Factor VII-Activating Protease Is Activated in Multiple Trauma Patients and Generates Anaphylatoxin C5a

This information is current as Sandip M. Kanse, Andrea Gallenmueller, Sacha Zeerleder, of September 30, 2021. Femke Stephan, Olivier Rannou, Stephanie Denk, Michael Etscheid, Guenter Lochnit, Marcus Krueger and Markus Huber-Lang J Immunol 2012; 188:2858-2865; Prepublished online 3

February 2012; Downloaded from doi: 10.4049/jimmunol.1103029 http://www.jimmunol.org/content/188/6/2858

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Factor VII-Activating Protease Is Activated in Multiple Trauma Patients and Generates Anaphylatoxin C5a

Sandip M. Kanse,*,† Andrea Gallenmueller,‡ Sacha Zeerleder,x,{ Femke Stephan,x Olivier Rannou,* Stephanie Denk,‡ Michael Etscheid,‖ Guenter Lochnit,* Marcus Krueger,# and Markus Huber-Lang‡

Severe tissue injury results in early activation of systems including the and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding in plasma. Anaphy- latoxin generation was measured by ELISA, Western blotting, sequencing, and assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and . Among others, we found complement com- Downloaded from ponents C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link http://www.jimmunol.org/ between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflam- mation. The Journal of Immunology, 2012, 188: 2858–2865.

mmediately after severe tissue trauma the coagulation system munity and the hemostasis system share some common factors as is massively activated, resulting in an extensive fibrin de- reviewed in detail (6, 7). I position and a concomitant activation of fibrinolysis (1, 2). Factor VII-activating protease (FSAP) is a circulating serine Uncontrolled systemic inflammatory response develops, as exem- protease that can activate factor VII as well as prourokinase (8). A plified by systemic mediator release and complement activation single nucleotide polymorphism (SNP) in the FSAP (Mar- by guest on September 30, 2021 (3). Among others, factors released by necrotic cells such as burg I [MI] SNP, G534E, 1601G/A) results in a protein with re- nucleic acids, histones, high mobility group box 1, and other duced enzymatic activity (9). Moreover, the MI-SNP was found to danger-associated molecular patterns contribute to the inflamma- be strongly linked to carotid stenosis (10), cardiovascular disease tory spiral (4, 5). Early defense mechanisms of both innate im- in general (11), stroke and its related mortality (12), and plaque thickness and calcification (13, 14). Recent studies show that FSAP zymogen in plasma can be activated by histones and nu- *Department of Medicine, Institute for Biochemistry, Justus Liebig University, D- cleosomes arising from necrotic and/or apoptotic cells (15, 16). 35392 Giessen, Germany; †Department of Biochemistry, Institute of Basic Medical This leads to the concept that cell injury may result in FSAP ‡ Sciences, University of Oslo, 0317 Oslo, Norway; Department of Traumatology, activation and that FSAP functions as a sensor of cell injury (16). Hand, Plastic, and Reconstructive Surgery, University Hospital–Ulm, 89075 Ulm, Germany; xDepartment of Immunopathology, Sanquin Research, 1066 CX Amster- To extend this concept we have investigated the activation of dam, The Netherlands; {Department of Hematology, Academic Medical Center, FSAP over time in polytrauma patients and have correlated this University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; ‖Paul Ehrlich # with circulating nucleosomes as a marker of tissue necrosis. Institute, 63225 Langen, Germany; and Max Planck Institute, 61231 Bad Nauheim, Germany Furthermore, to gain more insight into the functions of FSAP we Received for publication October 21, 2011. Accepted for publication January 4, have used a proteomics-based approach to identify potential FSAP- 2012. interacting partners in the plasma. Using this strategy we have This work was supported by grants from the Deutsche Forschungsgemeinschaft, discovered that complement proteins form complexes with FSAP Behring Ro¨ntgen Stiftung, and the Excellence Cluster in Cardiopulmonary Sciences and that FSAP can cleave C3 and C5 to generate anaphylatoxins (to S.M.K.) and by Deutsche Forschungsgemeinschaft Grant KFO200 HU 823-3/2 (to M.H.-L.). C3a and C5a. Hence, FSAP functions as a sensor of tissue injury Address correspondence and reprint requests to Prof. Sandip M. Kanse or Prof. and in turn activates anaphylatoxin production and the regulation of Markus Huber-Lang, Department of Medicine, Institute for Biochemistry, Justus the inflammatory process. FSAP may be considered as a novel Liebig University, Friedrichstrasse 24, D-35392 Giessen, Germany (S.M.K.) or De- circulating danger-associated molecular pattern that can partment of Traumatology, Hand, Plastic and Reconstructive Surgery, University Hospital Ulm, Steinhoevelstrasse 9, 89075 Ulm, Germany (M.H.-L.). E-mail ad- modulate the inflammatory response. dresses: [email protected] (S.M.K.) and Markus.Huber- [email protected] (M.H.-L.) Materials and Methods The online version of this article contains supplemental material. Human plasma/FSAP interactions Abbreviations used in this article: FSAP, factor VII-activating protease; MI, Marburg I; SNP, single nucleotide polymorphism. To identify proteins in the circulation that form complexes with FSAP, citrate plasma samples from three different healthy normal subjects were Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 subjected to immunoprecipitation with anti-FSAP (mAb 677; American www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103029 The Journal of Immunology 2859

