(Received for publication, December 7, 1995; and in revised form, February 20, 1996)
From the
The ability of kininogens to modulate thrombin-induced
aggregation of human platelets has been assigned to domain 3 (D3) in
the common heavy chain coded for by exons 7, 8, and 9 of kininogen
gene. We expressed each of the exons 7, 8, and 9, and various
combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7
(Lys-Gln
), 8
(Val
-Gly
), and 9
(Gln
-Met
), and their combinations
were evaluated for the ability to inhibit thrombin induced platelet
aggregation. Only products containing exon 7 inhibited platelet
aggregation induced by thrombin with an IC
of >20
µM. A deletion mutant of exon 7 product, polypeptide 7A
product (Lys
-Lys
) did not block
thrombin-induced platelet aggregation, while 7B product
(Thr
-Gln
) and 7C product
(Leu
-Gln
) inhibited aggregation.
These findings indicated that the inhibitory activity is localized to
residues Leu
-Gln
. Peptides
Phe
-Ile
and
Phe
-Gln
did not block thrombin, and
Asn
-Phe
had only minimal inhibitory
activity. A heptapeptide Leu
-Ala
inhibited thrombin-induced aggregation of platelets with an IC
of 65 µM. The effect is specific for the activation
of platelets by thrombin but not ADP or collagen. No evidence for a
thrombin-kininogen complex was found, and neither HK nor its
derivatives directly inhibited thrombin activity. Knowledge of the
critical sequence of kininogen should allow design of compounds that
can modulate thrombin activation of platelets.
Platelets are critical cells in physiological hemostasis and
pathological thrombosis. Platelet activation can be defined by distinct
cellular events initiated by thrombin, as well as other agonists such
as collagen, ADP, and thromboxane A. Stimulation of
platelets results in a orderly process consisting of shape change,
followed by aggregation and/or secretion, finally resulting in the
formation of a platelet plug (Holmsen, 1994). Thrombin has been shown
to bind to two receptors on the platelet surface: platelet glycoprotein
Ib-IX complex (Takamatsu et al., 1986) and a seven-membered
transmembrane receptor coupled to G proteins. The latter polypeptide is
cleaved to expose a new NH
-terminal sequence SFLLRN (Vu et al., 1991), which can bind to distal sequences in the
receptor to initiate signal transduction. Glycoprotein Ib-IX complex
modulates thrombin action since, in its absence in Bernard-Soulier
disease, 10 times as much thrombin is required for aggregation of
platelets (Jamieson and Okumura, 1978).
The multifunctional protein
high molecular weight kininogen (HK) ()serves as the
procofactor of the kallikrein-kinin system (also known as the contact
system), a surface-mediated defense system (Colman, 1984). HK binds to
platelets (Gustafson et al., 1986), neutrophils (Gustafson et al., 1989), and endothelial cells (Schmaier et
al., 1988) and serves as a source of bradykinin after cleavage by
a number of plasma proteases (Scott et al., 1984, 1985). HK
and low molecular weight kininogen (LK) are derived from the same gene
by alternate splicing of the primary transcript and have identical
heavy chains, which consist of three segments (domains 1, 2, and 3),
each highly homologous to cystatin (Salvesen et al., 1986;
Ishiguro et al., 1987). The sequence HK diverges from LK at a
position on their light chains 12 residues COOH-terminal of the
bradykinin moiety. Each molecule then exhibits a unique sequence
(Takagaki et al., 1985). Kininogens can inhibit papain and
cathepsins B, H and L, and are the most potent plasma inhibitors of the
calcium-activated cellular protease, calpain (Schmaier et al.,
1986). The concentration of the kininogens in plasma is 3.95 µM (Kerbiriou-Nabias et al., 1984) comprising HK (0.67
µM) and LK (3.28 µM).
HK (Puri et al., 1991) and LK (Meloni and Schmaier, 1991) both inhibit the aggregation of human gel-filtered and washed platelets by thrombin. The primary determinants for thrombin-induced platelet aggregation are present on the heavy chain, which is shared in both HK and LK. Unlike calpain, which exclusively requires domain 2 of kininogens (Bradford et al., 1990), and papain, which can be inhibited by ether domain 2 or 3, the inhibition of thrombin-induced platelet aggregation is uniquely found in domain 3 of kininogens (Jiang et al., 1992).
