifts from C3 to C5. Theresultant C5 convertases — C3bBbC3b for the alternative pathway andC4b2aC3b for the classical/lectin pathway— efficiently cleave C5 at argi-nine 751 (R751), liberating C5a and generating C5b, the initiating factorfor assembly of the lytic C5b-9 membrane attack complex (MAC).Although the C3/C5 convertases are well-recognized for their capacityto cleave C3 and C5, other serine proteases reportedly also exhibitconvertase activity (Huber-Lang et al., 2006; Amara et al., 2010; Wigginset al., 1981). Notably, thrombin was implicated in providing a “newpathway” to activate complement by cleaving C5 in a C3-independentmanner, thereby bypassing the bona fide C5 convertases (Huber-Langet al., 2006). However, C5 is a relatively poor substrate for thrombincleavage at R751 (Krisinger et al., 2012), raising questions as to its impor-tance in contributing to C5a generation during thrombus formationin vivo. We therefore explored the mechanisms by which C3a and C5aare generated using biochemical approaches and in vivo models ofvenous and arterial thrombosis.2. Materials and Methods2.1. MaterialsHuman complement C3a and C5a were measured using QuidelMicroVue C3a Plus or C5a ELISA kits (Cedarlane Laboratories, Burlington,Ontario). Murine thrombin–antithrombin (TAT) levels were measuredusing Enzygnost TAT micro ELISA (Siemens, Munich, Germany).Human complement proteins C3 and C5 were obtained from Comple-ment Technology, Inc. (Tyler, TX) and human hemostatic enzymes(plasmin, factor Xa and thrombin) were from Haematologic Technolo-gies, Inc. (Essex Junction, VT).2.2. ELISAs to Measure C3a and C5aMurine complement C5a levels were determined using the mousecomplement component C5a duoset and accompanying standard fromR&D systems (catalog #DY2150; Minneapolis, MN). An ELISA formurine complement C3a was established using a murine C3a standardand antibodies from BD Biosciences (Mississauga, Canada). The capturerat monoclonal anti-mouse C3a antibody (catalog # 55820, clone: I87-1162) was coated overnight onto 96-well plates in 100 μL of PBS at aconcentration of 2 μg/mL. Wells were washed ×3 with wash buffer(R&D catalog #WA126) followed by blocking for 2 h with 300 μl ofreagent diluent (R&D systems catalog #DY995). Plasma samples induplicate were diluted 1/50 and 1/125 in sample diluent to a finalvolume of 100 μl and incubated for 2 h. After 3 washes, 100 μl of thebiotinylated detection monoclonal rat anti-mouse C3a antibody (cloneI87-419, catalog #55821) 0.5 μg/mL in reagent diluent was incubatedfor 1 h. Wells were washed ×4 and incubated for 20 min at roomtemperature with 100 μl of streptavidin-biotinylated horseradish per-oxidase (1:3000), followed by 2 washes, and development with thesubstrate solution containing o-phenylenediamine using a plate readerset to 450 nm. A standard curve was generated with purified murineC3a (catalog # 558618). The sensitivity range of the assay was 0.1 nMto 2.5 nM. Intra-assay and inter-assay precision was 8–10%.2.3. Animal ModelsAll experiments with animals were approved by the University ofNorth Carolina at Chapel Hill Institutional Animal Care andUse Commit-tees. The mice (C57Bl/6) were male and between 6 and 8 weeks of age.The number of animals for eachmodel was determined based on previ-ous work that showed a broad range of TAT levels in the respectivemodels (Machlus et al., 2011a, 2011b). On each day of the experiments,animals were randomly assigned to receive the stated treatment or tobe used for baseline measurements. Quantification of biomarkers wasperformed in a blinded fashion wherein an experimenter, differentfrom the one who performed the procedures on the animals, carriedout the ELISAs on coded samples that were only de-coded after resultshad been generated.2.4. Murine model of Venous ThrombosisThe inferior vena cava (IVC) stasis model was performed as previ-ously described (Aleman et al., 2013). Briefly, mice were anesthetizedwith 1·5–2% isoflurane in oxygen and human prothrombin (to 300%,final, mouse plus human prothrombin) or vehicle was infused via tailvein injection. Prothrombin