Diagnostica, Pfungstadt, Germany) and control mAb. After SDS-PAGE, the boxyfluorescein acetoxymethyl ester; Molecular Probes, Eugene, OR) for whole gel was cut into small bands and subjected to in-gel digest with 30 min at 37˚C. HMC-1 ( line) was cultivated in basal Iscove’s . Reverse-phase nano-liquid chromatography and nanospray ioni- medium containing 10% FCS, L-glutamine, antibiotics, and monothio- zation mass spectrometry was performed by using an Agilent 1100/1200 glycerol. nanoflow LC system (Agilent Technologies, Karlsruhe, Germany) with a cooled 96-well autosampler. The LC system is coupled to an LTQ Chemotaxis assays Orbitrap XL instrument (Thermo Fisher Scientific, Schwerte, Germany) equipped with a nanoelectrospray source (Proxeon, Schwerte, Germany). To determine the chemotactic activity of the C3a and C5a generated by Mass spectroscopy data were analyzed with MaxQuant and Mascot soft- FSAP, mast (HMC-1) cells or human neutrophils were used. BCECF- 3 6 ware for peptide and protein identification. The results were arranged labeled neutrophils (5 10 cells/ml) were loaded into the upper cham- according to the number of recovered peptides from each protein as well as ber of a 96-well device (Neuro Probe, Gaithersburg, MD) and separated by m the intensity of the peptides as described before (17). For identification of a3- m polycarbonate filter. The lower chambers were loaded with peptides and proteins a false discovery rate of 1% was used, and only recombinant human C5a (50 ng/ml, positive control) or with indicated peptides with a minimum of 6 aa length were considered for identification. samples (e.g., C5 in absence or presence of increasing concentrations of After statistical analysis to determine which peptides show the highest FSAP). After incubation at 37˚C for 30 min, the number of cells that difference between immunoprecipitation with the FSAP Ab compared with migrated through the polycarbonate membrane was determined by cyto- the control Ab across the three samples, a ranked list of proteins was fluorometry (Cytofluor II; Per Septive Biosystems, Framingham, MA). obtained. Western blotting was performed to confirm the coimmunopre- Similarly, to evaluate C3a-dependent chemotaxis activation, HMC-1 cells m cipitation of various proteins with FSAP, and direct binding studies with (mast cell line HMC-1 cells) and a 5- m filter was used. Recombinant purified proteins were performed to further characterize these interactions. human C3a (100 ng/ml) served as a positive control. Cleavage of C3 and C5 in vitro by FSAP Polytrauma studies Downloaded from In vitro experiments were performed by incubating native C3 (100 mg/ml) The study was approved by the Independent Local Ethics Committee of the or C5 (100 mg/ml) (EMD, Darmstadt, Germany) in Dulbecco’s PBS in the University of Ulm (approval no. 44/06). A written informed consent was absence or presence of FSAP at 37˚C in a dose- and time-dependent obtained from all volunteers and patients where possible. If the patient was manner. Single-chain FSAP zymogen was isolated from human plasma unable to consent due to the injury pattern, sedation, or altered mental status, as described before (9). Dilution of FSAP into the end buffer leads to its informed consent was obtained after recovery. The polytrauma cohort rapid autoactivation into the enzymatically active two-chain form (9). consisted of 12 patients (10 men, 2 women) with a median age of 36 y (range, 6 Cleavage was followed by Western blotting with C3a and C5a Abs as well 19-74 y) suffering from multiple injuries (injury severity score, 30.3 2.9). as ELISA using commercially available ELISA kits (Quidel, San Diego, Ten patients (83%) had sustained fractures to extremities, pelvis, or spine. http://www.jimmunol.org/ CA and DRG Diagnostics, Marburg, Germany). For Western blotting, Eight multiple injured patients were diagnosed to have a blunt chest samples were separated by SDS-PAGE under reducing conditions and trauma (67%), four patients sustained abdominal injuries (33%), and one transferred onto a polyvinylidene difluoride membrane (Schleicher & patient had a major vascular injury. Eleven patients survived their injuries Schuell, Keene, NH). The blots were incubated overnight at 4˚C, using (92%) for at least 28 d. Four patients developed pneumonia (33%) and either polyclonal rabbit anti-human C3a IgG (EMD) or rabbit anti-human three patients showed signs of the sepsis syndrome (25%), whereas seven C5a IgG (EMD), and immunoreactive proteins were visualized using ap- patients developed signs of multiple organ dysfunction syndrome (58%). propriate secondary Abs (Jackson ImmunoResearch Laboratories, West Sex and age distributions of the healthy volunteers (n = 12; medium age, Grove, PA) and the ECL Plus detection kit (GE Healthcare, Heidelberg, 33 y) displayed a similar pattern in comparison with the trauma cohort. All Germany) or an alkaline phosphatase substrate color development buffer enrolled trauma patients showed clinical signs of hemorrhagic shock re- (Bio-Rad Laboratories, Hercules, CA). quiring RBC transfusions (average, 12 RBC concentrates) and catechol- For determining the cleavage sites, the nonreducing SDS-PAGE gels amine support within the first 24 h (11 of 12 patients). Patients received by guest on September 30, 2021 were run and blotted onto a polyvinylidene difluoride membrane, stained standardized critical care and surgical damage control principles. All pa- with Coomassie blue, and the protein bands were cut out for sequencing. tients were followed up for a 28-d observation period. Blood was collected The N-terminal sequences of the fragments were determined by automated from a central line in citrate at the indicated time points and immediately Edman degradation using an Applied Biosystems 492 pulsed liquid phase centrifuged at 2000 rpm for 10 min to obtain plasma. Plasma was frozen 2 sequencer equipped with an on-line 785A phenylthiohydantoin derivative and stored at 80˚C. analyzer (Applied Biosystems, Darmstadt, Germany). Seven cycles of Edman degradation were performed and the amino acids detected at each ELISA analysis of C3a, C5a, and FSAP Ag, FSAP activity, cycle were aligned with the C3/C5 sequence. FSAP/a2-antiplasmin complex, and nucleosomes Activation of endogenous FSAP in plasma and the concomitant For quantitative analysis of C3a and C5a, commercially available ELISA kits (Quidel, San Diego, CA and DRG Diagnostics, Marburg, Germany) generation of C3a/C5a were used according to the manufacturers’ instructions. FSAP/a2-anti- Human plasma was incubated for 90 min at 37˚C with various test sub- complexes and nucleosomes were measured as described before stances that potentially activate FSAP. After incubation, the samples were (16). FSAP Ag and activity were measured by ELISA as previously de- immediately mixed with SDS sample buffer, and Western blotting with scribed (19). anti-FSAP Ab was performed under nonreducing conditions. The test substance used included high-molecular mass (Sigma-Aldrich), Statistical analyses DNA (isolated from U937 and HEK 293 cells), dextran sulfate (average Data are shown as means 6 SEM. All in vitro experiments were performed molecular mass of 500 kDa; Sigma-Aldrich), poly-L-lysine (Sigma- in at least three independent replicates. Multiple significance testing was Aldrich), histones (mixture from calf thymus; Sigma-Aldrich), and chro- performed by ANOVA followed by a Tukey or Dunn posttest. For corre- matin, which was prepared from U937 and HEK 293 cells as described lation analysis, the Spearman rank correlation coefficient was determined. before (18) except that protease inhibitors were excluded since this would Results were considered statistically significant when p , 0.05. influence activation of FSAP. Activation experiments were also performed in the presence of protease inhibitors and in plasma of a person homo- zygous for the MI-SNP. C3a/C5a generation was determined using ELISA. Results Zymosan (Sigma-Aldrich) was used as a positive inducer of C3a and C5a Identification of FSAP-interacting proteins in human plasma generation. To further understand the functions of circulating FSAP, we used Cell isolation/culture a hypothesis-free screening strategy to search for FSAP-interacting For neutrophil isolation, whole blood from healthy human volunteers was proteins in normal human plasma. For this we used the method of drawn from the antecubital vein into syringes containing coimmunoprecipitation followed by mass spectrometry to identify citrate dextrose (1:10; Baxter Health Care, Deerfield, IL). Neutrophils were the proteins. Immunoprecipitation of FSAP from plasma of healthy isolated using Ficoll-Paque gradient centrifugation (Pharmacia Biotech, n Stockholm, Sweden) followed by a dextran sedimentation step. After hy- subjects ( = 3) led to the coimmunoprecipitation of a number of a a potonic lysis of residual RBCs, neutrophils were resuspended in HBSS and other proteins such as 2-macroglobulin, 1-, fluorescein-labeled with BCECF (29,79-bis(2-carboxyethyl)-5-(and 6)-car- heparin -2, C1 inhibitor, and a2-antiplasmin. Apart from 2860 FSAP AND COMPLEMENT IN TRAUMA

