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Division of Clinical Chemistry, Department of Laboratory Medicine, University Hospital Malmö, Lund University, Malmö, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Negatively charged phospholipid membranes, e.g., exposed on activated platelets, bind vitamin K-dependent coagulation factors, thus supporting the assembly of procoagulant enzyme-cofactor complexes (8, 9, 10, 11, 12). Several studies have also shown apoptotic cells to have procoagulant properties (13, 14, 15). However, there are also anticoagulant vitamin K-dependent proteins that bind negatively charged phospholipids, e.g., activated protein C (apc)3 and protein S (16). Protein S functions as cofactor to apc in the degradation of factors Va and VIIIa (17, 18). The importance of protein C and protein S is demonstrated by the fact that deficiency of either is associated with increased risk of thrombosis (19, 20). Because anionic membrane surfaces (such as that provided by activated platelets) have been shown to be equally competent in promoting pro- and anticoagulant reactions (21, 22), it is likely that the surface of apoptotic cells is also able to promote anticoagulant reactions.
In plasma, protein S exists in two forms; 
30–40% circulates as
free protein S, the remainder forming a 1:1 high-affinity
(Kd in a range of 0.1–0.6 nM) complex
with C4b-binding protein (C4BP; Refs. 23, 24, 25, 26). Only free
protein S works as a cofactor to apc (27). Protein S
contains multiple domains, including an N-terminal 
-carboxy glutamic
acid (Gla)-rich domain, which binds Ca2+ and is
responsible for the interaction with negatively charged phospholipids,
a thrombin sensitive region, four epidermal growth factor (EGF)-like
domains, and two laminin G-type domains. The G domains are involved in
the interaction with C4BP, especially residues 605–614 (28, 29), 420–434 (30, 31), and 447–460
(32). C4BP is an important regulator of the classical
pathway of complement. It functions as a cofactor to factor I in the
degradation of C4b (33). Furthermore, it inhibits the
formation of the C3 convertase (formed by C4b and C2a) and accelerates
its decay (34). The structure of C4BP resembles that of an
octopus. It consists of seven identical 
-chains, and one unique
-chain, the chains being held together in a central core. Several
complement control protein (CCP) modules compose each chain, the
-chains having eight CCPs and the -chain having three. Each
-chain binds a C4b, and a positively charged area on the interface
between CCP1 and 2 is important for the binding
(35). The -chain binds protein S, the main
binding site being localized to CCP1 and involving a large hydrophobic
patch (36, 37). CCP2 also seems to contribute slightly to
the interaction (38).
The physiological role of the complex between C4BP and protein S has remained an enigma. It has been suggested that protein S, due to its high affinity for negatively charged phospholipids, localizes C4BP to areas where such phospholipids are exposed (39). The complex binds to synthetic phospholipid vesicles, but not to platelet-derived microparticles, even though free protein S efficiently binds to these particles (40).
It has been demonstrated that complement components attach to apoptotic cells, C1 arguably being the most important. C1q deficiency may predispose to autoimmunity in systemic lupus erythematosus as a consequence of an impaired clearance of apoptotic cells (41, 42, 43, 44). Binding of C1 to apoptotic cells could result in activation of the early classical pathway unless efficient control mechanisms exist. A possible regulation could be achieved by the localization of C4BP to the apoptotic cell surface. The aim of the present investigation was to elucidate whether protein S has the capacity to localize C4BP to the apoptotic cell surface. Using Jurkat cells, a human T lymphocyte line, as a model system for apoptotic cells, we demonstrate protein S-mediated binding of C4BP to apoptotic cells. The surface-bound complex could bind C4b, the main target for C4BP in the complement system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Jurkat cells were cultured in RPMI, with 10%
heat-inactivated FCS, complemented with L-glutamine and
antibiotics (growth medium). For the induction of apoptosis, cells were
collected and resuspended at a concentration of 2 x
106 cells/ml. Apoptosis was induced by addition
of anti-Fas Ab CD95 (CH-11 clone; Immunotech, Marseille, France),
200 ng/ml, to the growth medium and culture was continued for 5 h.
