(Received for publication, June 26, 1995; and in revised form, September 7, 1995)
From the
To define the presence and potential role of platelet-associated
protease inhibitors, we initiated a study designed to characterize the
platelet components that are responsible for the formation of two
SDS-stable complexes of approximately 58 and 70 kDa initially observed
following the incubation of I-thrombin and human
platelets. We demonstrate that thermal-mediated unfolding of the 58-kDa
complex between
I-thrombin and a nonsecreted platelet
protein leads to an apparent molecular mass of 70 kDa. This platelet
component is functionally and immunologically indistinguishable from
the cytoplasmic antiproteinase (CAP), also known as placental thrombin
inhibitor, a recently cloned member of the ovalbumin family of
intracellular serpins (serine proteinase inhibitors). CAP-specific mRNA
and antigen were detected in human platelets, suggesting that CAP
synthesis occurs concurrent with platelet development. Utilizing
quantitative immunoblotting, CAP antigen was estimated at 1.014
± 0.181 µg/10
nonstimulated platelets. After
platelet activation with the calcium ionophore A23187, CAP antigen was
detected in released microparticles at approximately 0.195 ±
0.031 µg/10
platelets and a fraction of platelet CAP
was proteolytically modified. We provide evidence that these lower
molecular mass species arise by cleavage of CAP at or near the reactive
site loop. Most importantly, molecular sieving chromatography indicates
the presence of an approximately 68-kDa SDS-labile complex between
cleaved CAP and a cellular component in A23187-stimulated platelets,
suggesting a physiological target of this intracellular serpin and a
potential role for this inhibitor in regulating proteolytic activity
that may be formed during platelet activation.
Serine protease inhibitors or serpins are a ubiquitous
superfamily of homologous proteins that resemble
-proteinase inhibitor in overall structure and include
antithrombin III, protease nexin-1 (PN-1), (
)and
-antiplasmin, to name a few(1) . In general,
serpins contain a highly exposed reactive site loop near the carboxyl
terminus of the protein that interacts as a pseudosubstrate for the
target protease. Interaction of the serpin reactive site loop with the
substrate binding cleft of the target protease triggers a dramatic
conformational change in the serpin that results in a stochiometric 1:1
inhibitory complex that is typically stable to treatment with
denaturants, such as SDS(2) . In this manner, serpins play
crucial roles in neutralizing serine protease activities that are
involved in a wide variety of vital processes including blood
coagulation, fibrinolysis, complement activation, inflammation, and
cell migration (2) . In addition to the serpins that regulate
protease activity, several members of this superfamily lack a protease
inhibitory capability and have other physiological roles. These latter
serpins were originally identified by data base searching and include
thyroxine binding globulin, angiotensinogen, and
ovalbumin(1, 3) . Ovalbumin represents the parent
prototype of a unique family within the serpin superfamily that lacks a
typical cleavable signal sequence but has been found to reside either
intracellularly, extracellularly, or both(4) . Previously
identified members of the ovalbumin family of serpin proteins include
plasminogen activator inhibitor-2 (PAI-2)(5) , an elastase
inhibitor isolated from monocyte-like cells(6) , a squamous
cell carcinoma antigen(7, 8) , a tumor suppressor
called maspin(9) , and cytoplasmic antiproteinase or
CAP(10) , also known as placental thrombin
inhibitor(11) . CAP has been previously shown to inhibit a
broad spectrum of prototype extracellular serine proteases including
trypsin, thrombin, urokinase, and factor Xa(10) . However, the
intracellular physiological protease target(s) of CAP, or any other
intracellular serpin, remain unknown.
Previous studies analyzing the
interaction of thrombin with platelets detected an SDS-stable 77-kDa
complex between exogenously added I-thrombin and a form
of PN-1 that is present both on the platelet surface and in releasates
upon platelet activation (12, 13, 14, 15, 16, 17, 18, 19) .