was infused to give a broader range ofthrombin generation and clot weight. Following sterile laparotomy,the intestines were exteriorized, the IVC was dissected bluntly, andside branches were ligated with 8–0 prolene suture and lumbarbranches closed by cautery. The IVC was separated from the aorta byblunt dissection and completely ligated with 8–0 prolene suture. Afterreplacing the intestines, the muscle layer was closed with 5–0 vicrylsuture and skin closed with 8–0 prolene suture and skin glue. Mice re-covered with analgesia (buprenorphine, 0·05 mg/kg subcutaneous).After 12 h, blood was drawn from the IVC above the ligation site into3.2% sodium citrate and processed to platelet-poor plasma by centrifu-gation at 5000 ×g for 10 min. Thrombi were collected and weighed.Plasmas were stored at−80 °C for analysis of TAT, C3a and C5a levelsby a person that was blinded to the treatment group. Two samplesshowing hemolysis were excluded.2.5. In Vitro Generation of C3a or C5a by Hemostatic EnzymesThe relative efficiency, time course, and rate of complement cleavageby plasmin, factor Xa or thrombin were determined using a series ofassays. Briefly, complement C3 (20 μM) or C5 (2 μM) was incubatedwith 100 nM plasmin or 250 nM factor Xa or thrombin. Reactionswere quenched at various time points and C3a or C5a levels were quan-tified by ELISA. To determine the relative efficiency of cleavage, 2 μM ofC5 was incubated with 100 nM of plasmin, factor Xa or thrombin at37 °C. After 10 min the reaction was quenched with the appropriatechloromethylketone (Val-Phe-Lys-chloromethylketone (VFKck) forplasmin and Phe-Pro-Arg-chloromethylketone (PPAck) for factor Xaand thrombin). In similar experiments, aliquots of the reaction mixturewere sub-sampled into chloromethylketones at various time points todetermine the time courses of C3 and C5 cleavages by plasmin, factorXa and thrombin. The kinetics of C5a generation were determined byincubating 0–3 μM of C5 with 100 nM of plasmin at 37 °C. Reactionswere quenched after 1 min and C5a levels were quantified by ELISA.Similar experiments substituting factor Xa or thrombin for plasminwere conducted, but the amount of C5a generated in these assays over30 min (for factor Xa) or 1 h (for thrombin) was below the limit ofdetection for the assay.C5a generation occurring during clot formation and degradationwas assessed in vitro by incubating physiological concentrationsof fibrinogen (9 μM), plasminogen (2 μM), antiplasmin (1 μM)and C5 (400 nM) at 37 °C. High (1 μM) or low (10 nM) concentra-tions of thrombin were added to induce clot formation and 10 nMof tPA was used to induce plasminogen activation and clot lysis.The tPA concentration chosen resulted in complete clot lysis in30 min. At the end of the 30-minute incubation period, enzymeswere quenched with PPAck and VFKck, the sample centrifuged andsupernatant or sample stored at −80 °C for quantification of C5alevels. Background C5a signal when PPAck/VFKck was added att = 0 were subtracted.177J.H. Foley et al. / EBioMedicine 5 (2016) 175–1822.6. In Vitro Generation of C5b,6 by Hemostatic EnzymesComplement factors C5 (400 nM) and C6 (500 nM) were incubatedwith 100 nMof plasmin or 250 nMof factor Xa or thrombin. Parallel reac-tions were quenched over timewith an appropriate chloromethylketone.The amount of functional C5b,6 generated was subsequently quantifiedusing a chicken erythrocyte hemolytic assay (Wat et al., 2014). Since plas-min readily cleaves C5,we also incubated 500nMof C5b,6with 100nMofplasmin to determine if C5b,6 activity diminishes over time. In these ex-periments, plasmin was quenched with VFKck.2.7. Ferric Chloride Model of Arterial ThrombosisMice were anesthetized with 1·5–2% isoflurane in oxygen. Ferricchloride injury to the carotid artery was performed as described(Aleman et al., 2013). Briefly, the right common carotid artery was ex-posed, dried, and treated with ferric chloride (7·5 or 10% on0·5 × 1·0-mm filter paper) for 2 min. The artery was washed withwarm saline and bloodflowwas continuouslymonitored byDoppler ul-trasonic flow probe (Indus