This gave an 50% apoptotic population and 50% live population of
the cells gated (for gating details, see Analysis). After
apoptosis induction, cells were harvested by centrifugation and washed
in binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2). Cells
were then resuspended in binding buffer, to a final concentration of
5 x 106 cells/ml.
Proteins
Purified recombinant annexin V was purchased from BD PharMingen
(San Diego, CA). Human protein S (45), C4BP, and the
C4BP/protein S complex (46) and C4 (47) were
purified from plasma as described in their respective references. To
obtain C4BP without protein S, the purified C4BP/protein S complex was
dialyzed against 80% ethylenglycole, 10 mM Tris-Hcl (pH 7.5) before
being run through a heparin Sepharose (Amersham Pharmacia Biotech,
Uppsala, Sweden) column equilibrated in the same buffer. The column was
washed with 10 mM Tris-HCl (pH 8.0) and C4BP was eluted with a gradient
of 0–0.7 M NaCl in the same buffer. Fractions containing C4BP were
chosen and pooled after analysis using a screen agarose gel
electrophoresis under native conditions. The protein pool was then
dialyzed against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and finally
applied on a gel with coupled MK 104 as described previously
(48). In this fashion, -chain-containing C4BP can be
separated from protein S without losing its ability to bind protein S.
The concentrations were determined by measuring absorbance at 280 nm.
Extinction coefficients (1%, 1 cm) used were 9.5 (protein S), 14.1
(C4BP), and 8.3 (C4). To obtain C4b-like molecules, C4 was incubated
with methylamine as described before (35). The C4(met)
derivative was used throughout the study, but will be called C4b for
clarity. mAbs used were HPS 21 and 54, directed against human protein S
(49), and MK 104, against -chain CCP1 of human C4BP
(50). For the labeling with FITC (isomer I; Sigma-Aldrich,
St. Louis, MO), the proteins were dialyzed against 5 mM MOPS, 0.15 M
NaCl, and then diluted to 0.5–2.0 mg/ml in 5 mM MOPS, 0.15 M NaCl, 0.1
M sodium carbonate (pH 9.0), and 1 mM CaCl2. A
total of 50 µg of an FITC slurry (FITC at 10 mg/ml in DMSO) was added
for every milligram of protein and the mixture was incubated for
60–120 min at room temperature in the dark. The reaction was
terminated by the addition of 0.1 M NH4Cl (final
concentration). FITC-labeled protein was separated from free FITC on a
PD-10 column (Bio-Rad, Hercules, CA), equilibrated with 10 mM HEPES (pH
7.4), 140 mM NaCl. Final protein concentrations were determined by
measuring absorbance at 280 nm, and FITC-labeling was confirmed by
measuring absorbance at 495 nm. Rates of FITC to protein were within
recommended range (0.3–1.0; Ref. 51).
