(Received for publication, June 28, 1995)
From the
Phage displaying APPI Kunitz domain libraries have been used to
design potent and selective active site inhibitors of human plasma
kallikrein, a serine protease that plays an important role in both
inflammation and coagulation. Selected clones from two Kunitz domain
libraries randomized at or near the binding loop (positions
11-13, 15-19, and 34) were sequenced following five rounds
of selection on immobilized plasma kallikrein. Invariant preferences
for Arg at position 15 and His at position 18 were found, whereas His,
Ala, Ala, and Pro were highly preferred residues at positions 13, 16,
17, and 19, respectively. At position 11 Pro, Asp, and Glu were
favored, while hydrophobic residues were preferred at position 34.
Selected variants, purified by trypsin affinity chromatography and
reverse phase high performance liquid chromatography, potently
inhibited plasma kallikrein, with apparent equilibrium dissociation
constants (K*) ranging from
75 to
300 pM. From sequence and activity data, consensus mutants
were constructed by site directed mutagenesis. One such mutant,
KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A,
I18H, S19P, and F34Y), inhibited plasma kallikrein with a K
* = 15 ± 14 pM,
representing an increase in binding affinity of more than 10,000-fold
compared to APPI. Similar to APPI, the variants also inhibited Factor
XIa with high affinity, with K
* values
ranging from
0.3 to 15 nM; KALI-DY inhibited Factor XIa
with a K
* = 8.2 ± 3.5
nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa,
Factor XIIa, activated protein C, or tissue factor
Factor VIIa.
Consistent with the protease specificity profile, KALI-DY did not
prolong the clotting time in a prothrombin time assay, but did prolong
the clotting time in an activated partial thromboplastin time assay
>3.5-fold at 1 µM.
Plasma kallikrein plays a central role in the contact activation
and kinin generating pathways(1, 2, 3) . In
the surface-mediated contact activation pathway, plasma prekallikrein
(Fletcher Factor), the zymogen precursor, can be activated by Factor
XIIa (FXIIa). ()Activated plasma kallikrein can then
reciprocally activate Factor XII, which can activate Factor XI,
ultimately resulting in fibrin formation, as well as activate the first
component of the classical complement pathway. As a member of the
kallikrein-kinin system, plasma kallikrein cleaves high molecular
weight kininogen to produce bradykinin, a potent vasodilator. Plasma
kallikrein has fibrinolytic activities as well through activation of
prourokinase and plasminogen. Plasma kallikrein also stimulates
neutrophils to aggregate and degranulate, releasing their lysosomal
contents. Thus, plasma kallikrein is involved in both fibrin deposition
and lysis, modulation of blood pressure, complement activation and
support of the inflammatory response.
Protein inhibitors play
critical roles in the regulation of proteases in a wide variety of
physiological processes. The major physiological inhibitor of plasma
kallikrein is C1 inhibitor, a serpin that results in irreversible
inhibition. C1 inhibitor is also the major physiological inhibitor of
FXIIa, and the complement pathway proteases C1r and C1s.
-Macroglobulin, another major inhibitor of kallikrein,
inhibits the kinin-forming function while only partially inhibiting
esterolytic activity. Other serpins including antithrombin III, protein
C inhibitor, and
-antitrypsin also inhibit kallikrein
to varying extents(4, 5) .
Recently, ecotin, a
142-residue protein from Escherichia coli , has been shown to
potently inhibit plasma kallikrein with a K of
160 pM(6) ; however, ecotin is not
totally selective and also potently inhibits Factor XIIa, Factor Xa,
and human leukocyte elastase(7) . Bovine pancreatic trypsin
inhibitor (BPTI, aprotinin) is a well studied member of the Kunitz
domain family of serine protease inhibitors that moderately inhibits
plasma kallikrein with K
of
30
nM(8, 9, 10) . However, BPTI is a
more potent inhibitor of plasmin. Aprotinin has been used in a wide
variety of clinical states including acute pancreatitis, septic and
hemorrhagic shock, adult respiratory distress syndrome and multiple
trauma; recently it has shown promise both clinically and in models of
cardiopulmonary bypass(11, 12) . As a broad spectrum
Kunitz type serine protease inhibitor, aprotinin can prevent activation
of the clotting cascade initiated by the contact activation pathway. It
can also prevent activation of neutrophils and other inflammatory
responses resulting from tissue damage caused by ischemia and hypoxia.
