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W. D. CAMPBELL ET AL
bin inhibitor, Phe-Pro-Arg-chloromethyl ketone
(PPACK) were obtained from Calbiochem-Novabiochem
Corporation. All other chemicals were obtained locally
and were of reagent grade.
Results and Discussion
Using purified CPR and CPN, we showed that CPR is
more effective in removing carboxyterminal arginine
from C5a octapeptide than CPN, whereas CPN is more
effective towards C3a octapeptide (Fig. 1). In addition,
using plasma containing active CPN and serum with
active CPN and CPR, we showed that serum can remove
terminal arginine more rapidly than plasma (Fig. 2) and
that furthermore, this enhanced ability of serum is abro-
gated by potato carboxypeptidase inhibitor (PCI) which
inhibits CPR but not CPN.
Purification of ProCPR. ProCPR was purified to
homogeneity as described by Eaton et al. (5). The pure
enzyme showed a single band by SDS polyacrylamide
gel electrophoresis (PAGE) at around 60 kDa and was
converted by thrombin/thrombomodulin to the activated
form at around 35 kDa. CPN was purified to homo-
geneity as described by Plummer et al. (11). The enzyme
gave three bands on SDS-PAGE at 83 kDa, 53 kDa and
50 kDa corresponding to the two large identical glyco-
sylated subunits and the two smaller active subunits.
Peptide synthesis. Peptides were synthesised using an
AMS 422 Multiple Peptide Synthesiser (ABiMED, Lan-
genfeld, Germany) using standard solid-phase synthesis
techniques and 9-fluorenylmethoxycarbonyl (Fmoc)
amino acids (Watanabe Chem. Ind. Ltd., Hiroshima,
Japan). Peptides were purified by reversed phase HPLC
on a Waters Delta-Pak C18 column. MALDI-TOF mass
spectrometry on a Kompact Maldi II (Kratos Analytical,
Shimadzu, Japan) yielded the following results: C5a-
C, m/z 984.9 (calculated 984); C3a-C, m/z 828.2 (calcu-
lated 825); C5a-desR, m/z 827.3 (calculated 828); C3a-
desR, m/z 668.1 (calculated 668). The sequences of
the octapeptides were as follows: C5a (HKDMQLGR),
C3a (ASHLGLAR), C5a-desR (HKDMQLG), C3a-desR
(ASHLGLA).
Hydrolysis of octapeptides by plasma and serum.
Serum was generated by incubating plasma with 0.5
NIH units/ml of thrombin and 40 nM thrombomodulin in
the presence of 5 mmol/liter CaCl2 for 10 min at room
temperature followed by addition of PPACK (20
µmol/liter) to stop activation. Seventy-five microliters of
plasma or serum preparations and 25 µl of C3a or C5a
octapeptide were incubated for different time periods.
The reaction was stopped with 150 µl of 5 M HCl. After
incubation at 37 C and centrifugation at 12,000×g to
remove precipitates, 200 µl of supernatant were trans-
ferred to another tube followed by 120 µl of 5 M NaOH
and 100 µl of 1 M Tris pH 7.5. The solution was filtered
through a 0.22 µm filter and injected directly onto an
HPLC apparatus equipped with a C18 reverse phase
column. Peptides were separated with a linear gradient
from 0.1% trifluoroacetic acid to 0.1% trifluoroacetic
acid/65% acetonitrile. Ratios of hydrolyzed to unhy-
drolyzed peptides were determined by measuring peak
areas. For hydrolysis of peptides by pure CPN or CPR,
essentially the same procedure as above was followed
except for elimination of the centrifugation step.
In the complement system, continual low level hydrol-
ysis of the thioester bond in C3 generates C3(H2O)
which is in an activated form and generates an initial C3
convertase of the alternative complement pathway
C3(H2O)Bb reacting with factors B and D (10). This C3
convertase cleaves C3 into C3a and C3b, the latter of
which has the potential to generate another C3 conver-
tase, C3bBb. Although the continuous activation of
complement is immediately restricted in the absence of
foreign invaders such as microorganisms, C3a is con-
stantly generated. Therefore, it is reasonable to assume
that the C3a anaphylatoxin fragment is readily inactivated
by CPN which is present in the active form in plasma and
controls inflammation in the absence of infection. On the
other hand, C5a can be generated when the C3 convertase
escapes inactivation by factors H, I and membrane
inhibitors such as DAF (8), MCP (3), and gangliosides
(9). The surface of microorganisms provides an ideal
environment for complement activation due to the
absence of species specific membrane inhibitors such as
DAF (8) and MCP (3). The C3 convertase which escapes
restriction forms C5 convertase by fixing another C3b
molecule as an acceptor for C5. The extremely potent
anaphylatoxin, C5a (effective at as low a concentration as
－12
10 M), is generated at the site of infection and induces
local inflammation (12). ProCPR can be converted to
CPR by trypsin-like inflammatory proteases (14), as
well as coagulation proteases (1), and since it rapidly
inactivates C5a octapeptide compared to CPN, this dif-
ference is expected to reflect the activity of these two
enzymes on intact C5a. Our findings support the concept
that proCPR/TAFI is an acute phase protein which plays
an important role in vivo as an inactivator of inflamma-
tory mediators (13) in addition to its function as an
inhibitor of fibrinolysis. Although the carboxytermi-
nal octapeptides have been shown to be effective in
terms of biological activity (7), we are nevertheless
planning to compare the activities of CPR and CPN on
the intact anaphylatoxins in a future study.
Dains