A significant percentage of the complexes between
I-thrombin and platelet PN-1 (PN-1p) forms a 450-kDa
disulfide-linked ternary complex with thrombospondin detected under
nonreducing conditions(17, 20) . In the course of
these studies, two additional SDS-stable complexes of approximately 58
and 70 kDa were detected following the incubation of
I-thrombin with
platelets(13, 15, 16, 17) . The
observations of both Gronke et al.(15) and Miller et al.(17) suggest that disruption of the platelet
membrane integrity is required for the detection of the 58-kDa
thrombin-containing complex. In addition, no product-precursor
relationship between this complex and the 77-kDa
I-thrombin-PN-1p complex could be
demonstrated(17) , and the 58-kDa complex was not recognized by
polyclonal antibodies against human PN-1(15) . Because several
studies have established the presence of an emerging family of
intracellular serpins(2, 4) , we initiated the present
investigation to determine the relatedness of the platelet molecules
responsible for the 58- and 70-kDa
I-thrombin-containing
complexes to known serpins. We report that the platelet component of
the 58- and 70-kDa
I-thrombin complexes is
immunologically and functionally indistinguishable from CAP. The
mobility of the protease-CAP complex was dependent on the extent of
thermal unfolding in the presence of SDS. Most importantly, we provide
evidence that CAP interacts with a cellular protease upon platelet
activation, suggesting a physiological target of this recently
discovered serpin.
Figure 1:
Formation of SDS-stable complexes
between I-thrombin and proteins associated with
nonstimulated or PMA-stimulated platelets. Platelets were incubated (at
23 °C for 20 min) in the absence or the presence of PMA (160
nM). The platelets were pelleted by centrifugation, the
releasate from the PMA-stimulated platelets was harvested, and the
pelleted platelets were lysed by resuspension in the presence of 0.5%
Triton X-100. Duplicate samples (100 µl, representing 10
platelets) of the nonstimulated lysed platelets (lanes 1 and 5), the PMA-releasate (lanes 2 and 6), and the PMA-stimulated lysed platelets (lanes 3 and 7) were incubated with
I-thrombin (5
nM at 23 °C for 30 min). One set was incubated (at 100
°C for 3 min) in SDS sample buffer supplemented with 100 mM DTT (lanes 1-3), whereas the parallel set was
incubated (at 37 °C for 10 min) in SDS sample buffer in the absence
of reducing agents (lanes 5-7). Lane 4 contains
I-thrombin alone incubated (at 100 °C for 3 min) in
SDS-sample buffer in the presence of 100 mM DTT. SDS-PAGE on a
4%-15% linear gradient separating gel was followed by autoradiography.
Molecular mass markers are indicated in this and the following
figures.
Figure 2:
Thermal-mediated unfolding of the 58-kDa
complex between I-thrombin and a platelet protein results
in the migration of the complex at 70 kDa. A, nonstimulated (lanes 1-6) and PMA-stimulated (lanes
7-9) platelet pellets were lysed with 0.5% Triton X-100 and
incubated with
I-thrombin (5 nM at 23 °C for
30 min). Samples (100 µl, 10
platelets) were treated
with SDS sample buffer containing 100 mM DTT and incubated at
the indicated temperatures. SDS-PAGE was carried out using a 9%
separating gel followed by autoradiography. Lane 10 contains
I-thrombin alone (at 100 °C for 3 min). B,
PMA-stimulated platelets (500 µl, 5
10
platelets) were pelleted, lysed (0.5% Triton X-100), and
incubated with
I-thrombin (5 nM at 23 °C for
30 min), and the reaction was terminated by the addition of reducing
sample buffer. This sample was heated to 65 °C for 5 min and
subjected to SDS-PAGE. After electrophoresis, proteins were immobilized
in the gel by staining with 0.3 M CuCl
. The 58-kDa
radioactivity-containing band was localized by phosphor imaging of the
wet gel, the band was excised, and the gel slice was destained (250
mM Tris, pH 8.0, 250 mM EDTA) for 30 min with four
changes of the destaining solution. Proteins were electroeluted (25
mM Tris, 192 mM glycine, pH 7.4, 0.025% SDS) from the
gel slice at 50 V for 8 h(32) . Samples of the eluted proteins
were reanalyzed by reducing SDS-PAGE after heating to 65 °C for 5
min (lane 2) and 100 °C for 3 min (lane 3). A
sample of the PMA-stimulated platelet pellet/
I-thrombin
mixture (starting material) was treated at 65 °C for 5 min and
loaded in lane 1.