classical or alternative pathway of complement activation. Using an in vitro test, dependent on the generation and measurement of the C5b-9 complex (20), we found that the activation of the classical as well as the alternative pathway in serum was not influenced by the addition of exogenous FSAP or blocking endogenous FSAP by using a specific mAb or . Similarly, no difference was observed in complement activation in the serum of a homozygous MI-SNP carrier in comparison with the normal genotype (data not shown). Because C3a and C5a anaphylatoxins can be generated by serine proteases of the coagulation and fibrinolysis system such as and plasmin (21), we tested whether FSAP also had a similar activity. Incubation of C3/C5 with FSAP led to the generation of the corresponding anaphylatoxin in a time- and dose-dependent manner as determined by ELISA and Western blot analysis (Fig. 2). The generation of C5a was faster and achieved with lower concentrations of FSAP as compared with that of C3a. Blocking the enzymatic activity of FSAP with a specific FSAP- blocking mAb could diminish C3a/C5a generation (Fig. 3).

To check the precise nature of C3a and C5a generated by FSAP, Downloaded from N-terminal sequencing was performed. Both C3 and C5 are com- posed of an a- and b-chain, and internal cleavage of the a sub- unit releases the anaphylatoxin and the remainder is denoted C3a9 or C5a9, respectively. Cleavage of C3 with FSAP led to the gen- eration of an ∼10–14 kDa peptide with the N terminus of the ex-

pected C3a, whereas the C5a was truncated at the N terminus by http://www.jimmunol.org/ 4 aa (Fig. 4A). In a next step the biologic activity of the anaphylatoxins C3a/C5a FIGURE 1. Immunoprecipitation of FSAP and FSAP/protein complexes generated by FSAP was tested in a chemotaxis assay. Incubation of from plasma and its analysis by proteomics. (A) Normal human plasma both C3 and C5 with FSAP led to an increase in the chemotactic was subjected to immunoprecipitation with a mAb against FSAP as well as activity that paralleled the generation of the respective anaphyla- a control mAb. Immunoprecipitates were run on SDS-PAGE and stained with Coomassie blue. (B) The immunoprecipitates were analyzed by Western blotting with Abs against FSAP, C3, C5, plasminogen (PLSG), C1 inhibitor (C1-Inh), and a -Antiplasmin (a2-AP). (C) The gel from (A)

2 by guest on September 30, 2021 together with two other samples was then cut into pieces and trypsinized and the peptides were eluted. Reverse-phase nano-liquid chromatography and nanospray ionization mass spectrometry was performed and was coupled to a LTQ Orbitrap instrument equipped with a nanoelectrospray source. Mass spectroscopy data were analyzed for peptide and protein identification. The ratio between immunoprecipitation with the FSAP Ab compared with the control Ab (x-axis) as well as the significance (p) value of this ratio (2log thereof represented on the y-axis) gave a ranked list of proteins. In the scatter plot, black dots indicate proteins that were signif- icantly coimmunoprecipitated with FSAP (p , 0.01) and the others are indicated with gray dots. Only those proteins that showed a .2-fold log10 ratio of FSAP mAb/control mAb and a high significance (p , 0.002) are annotated. protease inhibitors, other proteins found in the complexes included plasminogen, clusterin, ceruloplasmin, serum paraoxanase, as well as the central complement proteins C3 and C5 (Fig. 1, Supple- mental Data 1). Western blotting for FSAP, C3, C5, plasminogen, C1 inhibitor, and a2-antiplasmin in the immunoprecipitates was performed to confirm these interactions (Fig. 1). Robust coim- munoprecipitation of FSAP with C3, plasminogen, C1 inhibitor, and a2-antiplasmin was observed whereas the coimmunoprecipi- tation of C5 was weaker. Strong direct binding between FSAP and FIGURE 2. Concentration-dependent effect of FSAP on the generation immobilized plasminogen as well as fibrinogen was observed but of C3a and C5a. C5 (A) and C3 (B) were incubated with varying concen- there was no direct binding between FSAP and C5 or C3 (data not trations of FSAP for 90 min at 37˚C and the generation of active C5a/C3a shown). was measured with a specific ELISA. Values are given in ng/ml (mean 6 Role of FSAP in complement activation and C3a and C5a SEM, n = 3). Similarly, the reaction mixtures were analyzed by Western C D anaphylatoxin generation blotting with specific Abs against C5a ( )andC3a( ). Values are given in ng/ml (mean 6 SEM, n = 3). Statistical significance was determined by The interaction of FSAP with a number of complement proteins in ANOVA and a Tukey posttest. Similar results were obtained in three in- the plasma led us to hypothesize that FSAP could interact with the dependent replicates. *p , 0.05. The Journal of Immunology 2861

