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Published as doi: 10.1096/fj.08-110429.
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(The FASEB Journal. 2008;22:4077-4084.)
© 2008 FASEB

Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP)

Sacha Zeerleder*,1, Bas Zwart*, Henk te Velthuis{dagger}, Femke Stephan*, Rishi Manoe*, Irma Rensink* and Lucien A. Aarden*

* Department of Immunopathology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and

{dagger} Business Unit Reagents, Sanquin, Amsterdam, The Netherlands

1Correspondence: Department of Immunopathology, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: s.zeerleder{at}sanquin.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma proteins such as early complement components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleosome-releasing factor (NRF) activity. However, recalcified plasma (r-plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size-exclusion and anion-exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS-PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII-activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to completely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single-chain (sc) FSAP from r-plasma. Purified scFSAP efficiently removes nucleosomes from sn cells. We report that factor VII-activating protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.—Zeerleder, S., Zwart, B., te Velthuis, H., Stephan, F., Manoe, R., Rensink, I., Aarden, L. A. Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP).


Key Words: apoptosis • hyaluronan binding protein • SLE • plasmin • monoclonal • single chain


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN MULTICELLULAR ORGANISMS, the process of apoptosis is essential for the control of tissue homeostasis (1) . Apoptotic cells are efficiently removed by professional scavenger cells to prevent the release of potentially harmful cytotoxic and immunogenic cellular content due to loss of membrane integrity on secondary necrosis (2) . Apoptotic cells not efficiently removed become late apoptotic or secondary necrotic (sn). Secondary necrotic cells are reported to bind various plasma proteins, such as soluble IgM, serum amyloid P, C-reactive protein, and complement proteins (3 4 5 6 7) . Binding of these plasma proteins enables professional phagocytes to recognize and eliminate sn cells (3 , 6 7 8) . The importance of plasma proteins in the removal of sn cells is illustrated by the fact that a deficiency of IgM or of the early complement components is associated with a defective or delayed clearance of apoptotic cells (9 , 10) . Delayed removal of sn cells exposes the immune system to intracellular contents, which may lead to autoantibody formation and finally to the development of autoimmune disease, such as systemic lupus erythemathosus (SLE) (11 , 12) .

We have previously shown that induction of apoptosis leads to swift fragmentation of DNA in human T-cell line cells, but that in the absence of serum or plasma it takes days for the nucleosomes to be released from these sn cells (13) . In the presence of plasma, DNA release starts immediately and is complete within minutes (14) . This release of nucleosomes is independent of the method to induce apoptosis and was found to occur with different cell types, including primary cells (14) . The DNA is released in the form of nucleosomes as indicated by its reactivity in a nucleosome ELISA (14) . This sandwich ELISA uses a monoclonal antibody to histone H3 in combination with a monoclonal antibody recognizing an epitope formed by a combination of DNA and histones H2B and H2A (13) . We showed that the capacity of plasma to induce the release of nucleosomes from sn cells’ nucleosome-releasing factor (NRF) was due to a plasmin-like protease because it was inhibited by the protease inhibitor aprotinin (14) . Indeed, purified plasmin added to sn cells showed NRF activity, whereas plasminogen was inactive. Also, other investigators recently demonstrated chromatin breakdown by DNAseI in necrotic cells to be dependent on plasminogen (15) . However, we still found undiminished NRF activity in plasmin and plasminogen-depleted plasma, suggesting another protease to be involved. The aim of this study was to identify the protease responsible for NRF activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Etoposide, RNase, propidium iodide (PI), plasmin, and β-mercaptoethanol were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). NuPAGE 4–12% polyacrylamide gels, polyvinylidene fluoride (PVDF) and nitrocellulose blotting membranes were purchased from Invitrogen (Breda, The Netherlands). Aprotinin (Trasylol®) was obtained from Bayer (Mijdrecht, The Netherlands). Q-sepharose and Superdex 200HR 10/30 columns were obtained from Amersham Biosciences (Uppsala, Sweden). ProtG sepharose (4 fast flow) was from GE Healthcare Europe GmbH (Diegem, Belgium). Biogel A 1.5 m was obtained from Bio-Rad Laboratories B.V. (Veenendaal, The Netherlands). Ultragel ACA34 column was obtained from Pall Medical USA (Ann Arbor, MI, USA). Iscove’s Modified Dulbecco’s Medium (IMDM) and biotin (NHS-LC biotin II, Pierce) were purchased from Perbio Science Nederland BV (Etten-Leur, The Netherlands), and YM 10 filters were obtained from Millipore International Holding Company BV (Amsterdam, The Netherlands). Anti-IL6/8, rat anti-mouse {kappa} (RM19) antibodies and the high-performance ELISA buffer were obtained from Sanquin (Amsterdam, The Netherlands). Vacutainer tubes were obtained from BD (Alphen aan den Rijn, The Netherlands). Penicillin and streptomycin were delivered by Gibco, Invitrogen, and 3,3',5,5'-tetramethylbenzidin (TMB) was provided by Merck (Darmstadt, Germany).