In the present study, we have expressed kininogen domain 3 and several fragments of D3, in E. coli as glutathione S-transferase (GST) fusion proteins, and then tested each polypeptide for its ability to inhibit thrombin-induced platelet aggregation. Recombinant fragments that demonstrated inhibition were further dissected for critical regions by deletion mutagenesis. Smaller peptides were synthesized and tested for functional inhibition of platelet aggregation. Identification of the inhibitory sequences of kininogen should yield new insights into the structural requirements for the modulation of thrombin-induced platelet aggregation by kininogens.
Figure 1:
Primary structure of kininogen domain 3.
Domain 3 (amino acids 235-357) is coded for by exons 7
(Gly-Gln
), 8
(Val
-Gly
), and 9
(Gln
-Met
). Boxed N is N-linked carbohydrate. The shaded area indicates the
site for cysteine protease binding. Figure is modified from DeLa Cadena et al. (1994a).
Figure 2:
Recombinant fragments of kininogen domain
3. The schematic representation of the 10 recombinant HK D3 GST-linked
constructs. The corresponding amino acid sequence location for each
construct is appropriately indicated. The resulting recombinant protein
product identification is listed for each exon product construct. The
position of the thrombin cleavage site located between GST and the
NH terminus of each construct is indicated with an arrow.
Figure 3:
SDS electrophoretic analysis of
recombinant HK D3 exon products. Coomassie-stained 14%
SDS-polyacrylamide gel electrophoresis of the recombinant GST-linked HK
D3 exon products are shown. The samples from left to right are
molecular weight markers (STD), glutathione S-transferase (GST), recombinant HK domain 3 (D-3), exon 9 (E-9), exon (E-8),
Lys-Gln
(7C),
Thr
-Gln
(7B),
Gly
-Leu
(7A), and exon 7 (E-7). All recombinant products migrate at a higher molecular
mass than the 27-kDa GST marker prior to thrombin cleavage of the
fusion protein. Some samples contain free GST that leaches from the
column during the purification process.
Figure 4:
Inhibition of the thrombin-induced
platelet aggregation by D3-derived peptide products. Thrombin-induced
platelet aggregation was evaluated in the presence of three
polypeptides derived from exon 7 of HK. Gel-filtered platelets were
incubated for 2 min with increasing amounts of each polypeptide and
then activated with 2 nM thrombin to determine the relative
IC inhibitory potencies. The recombinant polypeptide 7C
(Gly
-Gln
) resulted in an IC
of 13.4 µM (
). The COOH-terminal half of the
polypeptide coded for by exon 7, 7B
(Lys
-Gln
), has an IC
of
30 µM (
), and the synthetic peptide LNAENNA
(Leu
-Ala
) inhibited thrombin-induced
platelet aggregation with an IC
of 65 µM (
).
Figure 5:
Thrombin inhibitory sequence of HK. Figure
is a three-dimensional homology model of D3 based on cystatin with the
position of the peptide sequence LNAENNA highlighted and displayed as a
hydrated region. The amino terminus and carboxyl terminus are indicated
on the left and in the center of this model,
respectively. The LNAENNA peptide sequence exhibits a helix-loop
conformation (when viewed as an carbon trace) and is completely
exposed on the exterior surface of the molecular
model.
Figure 6: Specificity of LNAENNA on platelet agonists. Panels 1-4 demonstrate the specificity of 26 µM LNAENNA on three platelet agonists. Panel 1 shows the inhibition by LNAENNA of gel-filtered platelets stimulated by the addition of 2 nM thrombin. Panel 2 shows an apparent increase in inhibitory potency with the same concentration of inhibitor but using thrombin at 1 nM instead, suggesting that both inhibitor and agonists compete for the same site on the platelet surface. Panel 3 indicates that LNAENNA has no effect on ADP-stimulated platelets (10 mM). Panel 4 shows that the aggregation by collagen (2 mg/ml) was unaffected by this peptide sequence.
I-Thrombin was
reacted with kininogen at 1/0.5, 1/1, and 1/2 molar ratios and run on
10% native gels (no SDS), then autoradiographed to determined if a
mobility shift could be observed or the presence of a new band could be
seen. No difference could be found between the thrombin control and the
lanes containing thrombin and kininogen mixtures (data not shown).
These experiments provide further evidence against the formation of a
thrombin-HK complex to explain the ability of HK to inhibit
thrombin-induced platelet aggregation.