Figure 3:
Formation of SDS-stable complexes between
a platelet pellet-associated protein and I-trypsin and
I-urokinase. The lysed (0.5% Triton X-100) pellet of
PMA-stimulated platelets (10
platelets/ml) was incubated at
23 °C for 30 min with either 5 nM
I-trypsin (A) or with 5 nM
I-urokinase (B). Reactions were terminated by the addition of SDS-sample
buffer (100 mM DTT), and samples (100 µl) were heated to
65 °C for 5 min (lanes 2) or 100 °C for 3 min (lanes 3), followed by SDS-PAGE and autoradiography. The
radioiodinated proteases alone were loaded in lanes
1.
Figure 4:
Effect of antibodies directed against CAP
and PN-1 on the formation of the 58- and 77-kDa SDS-stable complexes
composed of I-thrombin and platelet proteins. Releasate (lanes 1, 3, and 5) and pellet (lanes
2, 4, and 6) of PMA-stimulated platelets was
incubated (at 23 °C for 30 min) with preimmune rabbit IgG (lanes 1 and 2), polyclonal rabbit anti-PN-1 IgG (lanes 3 and 4), and CAP-specific anti-MBP/CAP IgG (lanes 5 and 6). All antibodies were used in a final
concentration of 100 µg/ml. After incubation (at 23 °C for 30
min) with
I-thrombin (5 nM), samples were heated
to 65 °C (5 min) in the presence of 100 mM DTT and
analyzed by SDS-PAGE followed by autoradiography. Lane 7 shows
I-thrombin alone.
To investigate whether CAP is synthesized in megakaryocytes during the development of platelets, vestigial mRNA from platelets and mRNA isolated from megakaryocytic cell lines (i.e. PMA-induced HEL cells, DAMI cells) was reverse-transcribed and PCR-amplified utilizing specific primers for CAP, as well as von Willebrand factor as a control. The appropriately sized CAP- and von Willebrand factor-specific reaction products were detected in both megakaryocytic cell lines, as well as in platelets (Fig. 5). To confirm identiy of CAP at a molecular level, the complete coding region of CAP was PCR-amplified from PMA-stimulated HEL cell cDNA and sequencing of a cloned PCR product revealed identity of the nucleotide sequence with CAP.
Figure 5: PCR analysis of reverse-transcribed RNA from two megakaryocytic cell lines and peripheral blood platelets. RNA was isolated from PMA-stimulated HEL cells (lanes 2 and 3), DAMI cells (lanes 4 and 5), and platelets (lanes 6 and 7). The RNA was reverse-transcribed and amplified using PCR and specific primers (refer to ``Experimental Procedures'') to amplify the cDNA of CAP (lanes 2, 4, and 6) and von Willebrand factor (lanes 3, 5, and 7). PCR products (20 µl; one-fifth of the reaction mixture) were subjected to electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. The size of markers (lanes 1 and 8) is shown on the right in base pairs.
Figure 6:
Quantitative immunoblotting of CAP in
platelets. A, proteins in the releasate (lanes 1 and 3) and pellet (lanes 2 and 4) from
PMA-stimulated platelets were separated by reducing SDS-PAGE (10
µl, corresponding to 10 platelets per lane; at 100
°C for 3 min) and electroblotted onto an Immobilon polyvinylidene
difluoride membrane, which was then stained with 15 µg of
CAP-specific anti-MBP/CAP IgG (lanes 1 and 2) or with
15 µg of the same antibody preincubated (at 37 °C for 20 min)
with 30 µg of recombinant 6xHis/CAP in a volume of 100 µl (lanes 3 and 4). B, different quantities of
recombinant 6xHis/CAP (12 ng, lane 1; 24 ng, lane 2;
48 ng, lane 3), as well as pellets of nonstimulated platelets (lane 4, representing 2
10
platelets),
pellets of A23187-activated platelets (lane 5, 5
10
platelets), and microparticles (refer to Table 1for experimental details) derived from the releasate of
A23187-activated platelets (lane 6, corresponding to 10
platelets) were subjected to reducing SDS-PAGE followed by
electroblotting and immunodetection with CAP-specific anti-MBP/CAP
IgG.
