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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1661-1667
By
From the Department of Clinical Immunology and Transfusion Medicine,
University Hospital, Uppsala; the Department of Natural Sciences,
University of Kalmar, Sweden; and the Laboratory of Protein Chemistry,
The Department of Pathology and Laboratory Medicine, University of
Pennsylvania, Philadelphia.
Recently, a C3-binding cyclic synthetic peptide (Compstatin) has
been identified that binds to complement component C3 and inhibits
complement activation. Here we have examined the influence of
Compstatin on complement activation and its indirect effects on
cellular responses in whole blood in two models for extracorporeal circulation. Compstatin effectively inhibited the generation of C3a and
sC5b-9 and the binding of C3/ C3 fragments to the polymer surface. As a
result of the inhibition of complement activation, the activation of
polymorphonuclear leukocytes (PMNs; as assessed by the
expression of CD11b) and the binding of these cells
(CD16+) to the polymer surface were almost completely
lost. In contrast, blood cell counts were not affected. Using surface
plasmon resonance technology, we have confirmed that Compstatin exerts
its inhibitory effect on complement activation by binding to native C3.
These data show that complement activation, leading to activation and binding of PMNs to the biomaterial surface, can be abolished by the
addition of Compstatin. The properties of Compstatin make Compstatin a
promising drug for use in extracorporeal circuits to avoid
bioincompatibility reactions, eg, during cardiopulmonary bypass, but
also a favorable precursor peptide for the development of an
anticomplement drug for oral use.
© 1998 by The American Society of Hematology.
THE COMPLEMENT SYSTEM is known to be
activated during extracorporeal circulation, eg, during dialysis,
plasmapheresis, and cardiopulmonary bypass surgery. In particular,
complement-related complications resulting from bioincompatibility
reactions accompanying cardiopulmonary bypass (CPB) have gained recent
attention. Two major areas of concern are neurological sequelae and
postperfusion inflammation reactions that may lead to lung
damage.1-5 During CPB, complement activation is likely to
contribute to both of these conditions by activating platelets and
leukocytes, through which the ongoing "whole body" inflammation
is supported.2,6 During CPB the blood in the oxygenator is
in direct contact with two phases: the polymer surface and the oxygen
gas surface. Both interfaces are known to activate complement by the
alternative pathway, thereby generating C3a and soluble
C5b-9.7,8 Classical pathway activation has also been
reported.9
New inhibitors of complement activation have been shown to decrease the
severity of bioincompatibility reactions during extracorporeal treatment.10 Several types of inhibitors have been
developed in recent years: recombinant natural inhibitors of
complement,11 antibodies to complement
components,10 and protease inhibitors.12,13 However, a specific small-molecular-weight complement inhibitor has not
been available until recently. A recent study has described a specific
peptide inhibitor with a completely different mechanism of
action.14 This peptide (Compstatin) was developed from a clone of a phage-displayed random peptide library that was screened for
binding to C3b, a fragment of complement component C3. Compstatin consists of 13 amino acid residues (ICVVQDWGHHRCT-NH2),
whose ring is maintained by a disulphide bond between the two
cysteines. Further studies on the substitution analogs and the solution
structure of the peptide have indicated that the type-1 In this study we have used two recently developed models for
extracorporeal circulation to test the ability of Compstatin to
alleviate the complement-related side effect of such procedures. The
results obtained in this study suggest that Compstatin can be used as
an effective complement inhibitor during extracorporeal circulation.
Peptide Synthesis and Purification
Complement Purification
Preparation of Blood Sixty milliliters of blood was collected from healthy blood donors and immediately mixed with 30-180 IU (approximately 210 to 1,300 µg) sodium heparin from porcine intestinal mucosa (Bioiberica, Sociedad Anònima, Spain). Heparin-coated equipment was used for this procedure. In analyses in which Compstatin and control peptide were used, they were used in concentrations up to 250 µmol/L (ie, a 46-fold molar excess assuming a C3 concentration of 1 g/ L).Plasmon Resonance Measurements The experiments were performed on a Biacore 2000 instrument (BiaCore AB, Uppsala, Sweden). An alternative pathway convertase was constructed as described elsewhere (manuscript in preparation); 300 µg per cm2 of C3b was coupled by amine bonds to CM5 chips according to the manufacturer's recommendation. Factor B (58 µg/mL) was injected into the flow cell. As a working buffer, phosphate-buffered saline (PBS) containing 1 mmol/L Ni2+ was used.22 When the binding of factor B to C3b had reached equivalance, factor D (10 µg/mL) was injected. This resulted in the formation of the alternative pathway C3 convertase C3b, Bb. To deposit additional C3b molecules, native C3 (132 µg/mL) was injected. The newly attached C3b was used for another cycle of factor B, factor D, and C3 addition. After a second or a third cycle, the convertase was incubated with 70 µmol/L of Compstatin followed by C3 in the presence of either Compstatin or control peptide (70 µmol/L). No attachment of C3 occured in the absence of ions (Ni2+).Experimental Models Tubing loops model.
