©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of the Cytoplasmic Antiproteinase (CAP) in Human Platelets
EVIDENCE FOR THE INTERACTION OF CAP WITH ENDOGENOUS PLATELET PROTEINS (*)

(Received for publication, June 26, 1995; and in revised form, September 7, 1995)

Matthias Riewald Kurt A. Morgenstern (1) Raymond R. Schleef (§)

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 Blood Systems Research Foundation Laboratory, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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/10^9 nonstimulated platelets. After platelet activation with the calcium ionophore A23187, CAP antigen was detected in released microparticles at approximately 0.195 ± 0.031 µg/10^9 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.


INTRODUCTION

Serine protease inhibitors or serpins are a ubiquitous superfamily of homologous proteins that resemble alpha(1)-proteinase inhibitor in overall structure and include antithrombin III, protease nexin-1 (PN-1), (^1)and alpha(2)-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.


EXPERIMENTAL PROCEDURES

Platelet Preparation

Six parts blood were drawn from healthy individuals, who denied the ingestion of any medications, into one part acid-citrate-dextrose (NIH formula A), and the upper two-thirds of the platelet-rich plasma were obtained without disturbing the buffy coat after centrifugation at 160 times g for 15 min. All steps during the platelet preparation were performed at 23 °C. Platelet-rich plasma was incubated in the presence of 0.3 µg/ml prostaglandin E(1) (Sigma) for 10 min and centrifuged at 660 times g for 10 min. The pellet was resuspended in wash buffer (137 mM NaCl, 4 mM KCl, 1 mM MgCl(2), 0.1% (w/v) glucose, 0.1% (w/v) bovine serum albumin, 10 mM HEPES, pH 6.8) and again centrifuged (660 times g for 10 min). This washing procedure was repeated twice, and the platelets were resuspended in a small volume of resuspension buffer (137 mM NaCl, 4 mM KCl, 1 mM MgCl(2), 0.1% (w/v) glucose, 10 mM HEPES, pH 7.4), counted using a hemocytometer, and diluted with resuspension buffer to a concentration of 10^9/ml. For the isolation of platelet mRNA, platelets from 2 units (900 ml) blood were washed by centrifugation and subsequently purified on Sepharose 2B (Pharmacia Biotech Inc.) columns according to the protocols described by Konkle et al.(21) . Giemsa-stained smears of the platelet preparation revealed an absence of blood leukocytes. For the activation of platelets, 1-ml samples of centrifuged-washed platelet suspension were incubated at 23 °C for 20 min in the presence or the absence of 162 nM phorbol myristate acetate (PMA; Sigma) or 1 µM of the calcium ionophore A23187 (Sigma). For activation with A23187, the platelet suspension was supplemented with 1 mM CaCl(2) and stirred during incubation with the agonist. In several experiments, the platelets were pelleted by brief centrifugation in a microcentrifuge (13,600 times g for 30 s), 900 µl of the supernatant (releasate) were harvested, and the remaining supernatant was carefully removed. The platelet pellets were lysed in 1 ml of resuspension buffer supplemented with 0.5% Triton X-100 (Sigma). The Triton X-100-insoluble fraction of the cytoskeleton was pelleted by centrifugation (13,600 times g for 2 min), and 900 µl of the supernatant were harvested. Platelet preparations were either used immediately or snapfrozen in liquid nitrogen and stored at -70 °C for up to 2 months. Incubations of platelet samples with I-thrombin were performed in the presence of 0.5 mM Gly-Pro-Arg-Pro (Calbiochem) to inhibit fibrin formation (22) .

Radioiodionation of Proteases

High purity human alpha-thrombin (Sigma T6759; 3000 units/mg protein), high molecular weight human urokinase (American Diagnostica Inc., Greenwich, CT), and trypsin (type III from bovine pancreas; Sigma T8253) were radiolabeled with I utilizing immobilized chloramine T (Iodo-Beads; Pierce) according to the standard protocol that is suggested by the manufacturer. A specific radioactivity between 0.8 times 10^4 and 1.4 times 10^4 cpm/ng protein was routinely obtained.