Instruments). The time to vessel occlusionwas defined as the time between FeCl3 administration and lack of flowfor 60 s. Blood was sampled into citrate from the IVC 5 min after stablevessel occlusion (defined as continuous occlusion for 1 min) or after40 min if no occlusion occurred.2.8. Thrombolysis ModelThrombolysis was assessed in mice subjected to FeCl3 carotid arterythrombosis. After 5 consecutive minutes of vessel occlusion, mice wereinfused with Tenecteplase (5 mg/kg, generous gift of Genentech, CA)through a saphenous vein intravenous catheter constructed of pulledPE-10 tubing (Braintree Scientific, Braintree, MA) with a 3·0-mil(0·076-mm diameter) cleaning wire (Hamilton Company, Reno NV)placed into the lumen as a stylet, as described (Machlus et al., 2011a),while continuously monitoring carotid blood flow. Blood was sampledfrom the IVC 5 min after the return of blood flow or 30 min afterTenecteplase infusion if the clot did not lyse.2.9. StatisticsThe relationships between prothrombotic and complement activa-tion markers were assessed using Spearman rank correlation. C3a andC5a levels were compared using t-tests or the Wilcoxon rank sum test(Tenecteplase-treated versus untreated mice). P b 0·05 was consideredstatistically significant.3. ResultsWe studied the role of thrombin in complement activation using anin vivomurine model of ligation (stasis)-induced IVC thrombosis (vonBruhl et al., 2012; Aleman et al., 2013). Plasma levels of activationmarkers of coagulation and complement were measured by ELISA. Base-line TAT levels were 3·8 ± 4·0 ng/mL (n = 5), as previously reported(Aleman et al., 2013). 24 h after IVC ligation, plasma TAT levels rose to21·2 ± 11 ng/mL (mean ± SD, n = 6). Prothrombin was infused insome mice to give a broader range of thrombin generation and clotweight (Aleman et al., 2013) (Fig. 1 — open circles —mice infused withprothrombin; solid circles — mice infused with vehicle). When pro-thrombin was infused just prior to IVC ligation (Aleman et al., 2013),TAT levels measured at 24 h were significantly higher (47·9 ±19 ng/mL, n=8, p b 0·009). As expected, clot weights directly correlatedwith TAT levels (r = 0·66, p b 0·01). Plasma levels of C5a and C3a in ve-nous thrombosis were elevated as compared to baseline (unchallenged)levels (C5a = 0·43 ± 0·15 nM; C3a b 0.1 nM; n= 6). Interestingly, cir-culating levels of complement activationmarkers C3a and C5a correlatedpoorly with TAT levels (Fig. 1a, b), suggesting that thrombin does notdirectly activate complement in this experimental model of venousthrombosis. Notably, C5a also correlated poorly with C3a (Fig. 1c), sug-gesting that C5a was generated to a large extent via C3/C5 convertase-independent pathways. Furthermore, there was no relationship betweenclot weight and C3a levels (Fig. 1d). There was, however, a strong directcorrelation between clot weight and C5a levels (Fig. 1e). Taken together,these findings show that processes triggered during venous thrombosisare associated with C5a generation, but suggest that thrombin is notthe major activator of C5 under these conditions.To determine the potential mechanisms of C5a generation in vivo,we used in vitro assays in purified systems to compare C5a generationfollowing cleavage of C5 by thrombin, factor Xa or plasmin. Plasminwas considered a likely candidate of complement activation in the set-ting of a fibrin clot because, 1) plasmin is known to cleave C5 to yieldchemotactically-active C5a in vitro (Amara et al., 2010), and 2) fibrinis an essential cofactor for tissue-type plasminogen activator (tPA)-mediated plasmin generation (Horrevoets et al., 1997).Incubation of C3 or C5 with thrombin, factor Xa or plasmin revealedthat plasmin ismuchmore effective than thrombin or factor Xa in cleav-ing C3 and C5 to generate C3a and C5a, respectively (Fig. 2a, b). Plasminmore readily cleaved C5 than C3, with ~30% of C5 (~700 nM) beingconverted to C5a, and only ~2% of C3 (~450 nM) cleaved to form intactC3a. The low turnover of C3 by plasmin, thrombin and fXa precludedfurther in vitro interrogation. The incomplete cleavage