FIGURE 3. Characteristics of active C3a and C5a generation by FSAP. C3 and C5 were incubated with FSAP (1 mg/ml) for various time points for 0–180 min, and C5a (A) and C3a (B) levels were determined by ELISA. To test the specificity of C5a (C) and C3a (D) generation by FSAP, the protease was neutralized with a blocking mAb (no. 570, filled bar) (50 mg/ml) against FSAP or an isotypic control mAb (cross-hatched bar, 50 mg/ml). Incubation with FSAP, Abs, and substrate was for 90 min. Values are given in ng/ml (mean 6 Downloaded from SEM, n = 3). *p , 0.05. http://www.jimmunol.org/

toxin as measured by ELISA (Fig. 4B, 4C). Hence, active C3a and ments. The generation of C5a was much lower in the plasma of an C5a were generated by FSAP from the precursor proteins. MI-SNP homozygous subject (Fig. 5B). Zymosan, which is a po- by guest on September 30, 2021 tent activator of anaphylatoxin generation, did not influence the Activation of endogenous FSAP in plasma and generation of FSAP activation status (data not shown), and its influence on C5a C3a and C5a generation was not affected by either aprotinin or the use of In the next step we investigated the effect of activating endogenous plasma of different genotypes (Fig. 5B). Hence, in MI-SNP FSAP in human plasma on C3a and C5a generation. Moreover, we plasma, C5a generation via FSAP was reduced but another also tested plasma from a subject homozygous for MI-SNP (9) in FSAP-independent pathway was intact. C5a generation was also which FSAP has very low enzymatic activity and is akin to a hu- measured in FSAP-depleted plasma. In this experiment FSAP man “knockdown.” Addition of factors released from dead cells was depleted with anti-FSAP Ab beads as well as control Ab such as histones and model polyanionic molecules such as dextran beads. We observed a strong C5a generation due to the process of sulfate into plasma could activate endogenous FSAP as deter- immunodepletion with both Abs, and hence this experiment did mined by the formation of the 130-kDa FSAP/inhibitor complex not provide unambiguous results. Generation of C3a after acti- as described before (15, 16) (Fig. 5A). The activation of FSAP vation with FSAP activators was low (,1.5-fold) but could also be was inhibited by aprotinin and it was weaker in plasma of a sub- reduced by aprotinin and was lower in MI-SNP plasma (data not ject homozygous for MI-SNP (Fig. 5A). High-molecular mass shown). These results show a clear generation of C5a in plasma heparin did not activate FSAP in plasma. FSAP activation was after activation of endogenous FSAP. also inhibited by a specific FSAP-blocking mAb (no. 570) but not a control mAb (Supplemental Data 2), whereas other general in- FSAP activity, nucleosomes, and anaphylatoxins in the hibitors such as corn trypsin inhibitor or ε-, circulation of polytrauma patients which inhibit factor XII or plasmin, respectively, did not influence In the above experiment endogenous plasma FSAP was activated FSAP activation. Although both FSAP and factor XII can be ac- with exogenously added histones. To further consolidate our hy- tivated by negatively charged polyanions in vitro, factor XII was pothesis we investigated whether, in humans, activation of en- not activated by histones or nucleosomes (Supplemental Data 3). dogenous FSAP correlates with nucleosome release and C5a gen- Hence, with respect to histones, FSAP activation is completely eration. We determined whether FSAP is activated in situations of independent of factor XII or plasmin activation. severe tissue injury such as in polytrauma patients. Hence, plasma Measurements of C5a in these plasma samples showed that at different time points after trauma from 12 polytrauma patients as FSAP activation with histone and dextran sulfate, but not heparin, well as 12 normal healthy controls was systematically analyzed for led to higher C5a generation, which was blocked in the presence of evidence of FSAP activation, including release of nucleosomes aprotinin (Fig. 5B). These activators or aprotinin did not interfere that are associated with cellular injury as well as anaphylatoxins with the C5a ELISA, but mAbs interfered with the ELISA and, C5a/C3a. Immediately after injury there was a ∼6-fold increase in hence, FSAP-blocking mAbs could not be used in these experi- FSAP activity (Fig. 6A) and a ∼2-fold decrease in FSAP Ag 2862 FSAP AND COMPLEMENT IN TRAUMA Downloaded from http://www.jimmunol.org/