Binding experiments
A total of 75 µl of cells were incubated with protein S, C4BP, plasma purified C4BP/protein S complex, or protein S preincubated with C4BP (at 37°C for 30 min, in the presence of 2 mM CaCl2) in binding buffer in a final volume of 100 µl. In time course studies, binding of protein S leveled of at 25 min at room temperature, and remained stable at least until 60 min (data not shown). Therefore, 25 min was chosen as incubation time. Cells were then pelleted by centrifugation for 2 min at 350 x g, and washed in binding buffer. Following a second centrifugation, 15 µg/ml FITC-labeled Abs, HPS 54 for protein S, and MK 104 for all protein containing C4BP, annexin V labeled with PE (annexin V-PE; BD Biosciences, Mountain View, CA), 5 µl 10x diluted, and 5 µl 7-amino actinomycin D (7-AAD; ViaProbe; BD Biosciences) were added in binding buffer at a final volume of 100 µl. In control experiments, binding of FITC-labeled HPS 54 and 104 to corresponding proteins leveled off around 8 µg/ml and remained stable up to 100 µg/ml (highest concentration tested). Following a 30-min incubation in the dark at room temperature, samples were diluted with 100 µl of ice-cold binding buffer and immediately analyzed in the flow cytometer (FACSort; BD Biosciences). We also tested binding of the C4BP-protein S complex in the presence of human serum. The serum had been depleted of protein S and C4BP using HiTrap columns (5 ml; Amersham Pharmacia Biotech) coupled with HPS 54 and MK 104. Amounts of remaining protein S and C4BP were tested using ELISAs for protein S and C4BP and were below detection levels. We then added back the C4BP-protein S complex (final concentration in serum 200 µg/ml) and incubated apoptotic cells for 30 min at 37°C with 20% human serum where C4BP-protein S had been added back. Cells were washed and binding detected in the flow cytometer using FITC-labeled MK 104 as described above.
The apparent Kd for free protein S
bound to the apoptotic cell surface was obtained from the plot of the
increase in fluorescence as a function of the protein S concentration.
The Kd was calculated by fitting the
data to the following equation for a single site-binding isotherm using
nonliner least square regression analysis:
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Preincubation using mAbs
C4BP/protein S complex was preincubated with HPS 21 or 54 for 30 min at 37°C at a molar ratio of 1:2. Then, the same protocol was followed as for binding experiments. For protein S a different approach was used. Protein S labeled with FITC was preincubated with HPS 21 or 54 for 30 min at 37°C at a molar ratio of 1:1. A total of 75 µl of Jurkat cells were then incubated for 25 min at room temperature, in the dark, with 100 nM of the protein S/mAb mixture in binding buffer in a final volume of 100 µl. This was then diluted in ice-cold binding buffer and immediately analyzed in the flow cytometer.
Annexin V inhibition
Apoptotic Jurkat cells, 75 µl, were incubated for 25 min at room temperature with protein S, 100 nM, and recombinant annexin V, 0, 3, or 100 nM in binding buffer at a final volume of 100 µl. Cells were then washed as described above and incubated with FITC-labeled HPS 54, 15 µg/ml, for 30 min at room temperature in the dark. Samples were then diluted in 100 µl of ice-cold binding buffer and immediately analyzed in the flow cytometer.
Binding of C4b
For the C4b-binding, the same protocol was used as for binding experiments, except that the C4BP/protein S concentration was kept to a final concentration of 250 nM, FITC-labeled C4b at increasing concentrations was added instead of MK 104-FITC, and annexin V-PE was not diluted. To see whether binding of C4b to C4BP/protein S on the apoptotic cell could be inhibited by a blocking Ab, unlabeled MK 104 (3.3-fold molar excess over C4b added) was added at the same time as C4b-FITC (300 nM) in a separate experiment.