Cell culture and induction of apoptosis
Jurkat cells were cultured in culture medium [IMDM-FCS+: IMDM containing 5% fetal calf serum (FCS), penicillin (50 IU/ml), streptomycin (50 µg/ml), and 50 µM β-mercaptoethanol]. Before apoptosis induction, Jurkat cells were washed twice with IMDM without FCS (IMDM-FCS–). To get sn cells, Jurkat cells were incubated for 24 to 48 h in IMDM-FCS– containing 200 µM etoposide.

Recalcified plasma
Blood was collected from healthy donors in siliconized tubes containing sodium citrate at a final concentration of 10 mM. The tubes were immediately centrifuged 2 times for 10 min at 1300 g at 4°C to obtain platelet-poor plasma. Plasma was transferred to a glass vial, recalcified with CaCl2 at a final concentration of 10 mM, and incubated for 15 min at 37°C, followed by incubation for 30 min at 4°C until a retracted clot had formed. The clot was removed, and the recalcified plasma (r-plasma) was stored at –20°C until use.

Assessment of NRF activity
Secondary necrotic Jurkat cells were washed twice with 10 mM HEPES (pH 7.2), 2 mM CaCl2, and 140 mM NaCl (HEPES buffer) containing 1% (w/v) bovine serum albumin and resuspended in the same buffer at a concentration of 2 x 106/ml. RNase was added to the cells to a final concentration of 40 µg/ml and incubated for 30 min at 37°C. Thereafter, 100 µl aliquots of either r-plasma in various dilutions (diluted in HEPES-BSA buffer) or chromatography fractions were added to 100 µl of cells and incubated for another 30 min at 37°C. Subsequently the cells were washed twice with FACS buffer [10 mM HEPES, pH 7.2, containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 0.5% (w/v) bovine serum albumin] and stained with PI at a final concentration of 0.5 µg/ml. The samples were analyzed by flow cytometry. The median fluorescence intensity (MFI) of PI was quantified with either a FACSCaliburTM flow cytometer or a BD LSRII flow cytometer (Becton Dickinson, Mountain View, CA, USA) by using WinMDI v. 8 free-downloadable software (FACSCaliburTM flowcytometer; http://www.facs.scripps.edu) or FACS Diva software (Becton Dickinson). NRF activity is expressed as the percentage decrease of PI signal-taking cells incubated with buffer control as maximum and cells incubated with 20% plasma as minimum signal. In the figure legends the maximum (max) and minimum (min) MFI are indicated.

Isolation of NRF
Size-exclusion chromatograph
R-plasma (1.5 ml) was applied on a Biogel A 1.5 m column that had been equilibrated with 20 column volumes (CVs) PBS containing 0.02% Tween 20 and 0.02% sodium azide. The fractions were collected and stored at –20°C until use. All fractions were tested for NRF activity.