of C5 to C5aunder the conditions employed may reflect competitive inhibition byan abundance of cleavage products (molecular weight N30–70 kDa)that were detected following SDS-PAGE (not shown and (Barthelet al., 2012)).We compared the efficiency of C5 cleavage by various con-centrations (0–100 nM) of each of the three enzymes (Fig. 2c). During a10-minute incubation period, plasmin generated substantially moreC5a than factor Xa or thrombin.In kinetic assays, the rate of C5a generation by plasmin in-creased linearly as the concentration of C5 increased (Fig. 2d).The catalytic efficiency, inferred from the slope of the plot, was2·3 ± 0·6 × 104 M−1 s−1 (Distelmaier et al., 2009). This is similar tothe published rate of C5 cleavage by the bona fide alternative pathwayC5 convertase and the soluble monomeric classical/lectin pathway C3/C5 convertase (Rawal and Pangburn, 1998, 2001). This rate of C5 cleav-age by plasmin is therefore consistent with the premise that plasminhas a physiologically relevant role in generation of C5a.Since plasmin is rarely, if ever, free in circulation, we next testedwhether plasmin is capable of generating C5a in the presence of physi-ological concentrations of antiplasmin and fibrinogen, that whenconverted to fibrin, binds plasminogen and plasmin with high affinity.We first confirmed that even at very high concentrations, thrombingenerates almost undetectable amounts of C5a (Fig. 2e, conditions i,ii). In the absence of plasminogen, tPA was incapable of cleaving C5to yield C5a (Fig. 2e, condition iii). When C5 was incubated with tPAand plasminogen in the presence of fibrin(ogen) (Fig. 2e, condition v),readily detectable amounts of C5a were generated, and this occurredeven in the presence of physiological concentrations of antiplasmin(condition iv). Absence of fibrinogen, a cofactor for tPA-mediatedconversion of plasminogen to plasmin, resulted in the generation ofmeasurable, but less, C5a than with fibrinogen (condition v versus vi).Overall, the data confirm that in the presence of physiological concen-trations of hemostatic proteins, C5a can be generated in a plasmin-dependent manner.Convertase-mediated release of C5a from C5 occurs in parallel withgeneration of C5b that is required for MAC formation. Using a terminalpathwayhemolytic assay (Wat et al., 2014),we showed that in the pres-ence of excess C6, cleavage of C5 by factor Xa or plasmin yielded a C5b,6complex that could assemble with C7, C8 and C9 to form a fully func-tional lytic MAC. The efficiency of C5b,6 generation by plasmin, factorXa and thrombin mirrored that observed for C5a (Fig. 3a). Over60 min, 100 nM of plasmin generated 3·0 ± 0.6 nM C5b,6, whereaseven 250 nM of factor Xa (which is ~2-fold higher than the plasma178 J.H. Foley et al. / EBioMedicine 5 (2016) 175–182concentration of factor X) generated only 1·1 ± 0·7 nM of C5b,6.Consistent with its inefficient cleavage of C5, thrombin (250 nM) didnot generate any detectable C5b,6. Thus, although plasmin can cleaveC5 at several sites (Barthel et al., 2012), exposure of C5b,6 to plasminfor 1 h did not appreciably decrease the functionality of theMAC (Fig. 3b).In view of these in vitro findings, we tested the association betweenthrombin generation and complement activation in a second, indepen-dent thrombosis/thrombolysismodel, and used thismodel to determinewhether C5 is activated by thrombolytic pathways in vivo. Using wild-type mice with stable carotid artery thrombi induced by ferric chloride,we first showed that 5 min after occlusion, TAT levels correlated poorlywith systemic levels of C3a and C5a (r = −0·36, p = 0·39 for C3a;r =−0·05, p = 0·91 for C5a; n= 8), consistent with our observationsin the venous thrombosis model. To determine the impact ofFig. 