SDS-PAGE and Autoradiography

Samples either were incubated in the presence of 2% SDS at 37 °C for 10 min (nonreducing conditions) or were heated to 100 °C for 3 min in the presence of 2% SDS and 100 mM dithiothreitol (DTT) for disulfide reduction, unless stated otherwise in the figure legends. Proteins were resolved by electrophoresis using 4% stacking and 9% separating polyacrylamide slab gels as described by Laemmli(23) . Molecular weight markers (Life Technologies, Inc.) included lysozyme (M(r) 14,300), beta-lactoglobulin (M(r) 18,400), carbonic anhydrase (M(r) 29,000), ovalbumin (M(r) 43,000), bovine serum albumin (M(r) 68,000), phosphorylase b (M(r) 97,400), and myosin (H-chain) (M(r) 200,000). Proteins were stained with silver nitrate, and gels were dried and exposed to XAR-5 film (Kodak, New Haven, CT) for 24-48 h utilizing intensifying screens. For quantification, the dried gels were exposed to a storage phosphor screen and analyzed using a PhosphorImager and Image Quant software (Molecular Dynamics, Sunnyvale, CA).

Tissue Culture

Human erythroleukemia (HEL) cells (ATCC TIB 180, HEL 92.1.7) and the megakaryocytic DAMI cell line (ATCC CRL 9792) (24) were obtained from the American Type Culture Collection (Rockville, MD). HEL cells were cultivated in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD) in a 5% CO(2) humidified incubator at 37 °C. HEL cells were induced to megakaryocytic differentiation by incubation with 10M PMA according to the protocol described by Long et al.(25) . DAMI cells were grown and cultivated in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) supplemented with 10% horse serum (Bio Whittaker) as described(24) .

Polymerase Chain Reaction

RNA was extracted from PMA-stimulated HEL cells (1.5 times 10^8), DAMI cells (1.5 times 10^8), and platelets (10) according the previously described protocols(26, 27) . RNA was reverse-transcribed utilizing oligo p-(dt)15 primers, avian myeloblastosis virus reverse transcriptase, and the commercially available First Strand cDNA Synthesis Kit (Boehringer Mannheim). Samples of the reverse transcriptase reaction mixtures were adjusted to PCR buffer conditions in a total volume of 100 µl with 60 pmol of each PCR primer and 2.5 units of Taq polymerase (Perkin-Elmer). PCR was performed for 30 cycles in an automated thermocycler (Perkin-Elmer) with cycle times of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C. Primers were prepared utilizing an automated oligonucleotide synthesizer (PCR-MATE 391 DNA Synthesizer; Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. Primers were based on the published nucleotide sequences and included the following: sense, TGCTTAGGGTGGCCAACAGG (base position 272-291), and antisense, AGGTTGCGCAGGACACTCTC (base position 878-859), for CAP(10) ; and sense, CTCACTGTGAACGTGGCCTCC (base position 7013-7033), and antisense, GGCAGCACGCTGAGGTCTTACA (base position 7714-7693), for von Willebrand factor(28) . To confirm the identity of CAP at a molecular level, primers were also designed that flanked the complete coding region of CAP(10) : sense, GTACTGCTCGAGGTCTGCCATCATGGATGTTC (base position 179-198), and antisense, GATGACGAATTCCTGCCCTGTCCTCACGGAGA (base position 1330-1311), with 12-base pair 5` overhangs containing the unique restriction sites XhoI and EcoRI, respectively. The primers were employed to amplify PMA-stimulated HEL cell cDNA utilizing the aforementioned PCR conditions. PCR products were subcloned into the pBluescript SK(+) vector (Stratagene, La Jolla, CA) using the EcoRI and XhoI sites. A cloned PCR product was sequenced by the dideoxy-mediated chain termination method (29) .