Plasma precipitation
Recalcified pooled plasma (25 ml) was dialyzed against 50 mM sodium acetate, pH 5.0, at 4°C overnight. The sample was then centrifuged at 2000 g for 20 min. The supernatant was removed and stored at –20°C until use. The pelleted precipitate was washed 3 times with 50 mM sodium acetate, pH 5.2, and resuspended in 5 ml 20 mM Tris-HCl, pH 8.0. The precipitate was stored until use at –20°C. Both the precipitate and the supernatant were tested for NRF activity.

Ion-exchange chromatography
The NRF-containing sample was further processed by means of high-performance liquid chromatography. After equilibrating an 8 ml Q-Sepharose column with 10 CV 20 mM Tris-HCl, pH 8.0, the NRF-containing sample, which had already been dialyzed against the starting buffer, was loaded. The column was washed with 80 ml 20 mM Tris-HCl, pH 8.0, 300 mM NaCl. Elution was performed using a continuous NaCl gradient from 300 mM up to 2 M. The elution fractions from the ion-exchange chromatography were collected (4 ml/fraction) and stored at –20°C until use. All fractions were tested for NRF activity. The fractions having NRF activity were pooled (28 ml) and concentrated to a final volume of 450 µl using an YM 10 filter. After dialysis against the HEPES buffer, 350 µl of the concentrated pooled elution was applied to a Superdex 200HR 10/30 column using HEPES buffer as running buffer. The fractions (0.5 ml) were collected and stored at –20°C until use. All fractions from the size-exclusion chromatography were tested for NRF activity.

SDS-PAGE
The various steps in the purification procedure were analyzed on SDS/PAGE using NuPAGE Novex 4–12% Bis-Tris polyacrylamide gels (Invitrogen) following the manufacturer’s instructions. After electrophoresis the gels were stained with silver. In some experiments samples were concentrated by 5% trichloroacetic acid precipitation. After washing with ethanol the samples were applied to SDS/PAGE NuPAGE Novex 4–12%. The proteins were then blotted onto a PVDF membrane following standard procedures. N-terminal sequences of excised blotted protein bands were analyzed at the Sequence Centre Utrecht (Institute of Biomembranes, University of Utrecht, The Netherlands) using an automated system (Applied Biosystems 476A). The obtained amino acid sequences were further analyzed using the BLAST network service of the Swiss Institute of Bioinformatics (SIB). The SIB BLAST network service uses a server developed at SIB and the NCBI (http://www.expasy.ch/tools/blast).

Preparation of monoclonal antibodies
Balb/c mice were hyperimmunized by repeated subcutaneous injections of 25 µg of 2-chain factor VII-activating protease (tcFSAP) purified from plasma (a kind gift of Prof. K. Mertens, Department of Plasma Proteins, Sanquin, Amsterdam, The Netherlands) using montanide as adjuvans. Fusion of spleen cells from immunized mice with Sp2/0 myeloma cells and hybridoma selection were performed as described in detail elsewhere (16) . The specificity of the antibodies produced by the hybridomas was analyzed by ELISA. Briefly, microtiter plate wells were coated with monoclonal rat anti-mouse {kappa} IgG (RM19, at 2 µg/ml) in PBS overnight. After 5 washes with PBS-0.02% (w/v) Tween 20 (PT), the supernatant of the hybridoma cells was diluted in PTG buffer (PBS, 0.2% gelatin, 0.02% Tween 20) and incubated for 60 min at room temperature. After 5 washes with PT, biotinylated FSAP (see below) diluted in PTG buffer was incubated for 60 min at room temperature. Plates were washed 5 times with PT, and streptavidin-polyhorseradish peroxidase (Sanquin) diluted 10,000 times in PTG buffer was added for 20 min. After washing 5 times with PT, plates were developed by adding 100 µg/ml TMB and 0.003% (v/v) hydrogen peroxide in 0.11 M sodium acetate buffer (pH 5.5) for 5 min, and the reaction was stopped by adding 2 M H2SO4. The absorbance was measured at 450 nm.