FIGURE 5. Activation of endogenous FSAP and generation of C5a in human plasma. (A) Endogenous FSAP was activated in human pooled plasma to varying degrees by the addition of histones (10 and 100 mg/ml), FIGURE 4. Characterization of sequence and chemotactic activity of m m A dextran sulfate (DS) (1 and 10 g/ml), and heparin (10 and 100 g/ml) for C3a and C5a generated by FSAP in vitro. ( ) SDS-PAGE and N-terminal by guest on September 30, 2021 120 min, and the plasma samples were analyzed by Western blotting with sequencing of small-molecular mass bands was performed after incubation a polyclonal Ab against FSAP to determine the formation of FSAP/in- of FSAP with C5 and C3 (100 mg/ml), respectively. The single letter codes hibitor complexes in the plasma. (B) Normal wild-type (WT)/WT human for amino acids are shown. All samples were processed together and an- plasma in the absence (dotted bars) and presence (hatched bars) of apro- alyzed on the same gel, which was then cut to eliminate unrelated samples. tinin (75 mg/ml) was used to determine the influence of protease inhibition C5 and C3 were incubated with varying concentrations of FSAP, and the of FSAP on C5a generation. Zymosan (10 mg/ml) was used as positive generation of active C5a/C3a was measured with a chemotaxis assay with control of C5 activation. Similarly, this was compared with plasma from human neutrophils (B) and HMC1 mast cells (C), respectively (mean 6 a person with MI/MI (closed bars) genotype (homozygous carrier of MI- SEM, n = 6). *p , 0.05. SNP). Data are shown as means 6 SEM. *p , 0.05. compared with normal controls (Fig. 6B). This drop in Ag may be explained by the loss of blood after multiple injury followed by extremely high, that is, 0.95 (p , 0.001), confirming that both RBC transfusion and fluid replacement. The activity to Ag ratio methods are robust measures of FSAP activity in the circulation. was 12-fold higher above controls within the first hour after The above data support the concept that tissue injury releases trauma. The FSAP Ag and activity levels slowly returned to nor- factors that influence FSAP activation. In fact, it has been shown mal levels during a period of 24–48 h. that in trauma patients there is an increase in circulating nucleo- It has been recently shown that FSAP activation in plasma leads somes (22) as well as nucleic acids (23), so we also measured to an increase in FSAP/a2-antiplasmin complexes (15, 16) and that nucleosomes in our samples to determine the correlation with these serve as a surrogate marker for FSAP activation. In poly- FSAP activity. Nucleosome concentration was high immediately trauma patients the initial concentration of these complexes was after trauma and returned to baseline level at 24 h (Fig. 6D). high and decreased to normal levels after 48 h (Fig. 6C). These Circulating C5a level was increased 10-fold immediately after results were further confirmed by performing Western blot anal- trauma and decreased progressively over time (Fig. 7A). In con- ysis of circulating FSAP. Authentic single chain FSAP with a trast, C3a concentrations showed a 2- to 3-fold increase that molecular mass of 64–70 kDa was observed and a band corre- remained elevated during 5–10 d (Fig. 7B). Correlation analysis sponding to 130–140 kDa was elevated immediately after trauma revealed that C5a, but not C3a, correlated strongly with FSAP and decreased rapidly after 24 h (Supplement Data 4). The ELISA activity and nucleosomes (Fig. 7C, 7D) (Table I). results matched the intensity of the protein bands on Western blot (Supplement Data 4). Parallel experiments with activation Discussion of FSAP in plasma showed that this band was also generated The hemostasis as well as the show very high after FSAP activation in vitro and corresponds to FSAP/inhibitor organizational similarities. Both consist of zymogens that are complexes (Supplemental Data 2). The correlation between linked in a cascade leading to sequential activation of proteolytic FSAP activity and FSAP/a2-antiplasmin complex (Table I) was activities and are often dependent on surface-mediated reactions The Journal of Immunology 2863