The central role of thrombin in the pathogenesis of venous thrombosis has emphasized the actions of thrombin not only to clot fibrinogen and form the fibrin plug, but also to convert procofactors factor V and VIII to active cofactors factor Va (Colman et al., 1970) and VIIIa (Eaton et al., 1986), and thereby enhance the formation of thrombin. In contrast, on the arterial side, coronary or cerebral vessels are occluded by thrombi composed of both platelets and fibrin. The importance of thrombin in re-occlusion (Puri and Colman, 1993) after angioplasty (Heras et al., 1989) or thrombolysis (Gash et al., 1986) has emphasized the role of this serine protease in activating platelets. Various strategies have been suggested to prevent this process, including preventing platelet aggregation by all agonists and by blocking the binding of fibrinogen to its platelet receptor glycoproteins IIb/IIIa (Bennett and Vilaire, 1979). Unfortunately, monoclonal antibodies to this platelet integrin, while effective in inhibiting platelet aggregation by thrombin, cause excessive bleeding (Hanson et al., 1988). This result is not unexpected, since the hereditary absence or functional impairment of glycoprotein IIb/IIIa results in a hemorrhagic disease, Glanzmann's thrombasthenia (Nurden and Caen, 1974; Phillips et al., 1975). A second strategy uses direct thrombin inhibitory polypeptides such as hirudin (Markwardt, 1991) or small organic inhibitors such as argatroban (Kikumoto et al., 1984). Since these compounds inhibit the active site of thrombin, they can prevent thrombin activation of platelets, which requires proteolytic activity. By inhibiting the actions of thrombin on the coagulation process, they, like heparin, also increase the possibility of hemorrhage.
Spurred by the cloning of a seven-transmembrane thrombin receptor (Coughlin et al., 1992) and discovery of a tethered peptide revealed after cleavage that initiated changes in intracellular calcium, investigators have attempted, thus far without remarkable success, to develop peptides that inhibit the binding of thrombin to this receptor. We found that the presence of physiological concentrations of HK, not only in purified systems but also in plasma, modulates thrombin-induced platelet aggregation by shifting the concentration dependence so that 10 times more thrombin is required to stimulate platelets than in washed platelets (Puri et al., 1991). Thus, we hypothesized that HK contained critical sequences that could modulate thrombin-induced platelet aggregation.
Jiang et
al.(1992) demonstrated that D3 of kininogen was able to prevent
binding of thrombin to platelets. Using monoclonal antibodies, they
found that they could distinguish two activities in D3, one of which
blocked HK binding to platelets and the other thrombin binding.
However, the location of their binding sites was unknown. Recently,
Herwald et al.(1995) reported that a peptide
Leu-Met
present in the polypeptide
coded for by exon 9 inhibited HK binding with an IC
= 60 µM. We have studied the similar oxidized
peptide Cys
-Cys
for its ability to
inhibit platelet aggregation induced by thrombin. At 84
µM, no inhibition was observed (data not shown). It should
be noted that the exon 7 peptides blocked thrombin-induced aggregation,
while the exon 9 peptides blocked HK binding, and therefore the
biological activities of the peptides were distinct.
Domain 3 is
coded for by 3 exons designated exons 7, 8, and 9 (Kitamura et
al., 1985). We reasoned that each exon might express distinct
functions. Therefore, we expressed each exon product as a GST fusion
protein and tested its ability to inhibit thrombin-induced aggregation
of platelets from normal human donors, and compared the potency to HK
and D3. Recombinant D3 was similar in potency to HK, with only a 2-fold
difference in the concentration needed to inhibit the maximum
aggregation by 50%. Only the recombinant polypeptide coded by exon 7,
Gly-Gln
, exhibited potency equivalent
to HK or D3. In fact, exon 7 product when used as GST fusion protein
showed an IC
= 0.2 µM, 10 times more
potent than D3. This increase in potency is probably due to more
favorable folding, since, after cleavage of the thrombin-sensitive
linker and purification of free exon product 7, the potency decreased
67-fold and was one-third as potent as D3 (Table 2). Cleavage of
the fusion protein to yield the free recombinant fragment of kininogen
did not always result in a less potent polypeptide. In fact, in the
other cases of exon product 7 fragment, potency was increased (see
below) by a factor of 2-3. Since two activities were
distinguished within D3, we also made all three combinations of the
exon product, 7+8, 8+9, and 7+9, to test whether the
difference between exon 7 product and D3 could be accounted for by
another site on a different exon product. No increase in potency was
found in products of exons 7+8 or exons 7+9, and the product
of exons 8+9 showed no activity.