A modification of the model previously described was
used.23,24 Nine-milliliter tubing loops
(diameter = 6.3 mm, length = 300 mm, surface area = 59.3 cm2) were filled with 5 mL of blood (area: volume = 11.9 cm2/mL) that had been mixed with heparin immediately after
the blood had been withdrawn from the blood donor. The samples were
then thereafter closed into circuits with connectors of stainless steel furnished with immobilized heparin,23 giving a gas volume
of approximately 4 mL. The tubing loops were rotated vertically (at 32 rpm, linear flow rate 0.3 L/min) during incubation in a water bath for
up to 60 minutes at 37°C. After incubation, 250 µL of 0.2 mol/L
EDTA was added to the blood to give a final EDTA concentration of 10 mmol/L. The 0-minute sample was not added to the tubing loop but was
instead immediately added to a tube containing EDTA, because 0-minute
samples that had been allowed to come into contact with the tubing had
previously been shown to give results similar to background
levels.23 The blood samples were centrifuged at 4°C
(3,290g, 20 minutes), and the plasma was collected. Each piece of tubing was washed 3 times with PBS containing 0.05% Tween 20 (vol/vol) and 0.02% Antifoam (vol/vol; Pharmacia Upjohn AB, Sweden). Antifoam was added to avoid bubbles in the tubing. The tubing loops
were stored at Microscope slide model.
This model is described in detail in a manuscript under preparation.
Slides were prepared as follows: two polymethylmethacrylate (PMMA)
rings with a height of 5 mm, outer diameter of 24 mm, and an inner
diameter of 19 mm (surface area = 8.1 cm2) were fixed onto
a polystyrene microscope slide. This device was coated with heparin, as
described previously.23 The two wells formed by the rings
were filled with 1 mL of whole blood containing 3 IU heparin. The wells
were closed with a polyvinyl chloride (PVC) microscope
slide (test slide), and the device was then mounted on the outer rim of
a plastic disc (30 cm in diameter), which was rotated at 32 rpm in a
37°C water bath. After incubation for up to 1 hour the blood was
removed and the loosely attached PVC test slide was washed twice in
veronal-buffered saline, pH 7.4, containing 0.75 mmol/L
Ca2+ and 2.5 mmol/L Mg2+. The test slide was
then dried at room temperature and stored at Enzyme Immunoassays (EIA) for Detection of C3a and sC5b-9 PBS containing 1% (wt/vol) bovine serum albumin (BSA) and 0.1% (vol/vol) TWEEN 20 was used as a working buffer, and PBS containing 0.1% (vol/vol) TWEEN 20 was used as a washing buffer in all three EIAs described below.EIA for detection of C3a. This assay is a sandwich EIA that uses the monoclonal antibody (MoAb) 4SD17.3 as the capture antibody. EDTA plasma was diluted 1/500 and analyzed as described previously.25 Bound C3a was detected with biotinylated rabbit anti-C3a followed by horseradish peroxidase (HRP)-conjugated streptavidin (Amersham, UK). Zymosan-activated serum,26 calibrated against a solution of purified C3a,21 served as standard and the values are given in ng/mL. EIA for detection of sC5b-9. A modification of the EIA, described by Mollnes et al,27 was used to measure sC5b-9. Plasma samples, diluted 1/5, were added to microtiter plates coated with anti-neoC9 MoAb MCaE11. sC5b-9 was detected by polyclonal anti-C5 antibodies diluted 1/500, followed by HRP-conjugated anti-rabbit Ig diluted 1/500 (both from Dako A/S, Glostrup, Denmark). Zymosan-activated serum defined as containing 40,000 arbitrary units (AU) per mL served as the standard. Heparin, at the concentrations used in this study, did not interfere with the performance of any of the EIAs.23 EIA for Determination of Surface-Bound C3/C3 Fragments Lengths of PVC tubing were cut into 3-cm pieces and a tube stopper was applied to one end of each tubing piece. One hundred microliters of HRP-labeled rabbit anti-human C3c (Dako A/S), diluted 1/250 in working buffer, was incubated in each tubing piece for 60 minutes at room temperature. The tubing pieces were then washed 3 times in washing buffer, and the color reaction was initiated by the addition of 100 µL of 1,2-phenylendiamine dihydrochloride (Fluka, Buchs, Switzerland) then stopped with 100 µL of 1mol/L H2SO4 after 5 minutes. Absorbance was measured at 492 nm. Binding of C3/C3 fragments in plasma, containing 10 mmol/L EDTA, was used as a control and was subtracted from the values obtained without EDTA. Bound C3/C3 fragments were presented as mean 492A ±SEM, obtained from three pieces of tubing.Immunochemical Staining of Microscope Slides The microscope slides (described above), which had been stored at 70°C, were immersed in ice-cold 50% (vol/vol) acetone
for 30 seconds, followed by another incubation for 5 minutes in
ice-cold 100% (vol/vol) acetone. All subsequent steps were performed
at room temperature. The slides were dried and washed twice with PBS
for 5 minutes, then incubated in 0.3% (vol/vol)
H2O2 in PBS for 15 minutes, washed twice with
PBS for 5 minutes, and blocked with 4% BSA in PBS for 5 minutes.