FIGURE 6. Analysis of FSAP and nucleosomes in the circulation of polytrauma patients. Plasma sam- ples were collected from 12 patients with multiple trauma at the indicated time points as well as 12 matched healthy controls. FSAP activity (A) was measured by a combination of ELISA and prour- okinase activation assay. FSAP Ag (B), FSAP/a2- antiplasmin complexes (C), and nucleosomes (D) were measured by ELISA. Results are shown as means 6 SEM (n = 10–12). *p , 0.05. Downloaded from http://www.jimmunol.org/

(6, 7). Particularly, the / arm of the contact to proteins such as plasminogen and fibrinogen, which then form pathway of the coagulation system might share activators as well a macromolecular complex responsible for binding other proteins as inhibitors with the complement system (24). Using a com- in the plasma. There is a significant overlap between proteins in pletely hypothesis-free screen to elucidate the function of FSAP in the FSAP complex and those known to bind to fibrinogen (26). In the circulation, we found evidence for a possible interaction with fact, direct binding assays showed a robust binding of FSAP to by guest on September 30, 2021 the complement pathway. Detailed analysis revealed that FSAP plasminogen and fibrinogen but not to C5 and C3. The screen could generate anaphylatoxins from the precursor complement could be refined by performing coprecipitation studies by using proteins C3 and C5. Furthermore, we provide quantitative evi- tagged FSAP. Nonetheless, the striking presence of complement dence for massive activation of the FSAP zymogen in the circu- proteins in the coprecipitates led us to investigate these inter- lation of patients with polytrauma. These findings fit well into our actions in further detail. Detailed analysis of the other candidates previously described concept of a noncanonical pathway for ana- might also lead to further insight into the functions of FSAP in phylatoxin generation based on the activation by coagulation and the circulation. fibrinolysis factors (21, 25). Comparison of C5a and C3a generation consistently showed It was surprising that the immunoprecipitation of FSAP led to that C5a was produced more rapidly (5 min) and at lower concentra- the coimmunoprecipitation of a large number of proteins includ- tions of FSAP (0.1 mg/ml) than was C3a. Both C5a and C3a gen- ing many protease inhibitors. There could be many reasons why eration was dose- and concentration-dependent and was inhibited protease inhibitors were found in complex with FSAP: 1) even by an FSAP-blocking Ab. Anaphylatoxin generation could be though the vast majority of circulating FSAP is in the zymogen shown by ELISA, Western blotting, chemotaxis assays, as well as form, a small fraction may be active and form complexes with protein sequencing. FSAP-generated C3a showed two species in inhibitors; 2) the zymogen FSAP could be activated by incubation the range 10–14 kDa with an identical N-terminal sequence in- with beads ex vivo; and 3) FSAP, in its zymogen form, may also dicating cleavage at two different sites in the C-terminal region of bind to inhibitors. A further explanation as to why so many pro- C3a. Only one species of C5a was produced with FSAP with a teins coprecipitated with FSAP could be due to binding of FSAP N-terminal sequence 4 aa downstream of the classical C5a. Be- cause the domain in C5a is responsible for binding and activation of C5aR residues in the C-terminal region (27), this would be a Table I. Correlations between FSAP activity, FSAP/ 2-antiplasmin expected to be a fully active ligand. The effect of FSAP was complex, nucleosomes, and C5a/C3a in human polytrauma patients compared with that of plasmin (data not shown), which in our previous study was shown to be the most effective protease in FSAP Activity Nucleosomes anaphylatoxin generation (21). Comparisons carried out under the FSAP activity — 0.71 (p , 0.0001) same conditions have shown that FSAP shows not only a similar a , , FSAP/ 2-antiplasmin 0.95 (p 0.001) 0.76 (p 0.001) activity as plasmin with respect to concentrations required but also C5a 0.72 (p , 0.0001) 0.64 (p , 0.0001) C3a 0.12 (p = 0.322) 20.09 (p = 0.488) the stronger propensity to generate C5a compared with C3a (21) (data not shown). FSAP activity and nucleosome concentrations were correlated with FSAP/a2- antiplasmin complexes, C5a, and C3a. A Spearman rank correlation coefficient Activation of endogenous FSAP in the plasma could be achieved and p values were determined. by histones and dextran sulfate but not heparin as described before 2864 FSAP AND COMPLEMENT IN TRAUMA