Since exon 7 product comprises
56 amino acids, we sought to localize the responsible sequence more
precisely. We generated three overlapping recombinant polypeptides.
Exon product 7A, Gly-Leu
, comprised
the NH
-terminal half of the exon product 7, while 7C
represented the COOH-terminal portion,
Lys
-Gln
. In order to avoid splitting
the active sequence, we also expressed 7B,
Thr
-Gln
. Since 7B and 7C both
inhibited thrombin-induced activation, but 7A did not, the functional
region lay in the COOH-terminal 23-mer,
Lys
-Gln
. Examination of the molecular
model of D3 revealed that the proximal portion of the sequence of 7C
was on the surface, but much of the rest was associated with the
protein core. To test the hypothesis that the NH
-terminal
portion of 7C contained all or part of the site responsible for
inhibition of thrombin-induced platelet aggregation, we synthesized
four peptides that subsumed the sequence
Lys
-Gln
. Only the peptide
Leu
-Ala
, which exists in a surface
loop at the junction of a helix-turn configuration in the molecular
model, inhibited platelet aggregation by thrombin. A search of the
protein data base indicates no significant homologies except to domain
3 of rat and bovine kininogen. the latter shows complete identity for
LNAENNA. The rat HK and LK contains an identical heptapeptide except
for substitution of histidine for Ala
. Rat T-kininogen,
which is a potent inhibitor of thrombin-induced platelet aggregation, (
)shows high homology with 5 identical amino acids. Gln and
His are substituted, respectively, for Glu
and
Asn
, which are extremely conservative substitutions.
We then addressed the mechanism of the action of exon product 7 and
its contained peptide, LNAENNA, on thrombin-induced activation. Several
possibilities were considered. HK might form a complex with thrombin.
HK is known to form complexes with prekallikrein and factor XI using
portions of domain 6 (Tait and Fujikawa, 1987), with plasminogen via
the light chain (Humphries et al., 1994), and with
thrombospondin (DeLa Cadena et al., 1994b), involving both HK
light and heavy chains. A peptide in D3 within exon 7 product is
involved in HK binding to thrombospondin on the activated platelet
surface (DeLa Cadena et al., 1993). However, it is contained
within exon product 7A, which did not inhibit thrombin-induced
aggregation. We found that HK did not inhibit thrombin cleavage of
fibrinogen or a peptide substrate (amidolytic activity). Moreover,
thrombin failed to bind to immobilized HK or to HK in the fluid phase
or to HK on native gel electrophoresis. While this study was in
progress, Hasan and colleagues (Hasan et al., 1995) also were
unable to demonstrate a thrombin-HK complex using several techniques
different from those used in this report. The second possibility is
that the sequence LNAENNA in D3 directly blocks the binding of thrombin
to the seven transmembrane receptor. Three pieces of evidence make this
unlikely. First, HK does not inhibit the tethered peptide SFLLRN from
aggregating platelets (Jiang et al., 1992) nor did LNAENNA in
our study. Second, higher concentrations of thrombin can reverse the
inhibition by HK (Puri et al., 1991). Finally, exon 7C product
and LNAENNA fail to block shape change induced by thrombin. Shape change is the earliest morphological change following
ligand binding and is associated with an increase in intracellular
Ca
and with phosphorylation of myosin light chain
(Daniel et al., 1984). The calcium increase has been shown to
be a consequence of the interaction of SFLLRN or thrombin with the
seven-transmembrane receptor. We cannot rule out an effect on this
receptor at a site distant from the tethered peptide, but consider this
unlikely. We are currently testing the hypothesis that HK may inhibit
the binding of thrombin to glycoprotein Ib.
Finally, our studies show that the HK D3 and its constituents inhibit neither ADP and collagen-induced aggregation nor thrombin protease activity. This specificity would allow selective inhibition of thrombin-induced platelet aggregation, thus modulating thrombin-platelet interaction without either compromise of platelet function or thrombin effects on blood coagulation. These properties make it an attractive template for construction of peptidomimetic drugs to inhibit reocclusion and thrombosis.