1. Coagulation and complement activation in venous thrombosis. Following stasis-induthrombin-antithrombin complexes (TAT), C3a, and C5a were measured by ELISA. C3a (a)correlated (c), implying the existence of C3-independent pathways to generate C5a. Clotrepresents a separate mouse, n = 14. Solid circles are untreated mice (infused with vehigeneration (see Materials and Methods section). Correlation coefficients (r) with 95% confidenplasminogen activation in this setting, we intravenously administeredthe tPA analog Tenecteplase (n=8) intomice that had been challengedwith arterial occlusion and measured C3a and C5a 5 min after restora-tion of blood flow, or 30 min after infusion if blood flow was not re-stored (Fig. 4a, b). Of these, 3 mice did not re-perfuse and 5 mice didre-perfuse. Regardless of outcome, all mice that received Tenecteplasewere included in the analysis. As compared to ferric chloride-challenged mice that were not infused with Tenecteplase (n = 9),Tenecteplase caused a significant (~2-fold) increase in C3a and C5alevels. Notably, this increase is in line with published studies in asmall group of patients treated with recombinant tPA following acutemyocardial infarction (Bennett et al., 1987). Our finding that C5 couldnot be cleaved by recombinant tPA (Fig. 2d, iii) makes it highly unlikelythat the elevated C5a levels were attributable to Tenecteplase-mediatedced thrombosis of the inferior vena cava (see Materials and Methods section), levels ofand C5a (b) levels correlated poorly with TAT levels. C3a and C5a levels also poorlyweight poorly correlated with C3a (d), but strongly correlated with C5a (e). Each dotcle); open circles are mice that were infused with prothrombin to increase thrombince levels and p-values are indicated on each panel.179J.H. Foley et al. / EBioMedicine 5 (2016) 175–182generation of C5. We further excluded this possibility by showingin vitro that exposure of C5 to high concentrations of tPA (up to200 nM) for 10 and 30 min, did not yield measurable amounts of C5a(data not shown). Taken together, these data are consistent with adirect effect of plasmin on complement activation during thrombosis.4. DiscussionThrombus formation leading to pathological vaso-occlusive events(e.g. acute coronary syndrome, stroke, deep vein thrombosis and pul-monary embolus) is a major cause of death worldwide (MozaffarianFig. 2. Hemostatic enzymes generate C5a. a, b, c. In a purified system, plasmin (100 nM) (▴), faperiods of time and C3a (a) or C5a (b) wasmeasured by ELISA. Plasmin efficiently generated C3(b) within 1 min, whereas factor Xa or thrombin took much longer to generate appreciable ameach of the three enzymes, after which C5a levelsweremeasured by ELISA. Plasminwas significby plasmin increased linearly as the concentration of C5 increased. The slope of the line0.6 × 104 M−1 s−1 (Distelmaier et al., 2009). e. Thrombin (10 nM, 1 μM; i, ii, respectivelyplasminogen (2 μM) to the system enabled plasmin generation and was associated with C5a(vi) is not essential for C5a generation when tPA, plasminogen and C5 are present, but it enhpresented in a, b are a single representative experiment and the data for all other experimentset al., 2015; Raskob et al., 2014). Initiation, propagation, and resolutionof a thrombus rely on the recruitment of platelets and inflammatorycells, and this is mediated partly by local release of the complementactivation factor C5a. C5a is a potent anaphylatoxic peptide, inducing arange of pro-inflammatory and pro-thrombotic effects via its cognateG-protein-coupled receptors, C5aR and C5L2. C5a activates platelets,leukocytes and endothelial cells, upregulates expression of adhesionmolecules, induces secretion of pro-inflammatory and procoagulantcytokines, promotes tissue factor expression by neutrophils and releaseof tissue factor-containing microparticles, induces the formation ofneutrophil extracellular traps (NETs), and amplifies complementctor Xa (250 nM) (•) or thrombin (250 nM) (■) was incubated with C3 or C5 for varyinga (a) (inset is highermagnification to show relative initial rates of C3a generation) and C5aounts of C3a or C5a. c. C5 (2 μM)was incubated for 10 min with varying concentrations ofantlymore efficient at cleaving C5 than factor Xa or thrombin. d. The rate of C5a generationimplies that plasmin cleaves C5, generating C5a with a catalytic efficiency of 2.3 ±) or tPA (10 nM) (iii) did not generate