Recombinant CAP

The expression, purification, and characterization of CAP expressed in Escherichia coli as a fusion protein with a peptide containing six consecutive histidine residues (6xHis/CAP) will be described in detail elsewhere. (^2)In essence, the complete coding region of CAP was PCR-amplified utilizing specific CAP primers and reverse-transcribed mRNA from PMA-stimulated HEL cells. Identity of the sequence was confirmed by sequencing of the cloned PCR product. The entire coding region for CAP was cloned into the bacterial expression vector pTrcHisB (Invitrogen, San Diego, CA) and transformed into E. coli, and the cells were induced by incubation for 4 h at 37 °C in the presence of 1 mM isopropylthio-beta-D-galactoside. Recombinant 6xHis/CAP was purified on Ni-charged Sepharose to homogeneity as determined by SDS-PAGE followed by silver staining. 6xHis/CAP was shown to inhibit the amidolytic activity of thrombin, trypsin, and urokinase and to be capable of forming SDS-stable complexes with these proteases, suggesting that this recombinant molecule folds correctly.

Antibodies

Polyclonal rabbit anti-PN-1 serum (30) was kindly provided by Dr. Daniel Knauer (University of California, Irvine, CA). Polyclonal antibodies against a fusion construct encoding maltose-binding protein/CAP (MBP/CAP) were provided by Dr. Walter Kisiel (University of New Mexico, Albuquerque, NM). Antibodies with reactivity against maltose binding protein were removed by passing the preparation over a maltose binding protein-AffiGel 15 column (Bio-Rad, Hercules, CA). Antibodies against CAP were also prepared by injection of 6xHis/CAP into a New Zealand White rabbit according to standard protocols. Immunoglobulin G (IgG) was purified by adsorption to protein A-Sepharose CL-4B (Pharmacia) according to the manufacturer's protocol. Affinity purified anti-6xHis/CAP was prepared by adsorption and elution of antibodies using 6xHis/CAP immobilized on nitrocellulose blots according to the protocols described by Smith and Fisher(31) . Briefly, recombinant 6xHis/CAP (30 µg) was separated by SDS-PAGE and transferred to nitrocellulose, and the region between 38 and 50 kDa was excised and used as an affinity matrix to adsorb antibodies present within a protein A-purified anti-6xHis/CAP IgG preparation (100 µg in PBS/0.1% Tween 20). Antibodies were eluted from nitrocellulose strips by three successive 30-s washes (1.5 ml each) with 5 mM glycine-HCl, pH 2.3, 500 mM NaCl, 0.1% Tween 20, and the eluate was immediately neutralized by the addition of 375 µl of 250 mM Na(2)HPO(4). The resulting eluate is subsequently referred to as ``affinity purified anti-6xHis/CAP.'' As a control, an aliquot of protein A-purified anti-6xHis/CAP IgG (100 µg in 3 ml of PBS, 0.1% Tween 20) was incubated (4 h at 23 °C) with nitrocellulose containing the 38-50-kDa region of electrophoresed recombinant 6xHis/CAP. This adsorption procedure was repeated twice on the solution phase antibodies, and the residual preparation that was depleted of antibodies to CAP is subsequently referred to as ``adsorbed anti-6xHis/CAP.''

Immunoblotting

After electrophoresis, samples were transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) for 90 min at 700 mA in a buffer containing 50 mM Trizma base, 95 mM glycine, 10% methanol, 0.01% SDS. The membranes were blocked for 1 h at 23 °C in 5% nonfat dry milk (w/v) in PBS, 0.1% Tween 20 and were incubated for 1 h at 23 °C in PBS, 0.1% Tween 20 in the presence of the primary antibody in a concentration of 1.5 µg of IgG/ml. After washing, the membranes were incubated for 30 min at 23 °C in a 1:4,000 dilution of biotin-conjugated goat anti-rabbit IgG (Cappel, Durham, NC), followed by avidin-peroxidase (Vectastain ABC kit, Vector, Burlingame, CA) according to the manufacturer's protocol, or in a 1:4,000 dilution of peroxidase-conjugated donkey anti-rabbit Ig (Amersham Corp.). The blots were washed extensively with PBS, 0.1% Tween 20, and bound antibodies were detected using the enhanced chemiluminescence system (Amersham Corp.) according to the manufacturer's instructions. Quantification of immunoreactive bands was performed using a laser densitometer and Image Quant software (both from Molecular Dynamics).