The concentration of CAP antigen in platelets was determined
by quantitative Western blotting utilizing recombinant 6xHis/CAP
antigen as the standard. Different concentrations of 6xHis/CAP antigen
in addition to the unknown samples were blotted onto the same membrane.
The blots were probed with CAP-specific anti-MBP/CAP IgG and bound
antibodies were determined by employing a chemiluminescence-enhanced
system followed by laser densitometry of the autoradiographs. A limited
dose response is shown in Fig. 6B (lanes
1-3) and indicates that the recombinant 6xHis/CAP migrates
with the expected molecular mass of 44 kDa. Fig. 6B (lane 4) demonstrates that the 39-kDa CAP antigen was
present in nonstimulated platelets. After platelet stimulation with
A23187, the 39-kDa CAP antigen and three additional smaller proteins of
37-, 35-, and 34-kDa were detected in platelet pellets, as well as
associated with released microparticles (Fig. 6B, lanes 5 and 6). Staining of these bands could be
abolished by preincubation of the CAP-specific anti-MBP/CAP IgG with
6xHis/CAP antigen, indicating that the lower molecular mass proteins
are proteolytically modified forms of CAP (results not shown). These
cleaved 37-, 35-, and 34-kDa CAP forms were also detected in platelets
stimulated with the physiological agonist thrombin and subsequently
lysed by the addition of SDS-sample buffer, although after stimulation
with thrombin these forms were not as prominent as in A23187-stimulated
platelets (data not shown). In contrast, during incubation of platelets
with PMA no low molecular mass CAP species were formed (Fig. 6A, lane 2). Similarly, no CAP cleavage
was detected after incubating platelets with PMA under conditions that
lead to platelet aggregation (i.e. stirring, 1 mM CaCl), indicating that CAP cleavage is not a
consequence of platelet aggregation (results not shown). Table 1shows a comparison of estimated concentrations of CAP
antigen in resting platelets (1.014 ± 0.181 µg/10
platelets) and in platelet cellular fractions after stimulation
with PMA and A23187. In platelets activated with A23187, CAP antigen
was detected in released microparticles at approximately 0.195 ±
0.031 µg/10
platelets.
Figure 7:
Detection of cleaved CAP in
A23187-stimulated platelets and after incubation of platelet lysates
with thrombin. A, nonstimulated (lanes 1, 3,
and 5) and A23187-activated (lanes 2, 4, and 6) platelets (10 per lane) were lysed in reducing
SDS-sample buffer, and CAP was detected after SDS-PAGE and
electroblotting by staining with protein A-purified anti-6xHis/CAP (lanes 1 and 2), affinity purified anti-6xHis/CAP (lanes 3 and 4), and protein A-purified
anti-6xHis/CAP adsorbed with 6xHis/CAP (lanes 5 and 6). B, nonstimulated (lanes 1-6) and
A23187-activated (lanes 7-12) platelets
(10
/lane) were solubilized (0.5% Triton X-100) and
incubated with increasing concentrations of thrombin (at 23 °C for
20 min): no thrombin (lanes 1 and 7) or 0.15 (lanes 2 and 8), 0.5 (lanes 3 and 9), 1.5 (lanes 4 and 10), 5 (lanes 5 and 11), and 15 nM thrombin (lanes 6 and 12). CAP was detected after reducing SDS-PAGE (at 100
°C for 3 min) by staining of blots with affinity purified
anti-6xHis/CAP.