Biotinylation of FSAP
FSAP (1 mg/ml in PBS) was diluted in 0.05 M NaHCO3, pH 9.6, and incubated with 600 µg NHS-LC biotin II (Pierce) for 2 h at room temperature in the dark. Thereafter FSAP was dialyzed overnight against PBS.

Immunoblot analysis of anti-FSAP antibodies
Western blotting was performed according to the manufacturers’ instruction. In brief, SDS/PAGE 4–12% gels were blotted on to a nitrocellulose membrane (Novex®) and blocked with Western blocking reagent 1% in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). Thereafter the blotting membrane was incubated with biotinylated mAbs to FSAP or with a biotinylated monoclonal antibody with the same isotype but with irrelevant specificity at a final concentration of 10 µg/ml followed by detection with streptavidin-alkaline phosphatase (1:500 v/v). Finally, the blotting membrane was stained using nitro blue tetrazolium and 5'-bromo-4'-chloro-3'-indolyl phosphate, and the reaction was stopped with distilled water.

Inhibition of NRF activity by antibodies against FASP
Preparation of sn Jurkat cells is described in detail above. All dilutions are made in HEPES buffer. Diluted r-plasma (50 µl; final dilutions: 20, 10, 5, 2.5, 0%) were incubated for 30 min at 37°C with either 50 µl antibodies against FASP at 10 µg/ml or with a monoclonal antibody of the same isotype but with irrelevant specificity. In another set of experiments, r-plasma and plasmin at final dilutions of 10% and 1 µM, respectively, were incubated for 30 min at 37°C with monoclonal antibodies to FASP (at a final concentration of 10 µg/ml) and aprotinin (40 kIU/ml). Thereafter 100 µl of Jurkat cells (2x106/ml in HEPES buffer-BSA) was added to the samples and incubated for another 30 min at 37°C. Finally, cells were washed with FACS buffer, stained with PI, and analyzed as described above in detail.

Sandwich ELISA to measure FSAP antigen levels
Microtiter plates were coated overnight at 4°C with 2 µg/ml monoclonal antibody to FSAP recognizing the light chain (anti-FSAP4) diluted in PBS. After 5 washes with PT samples diluted in high-performance buffer (HPE) were added and incubated for 60 min at room temperature. After 5 washes with PT biotinlylated anti-FSAP2 recognizing the heavy chain diluted in HPE (0.5 µg/ml) was added to the wells and incubated for 60 min. After 5 washes with PT streptavidin-polymerized horseradish peroxidase diluted 10,000 times in HPE was added to the wells and incubated for 20 min. After another 5 washes with PT, plates were developed by adding 100 µg/ml TMB and 0.003% (v/v) hydrogen peroxide in 0.11 M sodium acetate buffer (pH 5.5) for 10 min. The reaction was stopped by adding 2 M H2SO4. Absorbance was measured at 405 nm. Plasma was used as standard and arbitrarily set as 100%. ELISA detects both sc and tcFSAP.

Purification of single-chain FSAP from plasma
Size-exclusion chromatography
R-plasma (8 ml) containing 1 M NaCl was applied on a Ultragel ACA34 column, which was equilibrated with 10 mM HEPES, pH 7.2, containing 2 mM CaCl2 mM, 1 M NaCl, and 0.02% Tween20. FSAP contents of gel filtration fractions (7.7 ml) were measured by ELISA. The FSAP peak with an apparent molecular mass of 60 kDa was pooled and further purified by affinity chromatography.