significant levels of C5a from C5. Addition ofgeneration in the absence (iv) or presence (v) of 1 μM of antiplasmin. Fibrinogen (9 μM)ances C5a generation in the context of tPA induced plasminogen activation (v). The data(c–e) represent means ± SD for 3 replicates. n.d. = not detectable.Fig. 3. Hemostatic enzymes generate C5b,6. a. Cleavage of C5 by plasmin (▴), factor Xa (•), or thrombin (■) in the presence of excess C6, C7, C8 and C9, resulted in the generation offunctional C5b,6, measured by a terminal pathway erythrocyte hemolytic assay. Thrombin did not generate any measurable C5b,6 in this experimental system. b. The functionalintegrity of C5b,6, measured by the terminal pathway hemolytic assay, did not decay appreciably over a 1-hour period when incubated with plasmin. The data presented representmeans ± SD for 3 replicates.Fig. 4. Effects of plasminogen activator-mediated thrombolysis on C3a and C5a levels. a, b. Stable carotid artery thrombosis was induced in wild-type mice using the ferric chloridemodel(seeMaterials andMethods section), afterwhich the plasminogen activator Tenecteplasewas administered intravenously as noted. C3a (a) and C5a (b) levelswere significantly increasedby Tenecteplase infusion. Each dot represents a separate mouse (n = 9 controls, n = 8 infused with Tenecteplase).Fig. 5. Proposed contributions of plasmin-mediated C5 activation in thrombosis. During a. thrombus formation, progression, and b. resolution, fibrin deposition promotes plasmingeneration, which activates complement. The traditional complement activation pathways likely also participate (not shown). The effects of plasmin-mediated complement activationon thrombus growth and resolution depend on the timing and localization of C5a generation, and assembly of the membrane attack complex (MAC). a. Thrombus formation:Following endothelial cell activation/damage, C5 (and C3) is activated, generating C5a and C5b (1), the latter of which binds to C6–C9 to form the membrane-damaging, procoagulantMAC. C5a is a potent chemoattractant for platelets and neutrophils (2) and activates cells to express monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8 (3). Thesepathways cooperate to recruit and activate platelets, monocytes and neutrophils (4), with release of reactive oxygen species, proteases and nucleosomes, all of which enhancethrombus formation (5). b. Thrombus resolution: As plasmin degrades fibrin into fibrin degradation products (FDPs) (6), it also generates C5a and C5b (1). C5a induces the release offactors (e.g. MCP-1, IL-8) (3) that recruit macrophages and neutrophils (7), which promote clot resolution by augmenting plasmin generation (8), fibrin degradation, and phagocyticclearance of clot-associated debris (9).180 J.H. Foley et al. / EBioMedicine 5 (2016) 175–182paradox of heightened plasmin generation with a larger thrombus is inenhanced production of plasmin. These findings are consistent with,Although plasmin generation is increased acutely and to a lesser181J.H. Foley et al. / EBioMedicine 5 (2016) 175–182activation through positive feedback loops (Oikonomopoulou et al.,2012). Given the current findings, we propose a model in which plas-min, via liberation of C5a, contributes to leukocyte trafficking duringthrombus formation, propagation and/or resolution (Fig. 5). The preciselocal contribution of C5a (and C3a) to thrombus formation and resolu-tion, is difficult to ascertain, particularly since clearance of these pep-tides is short and likely dynamically changes in this setting.Nonetheless, with generation of C5a, terminal complement pathwaycomplexes form which also regulate coagulation. C5b-7 induces tissuefactor expression by monocytic cells (Langer et al., 2013), while theMAC induces VWF and P-selectin secretion, platelet microparticle re-lease, and endothelial cell and platelet membrane changes that favorprothrombinase assembly and thrombin generation (Hamilton et al.,1990; Wiedmer et al., 1986; Sims et al., 1988). Since C5 activation is as-sociated with many disease states, including acute lung injury, arthritis,sepsis (Huber-Lang et al., 2006; Kessel et al., 2014; Yan and Gao, 2012),and thrombosis (Distelmaier et al., 2009; Cheung et al., 1994), the pres-ent studies suggest that interventions at the level of plasmin may havebroad clinical utility.From studies with mouse models (Huber-Lang et al., 2006; Hothet al., 2014; Khan et al., 2013; Auger et al., 2012; Zecher et al., 2014;Borkowska et al., 2014), several groups have concluded that thrombinis the major coagulation enzyme that generates C5a under pathologicconditions. This role for thrombin was supported by observations thatthrombin generates C5 in vitro (Huber-Lang et al., 2006), and inhibitionof thrombin dampens severity of disease and reduces C5a levels inmurine models of disease (Huber-Lang et al., 2006; Hoth et al., 2014;Khan et al., 2013; Auger et al., 2012; Zecher et al., 2014; Borkowskaet al., 2014). How do we reconcile these findings with the fact that theresiduesflanking theR751C5 convertase cleavage site necessary to gen-erate C5a lack similarity to thrombin cleavage sites in all other classicthrombin substrates (e.g., protein C, PAR1, fibrinogen, factor V, factorVIII) (Krisinger et al., 2012), and that thrombin is an inefficient cutterof C5 at that site (Krisinger et al., 2012)?That thrombin participates in C5a generation during coagulation isnot challenged by the present findings. However, this reaction likelydoes not occur via direct C5 cleavage. Indeed, in our experiments, withthrombin concentrations that more closely approximate the dynamicsof thrombin generation in plasma and blood (Brummel et al., 2002;Dielis et al., 2008), C5a could not be measured. Moreover, the C5Tproduct that is generated by thrombin-mediated cleavage of C5 at thethrombin-sensitive R947 site (Krisinger et al., 2012), does not exhibitC5a-like chemotactic/migration properties in vitro (data not shown).The present studies are consistent with the concept that thrombin con-tributes to C5a generation, but indirectly via plasmin-mediated events.Thrombin is fundamentally important for the initiation of fibrinolysissince it generates fibrin, an important cofactor for tPA-mediated plas-min generation. Thrombin further amplifies plasmin generation byinducing endothelial secretion of tPA and expression of urokinase-type plasminogen activator (van den Eijnden-Schrauwen et al., 1995).Given the relative kinetics of thrombin versus plasmin in generatingC5a, it is unlikely that thrombin, alone, generates C5a. Rather, our datasupport the premise that, in combination with C5 convertase, plasmin(and/or downstream proteolytic effectors of plasmin) is the majormediator, and that the amount of thrombin only affects C5a levels inso-far as thrombin affects the kinetics of fibrinolysis/plasmin generation.The caveat to this premise is that very high thrombin concentrationsthat may transiently accumulate during thrombus formation (Brummel-Ziedins et al., 2005) could cleave C5 at R751 to generate C5a (Krisingeret al., 2012). Interestingly, reports of a role for thrombin in generatingC5a come from studies with mice lacking C3 (Huber-Lang et al., 2006;Khan et al., 2013; Auger et al., 2012; Borkowska et al., 2014). Thesemice, for unexplained reasons, have elevated levels of prothrombin(Huber-Lang et al., 2006) which can result in increased generation ofthrombin following activation of coagulation (Aleman et al., 2013; Kyrleet al., 1998). It is reasonable to consider that the high thrombin levelsextent, chronically, in patients with thrombotic disorders (Wada et al.,1989), the role of plasmin in complement-mediated events duringthrombus formation, propagation, and resolution is unknown. Ourfinding that plasmin drives C5a generation in mouse models of throm-bosis exposes plasmin and/or its downstream effectors as potentialtherapeutic targets for limiting production of procoagulant and pro-inflammatory effectors. These insights are likely to be applicable tothrombi in arteries and veins, in spite of the differences with regard toetiology of these presentations. Indeed, and most notably, both presen-tations involve activation of