Figure 8:
Gel chromatography of platelet lysates on
a molecular sieving column. Nonstimulated (A) and
A23187-activated (B) platelets (1 ml, 10 platelets) were lysed (0.5% Triton X-100) and passed over a
Sephacryl S-200 (Pharmacia) column (1.77 cm
110 cm)
that was eluted at 15 ml/h with PBS. Samples of the column fractions
were analyzed by SDS-PAGE, followed by Western blotting utilizing
protein A-purified anti-6xHis/CAP. Relative amounts (the total amount
of CAP antigen that eluted from the column = 1) of 39-kDa CAP (solid squares) and 37-34-kDa cleaved CAP (open
squares) were determined by laser densitometry of the
autoradiographs. The arrow indicates fraction number 43 that
was analyzed by Western blotting using affinity purified anti-6xHis/CAP (insets, lanes 1) and adsorbed anti-6xHis/CAP (insets, lanes 2). The elution of blue dextran 2000 (Void), bovine serum albumin (BSA) (68 kDa), and
ovalbumin (43 kDa) (all obtained from Pharmacia) is
indicated.
Our data indicate the presence of a single nonsecreted
protein in platelets that forms an SDS-stable complex with I-thrombin ( Fig. 1and Fig. 2). We
demonstrate that this platelet pellet-associated complex-forming
protein is functionally and immunologically ( Fig. 3and Fig. 4) indistinguishable from CAP, also known as placental
thrombin inhibitor(10, 11, 34, 36) .
Complexes between
I-thrombin and CAP are transformed to
an apparent molecular mass that corresponds to the combined molecular
mass of the protease (32 kDa for reduced thrombin) and native CAP (39
kDa) only upon heating to more than 65 °C in the presence of SDS (Fig. 2). The approximately 58-kDa complexes that have been
observed by SDS-PAGE in several studies analyzing the interaction
between platelets and
I-thrombin (13, 15, 16, 17) were probably
incompletely unfolded
I-thrombin-CAP complexes. This type
of interaction is not specific for thrombin and CAP because both
trypsin- and urokinase-containing complexes similarly require harsh
conditions to unfold them (Fig. 3). The detection of
CAP-specific transcript in reverse-transcribed platelet mRNA (Fig. 5) suggests that CAP is synthesized in megakaryocytes
during the development of platelets.
CAP was initially detected and
partially characterized by Kirschner et al. as a
trypsin-binding and -inhibiting factor in mouse
fibroblasts(37) . Eaton and Baker (33) subsequently
demonstrated a similar cytoplasmic (nonsecreted) thrombin-, trypsin-,
urokinase-, and plasmin-binding component in several cultured cell
lines. In 1993, this inhibitor was purified from placental extracts (36) and a monkey kidney epithelial cell line
(BSC-1)(34) , and the cDNA was cloned the following year in two
independent laboratories(10, 11) . CAP is a potent
inhibitor of trypsin (K = 8
10
M
s
),
thrombin (K
= 5
10
M
s
), factor Xa (K
= 1.3
10
M
s
), and several
other proteases but not elastase(34) , and it is a member of
the emerging ovalbumin family of highly homologous
serpins(4, 10, 11) . Presently, this family
includes two other human cytoplasmic protease inhibitors, i.e. PAI-2 (5) and leukocyte elastase inhibitor(6) .
Although CAP, PAI-2, and leukocyte elastase inhibitor are potent
inhibitors of several known extracellular serine proteases, no
intracellular physiological roles or target proteases are currently
known. PAI-2 is synthesized in secreted and cytosolic
forms(38) , whereas leukocyte elastase inhibitor and CAP have
not been observed to be secreted from nucleated cells, and we could not
detect release of either CAP activity (Fig. 1, lanes 2 and 6) or antigen (Fig. 6A, lane
1) after platelet stimulation with PMA. It is possible that CAP,
released from disintegrating platelets in a thrombus or from platelets
disrupted by mechanical injury or complement-mediated cytolysis, plays
a role in modulating the activity of extracellular proteases involved
in coagulation or fibrinolysis. However, CAP is rapidly inactivated by
oxidizing reagents(11) , suggesting that its action would
probably be limited to the microenvironment of the site of its release.