Affinity chromatography
Monoclonal anti-FSAP4 (27 mg) was coupled to 1.5 g CBNr-sepharose according to the manufacturer’s instructions. The affinity column was equilibrated with 30 ml 20 mM sodium citrate (pH 6.0) containing 1M NaCl and 0.02% Tween20. The FSAP peak from the size-exclusion column was applied to the anti-FSAP4 column. Column-bound FSAP was eluted with 0.1 M glycine (pH 2.5) containing 0.02% Tween20 (elution volume 4 ml). Directly thereafter, the eluate was neutralized with an equal volume of a modified McIlvain buffer (0.5 M Na2HPO4/1 M citric acid, pH 7.0). Finally, the pH-adjusted eluate was incubated at room temperature for 30 min with 1 ml packed protein G sepharose to remove contaminating IgG. Protein G sepharose was subsequently removed, the supernatant was analyzed on SDS-PAGE, and the activity of the purified material was tested in the NRF.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of NRF from r-plasma
To determine the molecular mass of NRF, r-plasma was subjected to size-exclusion chromatography, and the fractions were tested for NRF activity. The NRF activity was found to migrate at an estimated molecular mass of 60 kDa (data not shown). To further characterize the protease, r-plasma was subjected to cation-exchange chromatography. To this end, r-plasma was first dialyzed overnight at 4°C against 50 mM sodium acetate, pH 5.2. During dialysis, however, a precipitate was formed. The precipitate readily dissolved in 20 mM Tris-HCl, pH 8.0, and appeared to contain all NRF activity (Fig. 1 ). The dissolved precipitate was further purified on a Q-sepharose anion-exchange column. Initial Q-sepharose chromatography experiments showed that, at 300 mM NaCl, NRF activity bound to the column, whereas 90% of the dissolved precipitated plasma proteins did not. Therefore loading in further experiments was started at 300 mM followed by elution with a continuous NaCl gradient. A small protein peak following the broad protein peak eluted at 800–1000 mM NaCl (elution fractions 34–40) was found to contain NRF activity (Fig. 2 ).


Figure 1
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  		Figure 1. Sodium actetate-precipitated plasma proteins contain all NRF activity. After overnight dialysis of r-plasma against 50 mM sodium acetate, pH 5.2, at 4°C, proteins precipitated from solution were pelleted, washed 3 times, and dissolved in 20 mM Tris-HCl, pH 8.0, in a volume corresponding to one-fifth of the original volume of the plasma. Thereafter, NRF activity (MFI max, 2525; MFI min, 332) was measured in the r-plasma (open circles), in the precipitate (open triangles), and in the supernatant (open squares).


Figure 2
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  		Figure 2. Anion-exchange chromatography of acetate-precipitated plasma proteins. Acetate-precipitated plasma proteins, dissolved in 20 mM Tris-HCl, pH 8.0, were loaded on a Q-sepharose column, which was then washed with 20 mM Tris-HCl, pH 8.0, with 300 mM NaCl and eluted with a continuous NaCl gradient from 300 mM up to 2 M. Only the part of the elution starting at 500 mM NaCl is indicated. Fractions 35–38 in the small protein peak eluting at 800–1000 mM NaCl contained most of the NRF activity. Solid line, absorbance at 280 nm (mAU); dotted line, NaCl gradient; gray bars, NRF activity of the fractions (MFI max, 1382; MFI min, 542).

NRF-containing fractions 35–38 were pooled and then subjected to size-exclusion chromatography (Fig. 3A ). The NRF activity of the fractions was compared with the SDS-PAGE pattern. NRF activity in fraction 10–12 coeluted nicely with a 60 kDa protein under nonreducing conditions (Fig. 3B ). On reduction the 60 kDa band disappeared, and 2 protein bands at 45 and 28 kDa were clearly visible (Fig. 3C ). Surprisingly, the elution pattern of the pooled fractions containing NRF activity eluted at a higher molecular mass than predicted from the initial size-exclusion chromatography experiments with plasma.