RESULTS

Formation of an SDS Stable 58/70-kDa Complex between I-Thrombin and a Platelet Pellet-associated Protein

Incubation of solubilized, nonstimulated platelets (Fig. 1, lane 1) or the releasate of PMA-stimulated platelets (Fig. 1, lane 2) with I-thrombin followed by reducing SDS-PAGE and autoradiography revealed the formation of a 77-kDa complex between I-thrombin and a platelet component, previously identified as PN-1p(14, 15, 19) . Analysis in the absence of reducing agents demonstrated the presence of high molecular weight material (Fig. 1, lanes 5 and 6) that has been previously shown to contain a ternary complex between I-thrombin-PN-1p and thrombospondin(17, 20) . Additional SDS-stable complexes with an apparent molecular mass of 70 kDa under reducing SDS-PAGE and of 58 kDa under nonreducing conditions formed between I-thrombin and components in lysates of nonstimulated (Fig. 1, lanes 1 and 5, respectively) and PMA-activated (Fig. 1, lanes 3 and 7, respectively) platelets. These smaller complexes were not detected in the releasate after stimulation with PMA (Fig. 1, lanes 2 and 6). Because sample preparation for reducing SDS-PAGE involved heating to 100 °C for 3 min, whereas sample preparation for nonreducing SDS-PAGE involved a 10-min incubation at 37 °C, we analyzed whether the extent of thermal-mediated unfolding accounts for the differential mobilities of complexes between I-thrombin and a platelet pellet-associated protein. The 70-kDa I-thrombin complex in solubilized platelet pellets was detected only in samples that had been heated to 100 °C prior to SDS-PAGE (Fig. 2A, lanes 4-6, 8, and 9). If the samples were heated to 65 °C or less, the 58-kDa I-thrombin complex was observed (Fig. 2A, lanes 1-3 and 7). Further experiments revealed that the temperature dependence of the mobilities of these I-thrombin complexes was not affected by the presence or the absence of reducing agents in the sample buffer (data not shown). To determine whether a product-precursor relationship between the 70- and 58-kDa complexes exists, the 58-kDa complex (Fig. 2B, lane 1) was excised from a gel, electroeluted, and heated to either 65 °C (Fig. 2B, lane 2) or 100 °C (Fig. 2B, lane 3), and subsequent reanalysis by SDS-PAGE revealed that the complex heated to 100 °C migrated at 70 kDa (Fig. 2B, lane 3).


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 10^8 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, 10^8 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 times 10^8 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(2). 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.



The 58/70-kDa Complex-forming Protein Is Functionally and Immunologically Similar to CAP

Based upon the estimated molecular mass of the 70-kDa thermally unfolded I-thrombin complex, we hypothesized that the platelet component of the 58/70-kDa complex was related to CAP. In addition to thrombin, CAP has been shown to inhibit other serine proteases, including trypsin and urokinase(33, 34) . To further characterize the specificity of this platelet inhibitor, I-trypsin and I-urokinase were incubated with the detergent extracted pellet of PMA-activated platelets followed by SDS-PAGE and autoradiography. Fig. 3shows that the platelet pellet-associated component forms SDS-stable complexes with I-trypsin and I-urokinase. Furthermore, these complexes undergo a unfolding transition upon boiling that is similar to the I-thrombin-containing complex (Fig. 3, A and B, compare lanes 2 and 3). To determine if the nonsecreted platelet inhibitor is immunologically related to CAP, we examined the ability of polyclonal rabbit anti-CAP and anti-PN-1 IgG to block formation of the I-thrombin-inhibitor complexes. Fig. 4shows that formation of the I-thrombin-containing 58-kDa complex could be inhibited by preincubation with anti-CAP IgG generated against the CAP moiety of a maltose-binding protein-CAP (MBP/CAP) antigen, whereas preincubation with anti-PN-1 IgG or preimmune rabbit IgG at similar concentrations had no effect on the formation of the putative I-thrombin-CAP complex. Likewise, the CAP-specific anti-MBP/CAP IgG had no effect on formation of the 77-kDa I-thrombin-PN-1p complex in platelet releasates, whereas the anti-PN-1 IgG completely inhibited formation of this 77-kDa complex. These results demonstrate that the previously identified platelet protease-complexing activity is functionally and immunologically identical to CAP.