These benefits are believed to be derived from its kallikrein or
plasmin inhibitory activity; however, the fact that aprotinin is
neither very potent nor selective make it difficult to interpret these
effects.
Recently, we utilized the 58-residue Kunitz protease
inhibitor domain of the Alzheimer's amyloid -protein
precursor (APPI), which is structurally similar to BPTI(13) ,
as a scaffold for phage display of a large library of variants to
select potent and specific active site inhibitors of tissue
factor
Factor VIIa (TF
FVIIa)(14, 15) . In
this report we have used these libraries to select and aid in the
design of potent and specific active site inhibitors of human plasma
kallikrein.
where V/V
is the
fractional activity (steady-state inhibited rate divided by the
uninhibited rate), [E
] is the total
plasma kallikrein active site concentration, and
[I
] is the total inhibitor concentration.
The amino acid preferences
observed at various positions from random clones in Libraries I and III
are shown as a histogram in Fig. 1. From the sequence analysis
of Library I, Arg was always found at the P position
(residue 15) and His, Ala, and Pro were highly favored at positions 13,
17, and 19, respectively. At position 11, a preference for Pro, Asp, or
Glu was observed. From the sequence analysis of Library III, Arg was
again solely selected at position 15, and His was the only residue
found at position 18. At positions 16 and 17, Ala was the preferred
residue, whereas Val or Tyr was found most frequently at position 34.
While a limited set of other amino acids were observed at the positions
randomized in Libraries I and III, the frequency at which they occurred
was not statistically significant. No clear consensus developed in the
sequences from Library II, and contaminating phage from Library I were
beginning to take over the phage population (data not shown).
Figure 1:
The amino acid
preference observed at each position in Libraries I and III following 5
rounds of selection on plasma kallikrein. The preference of individual
amino acids found at each of the varied positions in Library I (A) or Library III (B) is plotted. The preference for
any amino acid is reported as the number of standard deviation units
() above a random chance occurrence of a given residue in the
library assuming a binomial distribution of amino acids. Scoring by
this method accounts for the expected codon bias and sampling
statistics when establishing a consensus(39) . The sequences of
18 clones from Library I and 9 clones from Library III were determined
after five rounds of selection on plasma kallikrein. Only amino acids
that were observed are shown. Clones with identical DNA sequence
(siblings) were only counted once. Only amino acids observed above
2
were considered significant.
To
further investigate amino acids selected in Libraries I and III,
variants were generated by site-directed mutagenesis, which combined
preferred amino acids selected from both libraries while exploring the
variation in the amino acids observed at positions 11 and 34 (Fig. 1). Thus, the incorporation of Pro, Asp or Glu at position
11 and Val or Tyr at position 34 were tested in the context of the
consensus residues His, Arg
,
Ala
, Ala
, His
, and
Pro
.
Figure 2:
Determination of the apparent equilibrium
dissociation constants of selected Kunitz domains with plasma
kallikrein and FXIa. The inhibitory activity is expressed as the
fractional activity (inhibited rate/uninhibited rate) at varying
inhibitor concentrations. The apparent equilibrium dissociation
constants were determined by nonlinear regression analysis of the data
to . Shown in A is the fractional activity of 0.5
nM plasma kallikrein remaining in the presence of: APPI
(), BPTI (
), KALI-10 (
), and KALI-DY (
). Shown
in B is the fractional activity of 3.5 nM FXIa
remaining in the presence of: APPI (
), BPTI (
), KALI-10
(
), and KALI-DY (
). K
*
values are reported in Table 1and Table 2.
The inhibition of
other relevant serine proteases found in human plasma was also measured
to determine the relative specificity of the Kunitz domain inhibitors.
Serine proteases (1-20 nM) were assayed in the presence
of 100 nM inhibitor. The fraction of remaining proteolytic
activity is reported in Table 3. All of the selected Kunitz
domains from Libraries I and III as well as the consensus mutants
inhibited FXIa, whereas none of them appreciably inhibited FXIIa, FXa,
thrombin, TFFVIIa, or activated protein C (Table 3). Most
of the selected Kunitz inhibitors inhibited plasmin only slightly, and
moderate (>60%) inhibition was observed for KALI-38, KALI-42, and
KALI-48; however, the consensus mutants did not appreciably inhibit
plasmin. The degree to which FXIa was inhibited by selected inhibitors
from Libraries I and III, and the consensus mutants was further
investigated by measuring the K
*. The inhibition
of FXIa by APPI, BPTI, KALI-10 and KALI-DY is shown in Fig. 2B, and K
* values are
reported in Table 1and Table 2. In general, the consensus
mutants are weaker inhibitors of FXIa than those selected from
Libraries I and III.