Figure 3
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  		Figure 3. Size-exclusion chromatography of partially purified NRF activity. Fractions 35–38 of Q-sepharose chromatography were pooled and concentrated by using an YM 10 filter and subsequently applied to a Superdex 200HR 10/30 column. Each fraction was tested for NRF activity (A, gray bars; MFI max, 1240; MFI min, 723) and was also applied to a NuPAGE Novex 4–12% Bis-Tris polyacrylamide gel under nonreducing (B) and reducing (C) conditions. Thereafter, the gels were stained with silver. Of 35 fractions, only fractions 8–15 are shown. Arrows point to the 60 kDa band in B and the 45 and 28 kDa bands in C, which coelute with the NRF activity.

FSAP is identical to NRF
The 28 kDa band visible on SDS gels stained with Coomassie was excised. N-terminal sequence analysis revealed the amino acid sequence shown in Fig. 4 . A homology search in the database of the SIB BLAST network service of the sequence IYGGFKSTAG revealed 100% homology with FSAP originally described as plasma hyaluronan-binding protein (UniProtKB/Swiss-Prot entry Q14520; EC 3.4.21.–) (17) . The sequence IYGGFKSTAG, corresponding to amino acids 291–300 of the full-length FSAP molecule, represents the first 10 amino acids of the N-terminal end of the light chain of FSAP, which has a calculated molecular mass of 27 kDa.


Figure 4
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  		Figure 4. The 28 kDa peptide in NRF-containing chromatography fractions is the light chain of FASP. The fractions from the size-exclusion chromatography containing NRF activity (fraction 10–12) were pooled and concentrated by 5% trichloroacetic acid precipitation. After washing with ethanol, the samples were applied to SDS/PAGE NuPAGE Novex 4–12% under reducing conditions and stained with Coomassie. The proteins were then blotted onto a PVDF membrane following standard procedures. N-terminal sequences of excised blotted protein bands were analyzed at the Sequence Centre Utrecht. The amino acids were determined to show 100% homology to those of the light chain of FASP (Swiss-Prot entry Q14520; EC 3.4.21.–).

Inhibition of NRF activity by monoclonal antibodies
Mice were immunized with full-length FSAP isolated from human plasma for the preparation of monoclonal antibodies. We obtained more than 20 monoclonal antibodies. One of these antibodies, anti-FSAP 4, inhibited NRF activity of plasma (Fig. 5A ) and was shown to recognize the light chain of FSAP as indicated by immunoblot analysis (Fig. 5B ). Anti-FSAP 2, which reacts with the heavy chain of FSAP as well as mAbs directed to unrelated antigens, did not inhibit NRF activity. The specificity of anti-FSAP 4 was confirmed by analyzing their effect on the NRF activity of plasmin. As expected, anti-FSAP 4 did not inhibit the NRF activity of purified plasmin (Fig. 6 ).


Figure 5
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  		Figure 5. Inhibition of NRF activity by antibodies against FSAP and characterization of antibodies to FSAP by immunoblot analysis. A) NRF activity (MFI max, 5787; MFI min, 1106) after incubation of r-plasma with monoclonal antibodies anti-FSAP 2 (open circles), anti-FSAP 4 (closed squares), and a monoclonal antibody (anti-IL6/8) of the same isotype with an irrelevant specificity as a control (closed circles). B) Characterization of antibodies to FSAP by immunoblot. Two-chain FSAP was applied on SDS/PAGE 4–12% gel under reduced conditions and blotted onto a nitrocellulose membrane. Detection was performed by using biotinylated antibodies (10 µg/ml) to FSAP. Anti-FSAP 4 (left panel) recognize the light chain, and anti-FSAP 2 (right panel) react with the heavy chain of FSAP. A biotinylated antibody with the same isotype and of an irrelevant specificity did not recognize FSAP (not shown).


Figure 6
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  		Figure 6. Blocking antibodies to FSAP do not inhibit the NRF activity of plasmin. Antibodies to FSAP (at a concentration of 10 µg/ml) and aprotinin (40 kIU/ml) were either incubated with 10% r-plasma or with 1 µM plasmin diluted in HEPES buffer. Anti-FSAP 4 ({alpha}FSAP 4, 10 µg/ml) abrogated NRF activity in plasma but had no effect on the NRF activity of 1 µM plasmin. In contrast, aprotinin inhibited NRF activity of both plasma and plasmin (MFI max, 2484; MFI min, 181). Results are means ± SD (n=3).