inflammatory pathways and liberation offibrinolytic activity in response to the thrombus (Engelmann andMassberg, 2013; Wolberg et al., 2012). The impact of any interventionwould likely be dependent on its timing in relation to thrombus forma-tion and resolution (Fig. 5). Thus, early dampening of C5a and MACassembly may thwart leukocyte accumulation and thrombus initiationand propagation. Later interventions may hinder resolution of the clot,but may also reduce long-term inflammatory sequelae of thrombosis,such as occurs in post-thrombotic syndrome. Intuitively, administeringagents to suppress fibrinolysis during a thrombotic event at any stagemay be unappealing, and thus an optimal intervention strategy/agentmight preservefibrinolysiswhile restrictingC5 activation. Direct blockingof C5 activation is highly efficacious in preventing complement-mediatedthrombosis in atypical hemolytic uremic syndrome and paroxysmalnocturnal hemoglobinuria (Wong and Kavanagh, 2015). Similar suc-cesses were not, however, observed for acute myocardial infarction(APEX AMI Investigators et al., 2007), which might be due to the factthat therapy was initiated early at presentation. The variable responsesin these reports underline the need for further study, using models thatrepresent different vascular disorders.ContributorsJHF, ASW and EMC designed experiments, analyzed data and wrotethe manuscript and managed the project. JHF, MMA, BLW, AMO, VLand MH performed experiments. KAF analyzed data and edited themanuscript.Declaration of InterestsWe declare no competing interests.Ethical Research ConductAll aspects of the work covered in this manuscript were conductedwith the ethical approval of all relevant bodies and these are acknowl-and indeed, extend previous in vitro studies inwhich high concentrationsof plasmin cleaved C5 (Amara et al., 2010). Attempts to measure murinelevels of plasma plasmin–antiplasmin complexes were confounded by alack of reliable assays. However, in vivo gain-of-function studies usingpharmacologic-intervention (Tenecteplase infusion), allow us to con-clude that plasmin plays a physiologically relevant role in the generationof C5a.line with our observation that IVC clot weights strongly correlated withC5a levels. By interfering with deposition of fibrin, a critical cofactor forplasmin generation, thrombin inhibition would reduce C5a generation.Overall, thrombin is important in C5a generation, but indirectly, viareached in C3-deficient mice might contribute to C5a generation. How-ever, with excess thrombin and fibrin deposition, plasmin generationwould also be markedly increased, and plasmin would be significantlyfavored over thrombin in generating C5a from C5. Indeed, this apparentedged within the manuscript.Role of the Funding SourcesNone of the funders had any input into the design of the experi-Howes, J.M., Richardson, V.R., Smith, K.A., et al., 2012. Complement C3 is a novel plasmaclot component with anti-fibrinolytic properties. Diab. Vasc. Dis. Res. 9 (3), 216–225.Huber-Lang, M., Sarma, J.V., Zetoune, F.S., et al., 2006. Generation of C5a in the absence ofC3: a new complement activation pathway. Nat. Med. 12 (6), 682–687.Kessel, C., Nandakumar, K.S., Peters, F.B., Gauba, V., Schultz, P.G., Holmdahl, R., 2014. A182 J.H. Foley et al. / EBioMedicine 5 (2016) 175–182of the manuscript, or the decision to submit it for publication.AcknowledgmentsJHF was supported by a Banting Fellowship. EMC is supported byoperating grants from the Canadian Institutes of Health Research(CIHR), the Natural Sciences and Engineering Research Council ofCanada (NSERC), and the Canada Foundations for Innovation (CFI). Heholds a CSL Behring Research Chair and a Tier 1 Canada Research Chairin Endothelial Cell Biology and is an Adjunct Scientist with the CanadianBlood Services. ASWwas supported by funding from the National Insti-tutes of Health (R01HL094740 and R56HL094740). BLWwas supportedby a training grant to the University of North Carolina (T32HL069768)andMMAwas supported byNIH grant F31HL112608. 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