KALI-DY prolonged the clotting time of the surface-mediated contact activation pathway in a concentration-dependent manner as measured by the APTT. A greater than 3.5-fold prolongation of the clotting time at 1 µM was observed with KALI-DY compared with a 2.8- and 1.8-fold prolongation observed with APPI and BPTI, respectively (Fig. 3A). In contrast, neither KALI-DY, BPTI, nor APPI appreciably prolonged the clotting time in a tissue factor-initiated PT assay (Fig. 3B).
Figure 3:
Prolongation of clotting time in normal
human plasma. The concentration of APPI (), BPTI (
), and
KALI-DY (
) are plotted versus the -fold prolongation
of clotting time upon initiation by ellagic acid in the APTT assay (A) or by TF membranes in the PT assay (B). The
uninhibited clotting times were 33.6 and 14 s for the APTT and PT,
respectively.
The preference for Arg as the P residue
is further supported by data for several Kunitz domain variants
differing only by having either Arg or Lys here. In BPTI, replacement
of the Lys at the P
position with Arg resulted in a 20-fold
increase in affinity for plasma kallikrein(26) . Furthermore,
we had measured the binding affinities for APPI variants that, in
addition to inhibiting TF
FVIIa, also inhibited plasma
kallikrein(15) ; a comparison of two of these variants (IV-54C
and IV-36B) showed that kallikrein was inhibited 95-fold more potently
when Arg was at the P
position instead of Lys.
There was a striking preference for His at the
P` position (residue 18) among all randomized positions (Fig. 1). Residues in plasma kallikrein that are proximal to
this His probably include Arg
and Asp
, which
could provide a favorable hydrogen bonding network and binding pocket.
In comparison, the presence of Ala
and Phe
in
FVIIa resulted in the selection of Ile at the P
` position,
whereas in human leukocyte elastase, Phe was selected to fit in the
area flanked by Gly
and
Ala
(14, 15, 28) .
At the
P` position (residue 17), the preference for Ala at
position 17 is consistent with a relatively small hydrophobic pocket,
which likely involves Ile
in plasma kallikrein. Support
for Ala as a preferred residue at position 17 for plasma kallikrein
also comes from variants of aprotinin, where changing Arg to Ala here
resulted in a 10-15-fold improvement in affinity(29) . It
is unclear why Pro was preferred at the P
` position
(residue 19); we previously suggested that a Lys at this position would
be disfavored because of charge repulsion with Arg
in
plasma kallikrein (15) . The observation of Ala as the
preferred residue at the P
` position (residue 16) was not
unexpected since either Ala or Gly are usually found here in Kunitz
domains (9) .
At the P position (residue 11),
which is right at the edge of the protease/inhibitor interface, the
preferred residues were Pro, Asp, and Glu (Fig. 1). However, the
affinity for kallikrein was highest when Asp was present (Table 2). It is tempting to propose a salt bridge here with
Lys
of plasma kallikrein. At the P
position
(residue 13), the preferred His may form hydrogen bonds with
Lys
or Glu
found in plasma kallikrein. In
general, our lack of ability to fully rationalize the residues that
were preferred lends credence to the selection strategy that we have
pursued.
The specific inhibition of
plasma kallikrein improved somewhat for the consensus mutants, where
inhibitory activity was completely lost toward plasmin and reduced for
FXIa (Table 1Table 2Table 3). For example, KALI-DY
inhibited plasma kallikrein over 500-fold more potently than FXIa. In
addition, neither the selected inhibitors nor the consensus mutants
significantly inhibited thrombin, FXa, FXIIa, activated protein C, or
TFFVIIa (Table 3).
Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporeal perfusion, as evidenced by a reduction in blood loss and kallikrein-C1-inhibitor and C1-C1-inhibitor complexes, as well as prevention of neutrophil degranulation, platelet activation and aggregation(11, 12) . It has been used during lipopolysaccharide-induced endotoxic shock in pigs and prevented arterial hypotension(36) . In patients with hepatic cirrhosis, aprotinin has resulted in improved renal function and filtration(37) . The plasma kallikrein Kunitz domain inhibitors described here may prove useful in these and related indications.
In summary, we have developed very potent and selective inhibitors of human plasma kallikrein. Because plasma kallikrein is involved in multiple biological pathways, these selective inhibitors may allow exploration into the relative importance of this protease in its many roles.