NRF activity of single-chain FSAP purified by means of affinity chromatography from plasma
In another approach we wanted to show that purified plasma-derived FSAP induces nucleosome release from sn cells. To obtain single-chain FSAP, r-plasma was first subjected to size-exclusion chromatography. The FSAP-containing fractions were pooled and further purified by means an anti-FSAP4 affinity column and protein G depletion. On SDS-PAGE the eluate of the affinity chromatography revealed only a 64 kDa band under reducing and nonreducing conditions, indicating that the FSAP obtained was in its single-chain form (Fig. 7A ). When tested in the NRF assay, purified plasma-derived FSAP was fully active (Fig. 7B ).


Figure 7
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  		Figure 7. Purified, plasma-derived scFSAP removes nucleosomes from apoptotic cells. Affinity-purified FSAP was applied to SDS/PAGE NuPAGE Novex 4–12% and stained with silver (A). Under nonreducing (NR) and reducing (R) conditions a single protein band at 64 kDa was visible, indicating that the FSAP was in its single-chain form after purification. The activity of the purified scFSAP was tested in the NRF assay (B). The purified scFSAP efficiently removed nucleosomes from sn cells (MFI max, 5354; MFI min, 827). Results are means ± SD (n=3). Open squares, plasma; open circles, scFSAP.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have identified the plasma protease that is responsible for the removal of nucleosomes from sn cells, NRF, as being FSAP. First, a monoclonal antibody to the light chain of FSAP completely blocks NRF activity of plasma. Second, purified scFSAP is fully active in the NRF assay. This identification is in agreement with earlier findings on the nature of NRF. Before we characterized NRF as a plasmin-like protease that is efficiently inhibited by aprotinin (14) . Aprotinin has been shown to be an efficient inhibitor of the amidolytic activity of FSAP (18 , 19) . The observed size (60 kDa) as determined by gel filtration is in reasonable agreement with the expected 64 kDa. Also, the finding that NRF activity elutes from an anion-exchange column at NaCl concentrations higher than 700 mM is in line with recently published FSAP purification protocols (19 20 21) . Given the reported isoelectric point in the range 4.9–5.5, the observed elution properties are surprising, but binding of FSAP to plasma hyaluronan, a negatively charged anion, might explain these unexpected chromatography characteristics (17 , 22) . Sequence analysis of the 28 kDa band that copurified with NRF activity revealed a 100% homology with the N terminus of the light chain of FASP. Surprisingly, size-exclusion chromatography of the pooled fractions of the anion-exchange chromatography containing NRF activity eluted at a higher molecular mass than the predicted 60 kDa based on the size-exclusion chromatography experiments in plasma. At the same time this procedure leads to conversion of sc to tcFSAP. Very likely this purification procedure leads to activation of FSAP and to complexation of the activated FSAP to protease inhibitors.

It has been reported that a plasma protease is instrumental in removing nucleosomes from DNase-treated necrotic cells (15) . Very likely this phenomenon is related to our finding that a plasma protease is involved in the removal of nucleosomes from sn cells. In the latter case, no DNase treatment is required because the apoptotic process has already taken care of DNA cleavage. However, the responsible protease was reported to be plasmin. Previously we showed that plasmin has NRF activity but that NRF is not plasmin because depletion of plasmin and plasminogen from plasma did not affect NRF activity (14) . The identification of FSAP being responsible for NRF activity is in line with these observations. It remains to be investigated whether FSAP is also involved in the removal of nucleosomes from DNase-treated necrotic cells. The neutralizing monoclonal antibody to FSAP could be useful in that analysis.