Subsequently, the slides were:
Flow Cytometry The expression of CD11b (CR3) on PMNs was quantitated using flow cytometry measurements. Briefly, 100 µL of blood was incubated for 15 minutes at 37°C with monoclonal anti-CD11b conjugated with phycoerythrin (Becton Dickinson, Franklin Lakes, NJ), followed by the addition of lysis reagent (Becton Dickinson). In a few experiments the cells were double-labeled in addition to the anti-CD11b label by adding 100 µL of anti-CD16 antibody conjugated with fluorescein isothiocyanate (FITC; Becton Dickinson) before lysis.Statistical Analyses Results were expressed as mean ±SEM. Statistical significance was calculated with Student's t-test, using Statview 4.0 (SAS Institute, Inc, Capy, NC) for Macintosh (Apple Computer, Inc, Cupertino, CA).
Inhibition of the Alternative Pathway Convertase-Mediated Cleavage of C3 by Compstatin An alternative pathway C3 convertase was formed on a CM5 chip in the BiaCore instrument. Cleavage of C3 by the convertase (C3b,Bb), resulting in deposition of C3b on the chip, was monitored by plasmon resonance. Alternative pathway convertase complexes, which were preincubated with 70 µmol/L of Compstatin, still cleaved native C3 (Fig 1). In contrast, the cleavage of native C3 was completely inhibited when C3 was added to the surface-bound convertase complexes in the presence of Compstatin, whereas the control peptide had no effect.
Dose-Dependent Inhibition of Complement Activation by Compstatin In three different experiments using three different blood donors, PVC tubing loops containing 5 mL of blood were rotated for 60 minutes at 37°C with 0 to 250 µmol/L (a 46-fold molar excess when compared with C3) of Compstatin or control peptide (Fig 2). In the loops containing 250 µmol/L control peptide, the levels of C3a increased from 141 ± 18 to 2,887 ± 603 ng/mL and those of sC5b-9 from 39 ± 6 to 491 ± 141 AU/mL (similar values were obtained for buffer controls). In contrast, in tubing loops containing 250 µmol/L Compstatin, the levels of C3a and sC5b-9 were reduced to 271 ± 106 ng/mL (5% of controls) and 83 ± 20 AU/mL (10%), respectively.
Time-Dependent Inhibition of Complement Activation by Compstatin
The Effect of Compstatin on the Binding of CD16+,
CD11b+ Cells to a PVC Surface
We have recently described a synthetic cyclic peptide, Compstatin, that
specifically inhibits C3 activation.14 This peptide has
been shown to affect both the classical and the alternative pathway by
inhibiting the C3 convertase-mediated cleavage of native C3 to C3a and
C3b. The inhibition did not involve direct blocking of the protease
cleavage site, because trypsin cleavage was not affected. A more likely
explanation is that the Compstatin bound to a site on C3 that is
important for interaction with the C3 convertase complexes. These
studies, however, did not completely exclude the possibility that the
peptide exerted its effect on C3 convertases. The conclusions of these
previous studies were confirmed in the present study by plasmon
resonance. An alternative pathway C3 convertase was formed by purified
components on a Biacore chip. The cleavage of purified C3 to C3a and
C3b was completely inhibited when the peptide was allowed to bind to
native C3, but preincubation of the convertase had no effect (Fig 1).
Submitted January 5, 1998;
accepted April 16, 1998.
We acknowledge the expertise of Dr Jerker Westin who performed the
BiaCore analyses. We also thank Lynn Spruce for peptide synthesis and
Dr Deborah McClellan for editorial assistance.
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This article has been cited by other articles:
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Abstract
Introduction
Abstract
-turn
segment of the peptide is critical for the preservation of its
conformational stability and probably forms the C3 binding
site.