Analysis
Flow cytometric results were analyzed using CellQuest software (BD Biosciences). Fluorescence was measured on a log scale and is expressed as geometric mean fluorescence, or percentage of maximal binding seen. A gate was set around cells where no dramatic size changes were discernible, i.e., forward and side scatter were unchanged as compared with cells where no anti-Fas Ab had been added. Early apoptotic cells were defined as cells staining single positive for annexin V-PE. Late apoptotic and necrotic cells were defined as cells staining double positive for annexin V-PE and 7-AAD; these cells were excluded from analysis. Live cells were defined as cells staining double negative for annexin V-PE and 7-AAD. There was no difference observed in the various binding studies between this live cell population and control cells, cultured without anti-Fas Ab. In all experiments, at least 10,000 cells were collected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat cells, induced to undergo apoptosis with anti-Fas Ab,
were mixed with protein S. Following incubation, cells were washed with
ice-cold binding buffer and mixed with FITC-labeled mAb HPS 54,
directed against the first EGF-like domain on protein S. Annexin V-PE,
which binds to negatively charged phospholipid and thus was used for
early detection of apoptosis, and 7-AAD were also added. Samples were
diluted in ice-cold binding buffer and FACS analysis was immediately
performed. Early apoptotic cells were defined as the population
staining positive for annexin V-PE (binding negatively charged
phospholipid) and negative for 7-AAD (nucleic acid dye), live cells
were defined as the population that was negative for both. As shown in
Fig. 1
A, apoptotic cells bound
protein S in a dose-dependent manner, whereas there was virtually no
binding of protein S to live cells. Due to variation between
experiments with regard to fluorescence intensity/cell, we have chosen
to plot binding as mean percentage of maximum binding seen in each
different experiment. The actual fluorescence intensity/cell (geometric
mean ± SD) seen at highest concentration of protein S was
2285 ± 291. Binding was saturable, which enabled us to calculate
an apparent Kd of the protein S
association to the apoptotic cell. Free protein S demonstrated a
Kd of 220 nM. For reference, the
concentration of free protein S in human plasma is 100 nM, which
constitutes 30% of total protein S (45, 46).
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C4BP binds to apoptotic Jurkat cells in the presence of protein S
To test whether C4BP binds to apoptotic cells and if protein S has
any influence on the interaction, FITC-labeled MK 104, directed against
the -chains of C4BP, was used for detection. Three different forms
of C4BP were tested. The first variant was the C4BP/protein S complex
purified from plasma. The second variant was C4BP isolated from the
purified C4BP/protein S complex on a heparin Sepharose column run in
ethylene glycol. This breaks the strong hydrophobic bond between C4BP
and protein S, and allows C4BP to be separated from protein S. This
C4BP retains the ability to bind protein S. The third variant tested
was C4BP incubated in vitro with protein S, to reconstitute the
complex. All three forms of C4BP; i.e., in complex with protein S,
free, and recomplexed with protein S, were tested for their ability to
bind apoptotic cells. The results presented in Fig. 1
B
clearly show that C4BP/protein S complexes binds to apoptotic, but not
to live Jurkat cells. Binding was dose-dependent, and C4BP by itself
demonstrated almost no binding. For reference, the concentration of
C4BP in human plasma is 300 nM, the majority being -chain
containing and thus circulating in complex with protein S
(52). Due to variation between experiments with regard to
fluorescence intensity/cell, we have chosen to plot binding as mean
percentage of maximum binding seen in each different experiment. The
actual fluorescence intensity/cell (geometric mean ± SD) seen at
the highest concentration of C4BP-protein S complex was 465 ±
175. To investigate whether the very slight binding observed by C4BP
alone was due to small amounts of contaminating protein S (although not
detected by Western blot), we repeated the experiment using
FITC-labeled HPS 54 instead of the anti-C4BP Ab. The results were
very similar to those obtained with the FITC-labeled MK 104, showing
that indeed the binding of C4BP was due to trace amounts of protein S
in the sample (data not shown). We also tested binding of the
C4BP-protein S complex to apoptotic cells in the presence of serum,
using detection with FITC-labeled MK 104. Also in the presence of serum
there was a clear binding of the C4BP-protein S complex as shown in
Fig. 2.