FIGURE 7. Analysis of C5a and C3a in the circu- lation of polytrauma patients. Plasma samples were collected from 12 patients with multiple trauma at the indicated time points as well as 12 matched healthy controls, and C5a (A) and C3a (B) were measured by ELISA. To determine the correlation between FSAP activity and C5a or C3a, respectively, for each time point and each patient the values for FSAP activity were plotted against the corresponding C5a (C) or C3a (D) concentrations. A Spearman rank correlation co- efficient test was used to determine the correlation Downloaded from between these parameters. http://www.jimmunol.org/

(15, 16). This activation could be inhibited by aprotinin as well as and fluid replacement with plasma expanders does not directly a specific FSAP-blocking Ab. Activation was strongly reduced in lead to FSAP activation, measurements were performed in a rabbit the plasma of a MI-SNP homozygous carrier. This activation of and pig model of blood loss followed by transfusion of erythro- endogenous FSAP led to the generation of C5a in the plasma, and cytes and plasma expanders. In both models there was a decrease this was inhibited in the presence of aprotinin as well as in MI in FSAP activity. Hence, FSAP activation can be considered to be by guest on September 30, 2021 plasma. Activation of C5a generation with zymosan, which does the result of the original traumatic event rather than hemodilution not activate FSAP, was identical in the presence of aprotinin and and the use of plasma expanders. in MI plasma. These results confirm the linkage between FSAP Therefore, trauma induces an increase in circulating nucleo- activation and C5a generation in plasma. In these experiments in somes that in turn correlates strongly with increased FSAP activity plasma as well, C3a generation showed the same pattern but was and C5a levels. Although a strong correlation does not necessarily much weaker. This highlights a consistent difference between C3a indicate a mechanistic link between the FSAP, nucleosomes, and and C5a generation in the in vitro experiments as well as in the C5a, our plasma studies where C5a generation by histones was plasma experiments. reduced in situations where FSAP was inhibited provide strong Previous studies have shown that in patients with various in- evidence for such a mechanism in vivo. This fits well into the fectious diseases or after LPS application in mice that there is an observation that various serine proteases of the coagulation and increase in FSAP/inhibitor complexes (15, 16). However, these fibrinolysis system are capable of generating anaphylatoxins C3a assays used did not allow for a quantitative analysis of FSAP and C5a (21). activation in these situations. We have now cross-validated the To the previously known substrates of FSAP such as coagula- assay to measure FSAP activity in plasma with the presence of tion factor VII and fibrinolysis factors (prourokinase plasminogen FSAP/inhibitor complexes and Western blotting and there is a very activator), as well as growth regulating factors such as - high correlation between all three parameters. In polytrauma pa- derived growth factor BB homodimer and basic fibroblast growth tients there was a robust increase in FSAP activity in the face of factor, we can now add the central complement factors C3 and C5. decreased FSAP Ag in the immediate aftermath of trauma, and This indicates that FSAP is a broad-spectrum protease with mul- these values reached their respective basal levels after 24 h. There tiple functions. These multiple functions may contribute to he- was a high correlation between FSAP activity and circulating mostasis, repair and regeneration, as well as inflammation. Ana- nucleosome concentrations, indicating that both of these param- phylatoxins are potent inflammatory mediators that are largely eters are casually linked. This pattern fits well with our observa- produced by the activation of various complement pathways after tion in patients with bacterial infections (15, 16). Plasma C5a trauma (30) and during the systemic inflammatory response. In levels correlated strongly with FSAP activity, but this was not the trauma patients the activation of anaphylatoxins may be harmful case with C3a. It is possible that activated FSAP also generates due to perpetuation of inflammation, and their inhibition could C3a in vivo but that the high turnover of C3a does not allow for be beneficial (31). Based on our results we hypothesize that the a significant increase in its circulating levels. outcome of trauma might be different in carriers of the MI-SNP, It has been suggested that the process of hemodilution can which is akin to a natural FSAP knockdown. One would predict nonspecifically activate coagulation factors (28) and support that generation of C5a might be lower in MI-SNP carriers, which thrombin generation (29). To test the possibility that blood loss might dampen the inflammatory response in trauma. In this case The Journal of Immunology 2865 routine determination of this SNP may guide the course of ther- 14. Zakai, N. A., L. 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