Kannemeier et al. (22) reported FSAP to circulate as a single-chain molecule (scFSAP) in plasma with an apparent molecular mass of 60 kDa. Most purification protocols without protease inhibitors result in activation hence in purification of tcFSAP. Purification of FSAP in high concentrations of urea without protease inhibitors allows purification of scFSAP (23) . The development of suitable monoclonal antibodies allowed us to purify scFSAP in the absence of protease inhibitors or urea. Incubation of scFSAP with sn cells leads to activation of FSAP. Immunoprecipitation revealed that interaction of scFSAP with sn cells leads to conversion to tcFSAP (unpublished results). Because FSAP circulates as a proteolytically inactive single-chain molecule in plasma, we propose that FSAP in plasma is activated on contact with sn cells, and in analogy, the same is expected for purified, plasma-derived FSAP. All the experiments to isolate and identify FSAP have been performed with r-plasma to avoid induction of coagulation on contact with sn cells. Because FSAP is susceptible for autoactivation, one would expect FSAP activation to occur on recalcification of plasma. Apparently recalification of plasma did not lead to activation of FSAP, which is evidenced by the fact that we purified scFSAP from r-plasma with high recovery. Hence the activation of FSAP observed in the initial fractionation of NRF was probably due to the low salt precipitation. The data presented in Fig. 7 suggest that purified scFSAP is even more active then when present in plasma. Possibly the absence of plasma protease inhibitors is responsible for the higher than 100% recovery of NRF activity in the purified FSAP. Recent work suggests that tcFSAP is mainly produced by autoactivation of scFSAP (22) . Glycosaminoglycans (GAGs), such as heparin, promote FSAP (auto) activation (22 , 23) . Thus, endogenous GAGs from sn cells might promote autoactivation of FSAP on sn cells. In addition, RNA and to a lesser extent DNA were reported to act as potent cofactors for FSAP autoactivation (24) . However, stimulation of FSAP autoactivation by RNA in our experiments seems unlikely because the sn cells in our experiments were pretreated with RNase. If GAGs are responsible for the activation of FSAP, it remains to be explained why only apoptotic cells do activate FSAP.

FSAP is abundantly present in plasma, with a reported concentration of 10.5 µg/ml (range 4.36–16.15 µg/ml) in healthy men and 11.15 µg/ml (range 3.6–16.4 µg/ml) in healthy woman (25) The name "factor VII-activating protease" refers to the ability of FSAP to activate factor VII in a tissue factor-independent pathway in vitro (20) . FSAP was also demonstrated to efficiently convert single-chain urokinase to 2-chain urokinase in vitro (18) . The in vivo relevance of FSAP in coagulation and fibrinolysis has not been established. Finally, FSAP has been reported to be a potent inhibitor of platelet-derived growth factor-mediated vascular smooth muscle cell proliferation and migration in vitro and in vivo (26 , 27) . We now report a novel role for FSAP. We demonstrate that FSAP efficiently releases nucleosomes from sn cells. Early apoptotic cells are rapidly cleared in vivo. Delayed removal of early apoptotic cells results in sn cells and exposes the immune system to their intracellular contents, which may lead to autoantibody formation and finally to the development of autoimmune disease, such as SLE (11 , 12) . Elevated nucleosome levels and antinucleosome antibodies can be measured in SLE patients (28 , 29) . Although nucleosomes were recently reported to be exposed on surfaces and apoptotic blebs of cells undergoing apoptosis, little is known on the mechanism of release of nucleosomes from sn cells (30) . Identification of FSAP as a crucial factor in the release of nucleosomes from sn cells has now provided the impetus for follow-up studies on the role of FSAP in the development of autoimmune disease.


   ACKNOWLEDGMENTS
 
S.Z. was supported by a grant of the Landsteiner Foundation for Blood Transfusion Research (LSBR 0422). B.Z. was supported by grant 97.402.01 of the Dutch Arthritis Association. We thank Piet Modderman for his helpful comments on the manuscript.

Received for publication April 7, 2008. Accepted for publication July 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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