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FITC-labeled protein S and unlabeled C4BP/protein S (plasma
purified complex) were preincubated with mAbs HPS 21 and HPS 54. HPS 21
is directed against the Gla domain of protein S and blocks binding of
protein S to phospholipid, whereas HPS 54 is directed against the first
EGF-like domain in protein S (49). The proteins were then
mixed with Jurkat cells induced to undergo apoptosis with anti-Fas
Ab as before. Following incubation, cells incubated with FITC-labeled
protein S were immediately analyzed in the flow cytometer while the
cells mixed with the C4BP/protein S complex were washed and incubated
with FITC-labeled MK 104 before analysis. Preincubation with HPS 21
blocked binding of both protein S and C4BP/protein S, strongly
suggesting that the Gla domain of protein S is what attaches both free
protein S and the C4BP/protein S complex to the apoptotic cell (Fig. 3, A and B). In
contrast, HPS 54 did not block binding. Rather, it seemed to enhance
the binding of FITC-labeled protein S (when the molar ratio between
protein and Ab was 1:1) as compared with binding of protein S without
preincubation with the mAb HPS 54 (data not shown). We have previously
reported a similar phenomenon when studying protein S binding to
synthetic phospholipid vesicles. The explanation was then found to be
cross-linking of two protein S molecules on the phospholipid surface by
the divalent Ab (49). In our binding experiments using
detection of cell-bound protein S using FITC-labeled HPS 54, saturating
concentrations of HPS 54 were used (see Materials and
Methods), thus, no cross-linking was to be expected in those
experiments. The binding of protein S to the apoptotic cell surface was
Ca2+-dependent, and addition of EDTA at the end
of the incubation of FITC-labeled protein S and cells completely
abrogated the binding (data not shown).
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As annexin V binds with a high affinity to the negatively charged
phospholipid exposed early in apoptosis (53), we tested
whether an equimolar concentration of annexin V could affect the
binding of protein S to the apoptotic cell. Annexin V, 100 nM was added
to cells together with protein S, 100 nM. In addition, we also tested
the effect of 3 nM unlabeled annexin V, which corresponds to the
concentration of PE-labeled annexin V used in our binding experiments.
Cells were washed and then incubated with FITC-labeled HPS 54
and 7-AAD. Cells were transferred to tubes containing ice-cold binding
buffer and immediately analyzed in the flow cytometer. In Fig. 4 we demonstrate that in the presence of
annexin V, protein S binding to the apoptotic cells is diminished,
strongly suggesting that the interaction is between phosphoplipid and
the protein S Gla domain. As annexin V-PE was not included in this
experiment, it was not possible to selectively gate for the apoptotic
population. Fig. 4B shows the same experiment where cells
had not been induced to undergo apoptosis. However, it was clearly
shown that annexin V is able to impair the protein S binding to
apoptotic cells, demonstrating that the binding sites for protein S and
annexin V on the apoptotic cells are the same.
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To investigate whether the C4BP that was localized to the
apoptotic cells could function to bind C4b, we preincubated cells
induced to undergo apoptosis with the C4BP/protein S complex. The cells
were washed, and FITC-labeled C4b was added in the presence of annexin
V-PE and 7-AAD. C4b-FITC was found to bind to apoptotic cells
preincubated with the C4BP/protein S complex as compared with the
apoptotic cells that were not preincubated with the C4BP/protein S
complex (Fig. 5). Clearly, C4BP is able
to bind C4b even when it is bound via protein S to apoptotic cells,
demonstrating the physiological relevance of the C4BP/protein S complex
binding. The mAb MK 104 directed against CCP1–2 of the C4BP -chain
(50), when added at the same time as C4b-FITC, could block
the binding between C4BP and C4b. We have previously described the
binding site of C4b on C4BP to be localized to the interface of
-chain CCP1–2 (35).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results demonstrate binding of C4BP/protein S complexes to apoptotic cells and provide new insight into the regulation of complement on apoptotic cells. In addition, binding of C4BP was totally dependent on protein S, because C4BP by itself did not display any binding. Protein S is shown to use the same binding site as annexin V, and binds to the cell surface exposed phosphatidylserine via its Gla domain. Furthermore, C4BP localized via protein S to the cell surface could still bind C4b, implying that C4BP retains its physiological role also when attached to an apoptotic cell surface, because C4b will be cleaved by factor I when C4b is associated with C4BP (61, 62). This is similar to our previous results, where we saw that phospholipid vesicle-associated C4BP/protein S complex still could bind C4b (63). In addition, we could still detect binding of the protein S-C4BP complex to apoptotic cells when they were incubated with physiological concentration of C4BP-protein S in the presence of human serum, demonstrating that the affinity is sufficiently high for the complex to associate specifically with the apoptotic cells also when serum proteins are present. We were interested to study the physiological consequences of the binding of the protein S-C4BP complex to apoptotic cells. However, our efforts to reliably and reproducibly induce and follow complement activation on these cells have been unsuccessful. Nonetheless, the present study demonstrates protein S-mediated binding of C4BP to apoptotic cells and suggests a potential physiological role for the complex in vivo, in localizing C4BP, via protein S, to apoptotic cells where C4BP can down-regulate complement activation.