Our finding of CAP in platelet-derived microparticles indicates a
mechanism for releasing CAP from activated platelets in an
oxidation-protected environment.
In this report, we have established
the levels of CAP antigen associated with nonactivated and PMA- and
A23187-activated platelets (Table 1). Platelets contain CAP
antigen in a concentration of approximately 760 ng/mg total protein (Table 1). Currently no information is available regarding the
CAP antigen content in other cell types. Using an activity assay based
upon complex formation with I-thrombin, Eaton and Baker (33) detected between 0.35 and 72.0 ng CAP/10
cells
in several tissue culture cell lines. The highest concentration,
detected in a monkey kidney epithelial cell line (BSC-1), corresponds
to 600 ng/mg cytosolic protein(34) . Using a similar activity
assay(33) , we have observed that about 60% of the total
platelet CAP is detected in either nonactivated or PMA-stimulated
platelets. (
)Thus, platelets represent a well characterized
and easily obtainable source of relatively large quantities of human
CAP and might be useful in the elucidation of the physiological role of
this protease inhibitor.
In view of the widespread tissue
distribution(11, 33) , intracellular location, and
absence of secretion from tissue culture cells(33, 36) and platelets, an intracellular physiological role of CAP
is likely. Although CAP in a complex with a protease (e.g. thrombin) can be readily detected by our antibodies following
SDS-PAGE and electroblotting, only cleaved forms (i.e. 37-,
35-, and 34-kDa) of CAP were detected following stimulation of
platelets with A23187 (Fig. 6B, lanes 5 and 6, and Fig. 7A). The 37- and 35-kDa CAP
species were also detected after incubation of lysates of resting
platelets with thrombin (Fig. 7B, lanes 5 and 6). A similar 35-kDa CAP species has been observed by other
investigators and has been proposed to be CAP that has been cleaved at
the reactive site loop(11, 36) , releasing an
4-kDa carboxyl-terminal peptide from the native inhibitor. It is
possible that a portion of the thrombin-CAP complexes dissociates under
the conditions of reduced SDS-PAGE or that a fraction of the platelet
CAP antigen may act as a substrate for thrombin, both situations that
have been observed for the interaction of the fibrinolytic protease,
tissue type plasminogen activator with its primary physiological
inhibitor, plasminogen activator inhibitor type 1 (39) . Jensen et al.(40) recently detected a form of PAI-2 in
myeloleukemia cells undergoing apoptosis that was cleaved in the
amino-terminal third of the molecule and functionally active. In
contrast, the cleaved forms of CAP after A23187-activation were unable
to interact with thrombin (Fig. 7B), thus further
suggesting that cleavage of CAP occurs at or near the reactive site
loop in the carboxyl-terminal region of the molecule. The low molecular
weight CAP species in A23187-stimulated platelets elute from a
molecular sizing column in fractions corresponding to a molecular mass
of about 68 kDa (Fig. 8). Thus, our data suggest the presence of
an SDS-labile complex of cleaved CAP with an approximately 30-kDa
platelet-associated protein in A23187-stimulated platelets, thus
suggesting a role of this protease inhibitor in regulating proteolytic
activity generated during platelet activation. Complexes between
serpins and certain proteases have been observed to be completely
dissociated under the conditions used for SDS-PAGE, resulting in the
release of reactive site loop-cleaved serpin and free protease. For
example, a cowpox virus-encoded serpin (i.e. the crmA gene product) has recently been shown to form an SDS-labile
inhibitory complex with interleukin-1
converting enzyme, a
cysteine protease(41) . Preliminary experiments revealed that
preincubation of platelets with the membrane-permeable thiol protease
inhibitor EST (E-64d,
epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester)
completely inhibited cleavage of CAP upon platelet stimulation with
A23187.
Experiments in our laboratory are currently
directed on the identification of the proteins (e.g. proteases) interacting with CAP in stimulated platelets.