From a number of studies (41, 42, 43, 44, 54), it appears that early complement components are important for the rapid clearance of apoptotic cells, but that the cell must be protected from assembly of later components in order not to provoke an inflammatory response triggered by the complement system. Apart from forming the cell lytic membrane attack complex, activation of complement yields an inflammatory response as anaphylatoxins (mainly C5a and C3a) and opsonins (mainly C3b) are generated. Thus, the presence of C4BP, a major inhibitor of the classical pathway, may be of utmost importance to inhibit inflammation close to apoptotic cells. As we have shown in the present study that C4BP cannot bind in the absence of protein S, our results point toward the physiological relevance of the C4BP/protein S complex formation. Although it has been suggested before, this is the first time we show that protein S is crucial to localize C4BP to an area where complement should be prevented.
Serum amyloid P component (SAP) has been shown to bind apoptotic cells (64). It is conceivable that SAP may modulate the function of apoptotic cell surface-bound C4BP, because SAP has been shown to have an effect on C4b-binding/factor I cofactor function of C4BP (65). However, the interaction between C4BP and SAP is inhibited by the presence of phopshoethanolamine-containing compounds (63, 65). Because SAP was shown to bind to the apoptotic cells via phosphatidylethanolamine, it is possible that SAP does not interact with C4BP bound to the apoptotic cell surface, but rather binds directly to the cell surface. In the present investigation, we observed that the C4BP-protein S complex could bind to apoptotic cells in the presence of serum, indicating that the complex can bind in the presence of SAP. Moreover, addition of serum concentrations of SAP in the binding studies did not affect binding of the C4BP-protein S complex to apoptotic cells (data not shown).
It has also been suggested that the death of T lymphocytes in atherosclerotic plaques may be considered beneficial if apoptosis is not accompanied by an inflammatory reaction (66). Our results and previous studies on Jurkat cells (42, 43) imply that strictly regulated complement could be essential in the clearance of such cells.
C4BP by itself did not bind apoptotic cells, while protein S did. As apoptotic cells expose negatively charged phospholipids, as do activated platelets, the apoptotic cell surface has been considered, and also shown, to be procoagulant (13, 14, 15). Given our results and the fact that protein S has among the highest affinities of the vitamin K-dependent proteins for negatively charged phospholipids (67), the surface of the apoptotic cell may not be solely procoagulant, but also anticoagulant. In short, where there is coagulation, there is also anticoagulation. Casciola-Rosen et al. (15) showed that surface blebs of apoptotic cells are sites of enhanced procoagulant activity. These surface blebs resemble microparticles formed by activated platelets (68) to which we have shown protein S to bind. We saw no binding of the C4BP/protein S complex to these platelet-derived microparticles. The reason why these microparticles differed from apoptotic cells in this regard is unclear. Considering that they both expose negatively charged phospholipids, one could expect that they would display similar binding of not only free, but also C4BP-bound protein S. The explanation to this may lie in the physiological role of the platelet microparticles, foremost providing an area for coagulation or anticoagulation rather than complement activation. Because protein S loses its anticoagulant cofactor function when in complex with C4BP, binding of the complex to platelet microparticles would be without gain. In contrast, on the apoptotic cell surface where complement components have associated, binding of the complex can be of great importance. It is conceivable that this surface may harbor both free protein S, performing its anticoagulant function, and protein S with C4BP, regulating complement action. The potential role of protein S in controlling coagulation on apoptotic cells is an important and interesting area of investigation, especially as patients suffering from protein S deficiency are at an increased risk of thrombosis.
In conclusion, we show that apoptotic cells bind protein S, and that protein S can localize the complement regulatory protein C4BP to the surface of apoptotic cells, thus providing the potential for local regulation of complement activation. In addition, the cell-bound C4BP could still associate with C4b, strongly suggesting a physiological role for the C4BP/protein S complex in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Björn Dahlbäck, Division of Clinical Chemistry, Department of Laboratory Medicine, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden. E-mail address: Bjorn.Dahlback{at}klkemi.mas.lu.se
3 Abbreviations used in this paper: apc, activated protein C; C4BP, C4b-binding protein; CCP, complement control protein; 7-AAD, 7-amino actinomycin D; SAP, serum amyloid P component; EGF, epidermal growth factor; Gla, -carboxy glutamic acid.
Received for publication April 4, 2002. Accepted for publication June 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1H in proteolysis of C4b and C3b. J. Exp. Med. 150:267.-chain is crucial for C4b binding and factor I cofactor function. J. Biol. Chem. 274:19237.-chain contains the protein S-binding site. J. Biol. Chem. 271:20861.-chain of complement regulator C4b-binding protein. J. Biol. Chem. 276:4330.-chain short consensus repeats. J. Biol. Chem. 274:15144.-chain is crucial for C4b binding and factor I cofactor function. J. Biol. Chem. 274:19237.
-chain is crucial for C4b binding and factor I-cofactor function. Biochem. J. 323:469.
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B. Dahlback and B. O. Villoutreix Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway: Novel Insights Into Structure-Function Relationships and Molecular Recognition Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1311 - 1320. [Abstract] [Full Text] [PDF]
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 L. A. Trouw, S. C. Nilsson, I. Goncalves, G. Landberg, and A. M. Blom C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation J. Exp. Med., June 20, 2005; 201(12): 1937 - 1948. [Abstract] [Full Text] [PDF]
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 T. Meri, A. M. Blom, A. Hartmann, D. Lenk, S. Meri, and P. F. Zipfel The Hyphal and Yeast Forms of Candida albicans Bind the Complement Regulator C4b-Binding Protein Infect. Immun., November 1, 2004; 72(11): 6633 - 6641. [Abstract] [Full Text] [PDF]
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T. Nordstrom, A. M. Blom, A. Forsgren, and K. Riesbeck The Emerging Pathogen Moraxella catarrhalis Interacts with Complement Inhibitor C4b Binding Protein through Ubiquitous Surface Proteins A1 and A2 J. Immunol., October 1, 2004; 173(7): 4598 - 4606. [Abstract] [Full Text] [PDF]
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L. Kask, L. A. Trouw, B. Dahlback, and A. M. Blom The C4b-binding Protein-Protein S Complex Inhibits the Phagocytosis of Apoptotic Cells J. Biol. Chem., June 4, 2004; 279(23): 23869 - 23873. [Abstract] [Full Text] [PDF]
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 S. M. Rezende, R. E. Simmonds, and D. A. Lane Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex Blood, February 15, 2004; 103(4): 1192 - 1201. [Abstract] [Full Text] [PDF]
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B. Dahlback Successful Hunt for Quantitative Trait Locus in Thrombophilia Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 376 - 377. [Full Text] [PDF]
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, Live cells; 
, apoptotic cells. B,
Binding of C4BP. 
, apoptotic cells, C4BP preincubated
with protein S; 
, live cells, C4BP without protein S; 
, apoptotic
cells, C4BP without protein S.