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 (10^9 platelets/ml) was incubated at 23 °C for 30 min with either 5 nMI-trypsin (A) or with 5 nMI-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.



Immunoblotting Analysis of CAP Antigen in Platelets and Platelet-derived Microparticles

Because the utilization of radiolabeled proteases to detect CAP provides information only about the functionally active pool of CAP, Western blotting was subsequently utilized to investigate total CAP antigen associated with platelets and their products (i.e. releasate and microparticles) following agonist-induced activation. Fig. 6shows the molecular species of CAP following activation of platelets with compounds that either stimulate (calcium ionophore A23187) or do not stimulate (PMA) the shedding of platelet microparticles. Fig. 6A (lane 2) demonstrates the detection of a 39-kDa antigen in the pellet of PMA-activated platelets using CAP-specific anti-MBP/CAP IgG. The apparent size of this antigen is in agreement with the reported size of native CAP(34) . Preincubation of CAP-specific anti-MBP/CAP IgG with a recombinant fusion protein of a six histidine residues-containing peptide and CAP (6xHis/CAP) significantly reduced the staining of the 39-kDa protein (Fig. 6A, lane 4), demonstrating that this platelet-associated protein is CAP. CAP-specific antigen was not detected in the releasates after platelet stimulation with PMA (Fig. 6A, lanes 1 and 3). A strong doublet with a molecular mass of approximately 74 kDa was also detected in the platelet pellet by Western blotting and was nonspecifically recognized by the secondary antibody (data not shown).


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 10^7 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 times 10^7 platelets), pellets of A23187-activated platelets (lane 5, 5 times 10^7 platelets), and microparticles (refer to Table 1for experimental details) derived from the releasate of A23187-activated platelets (lane 6, corresponding to 10^8 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(2)), 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/10^9 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/10^9 platelets.

Characterization of Proteolytically Modified Forms of CAP in A23187-stimulated Platelets

Because the supplies of CAP-specific anti-MBP/CAP IgG were limited, we obtained a rabbit antiserum against recombinant 6xHis/CAP and subsequently isolated a 6xHis/CAP affinity purified IgG fraction to further characterize the various CAP species in platelets. Fig. 7A shows the detection of CAP associated with nonstimulated and A23187-stimulated platelets using protein A-purified anti-6xHis/CAP prior to (lanes 1 and 2) and following adsorption with 6xHis/CAP (lanes 5 and 6) and affinity purified antibodies to 6xHis/CAP (lanes 3 and 4). Therefore, the 6xHis/CAP-specific IgG behaved similar to the CAP-specific anti-MBP/CAP IgG and was employed in the following experiments. Fig. 7B indicates that incubation of solubilized A23187-activated platelets with increasing concentrations of thrombin led to the detection of significantly less 70-kDa thrombin-CAP complex (Fig. 7B, lanes 7-12) in comparison with the amount of complex detected in the solubilized nonstimulated platelets (lanes 1-6). In addition, the 37- and 35-kDa forms of CAP appeared after incubation of platelet lysates with thrombin and were indistinguishable from the 37- and 35-kDa CAP forms in A23187-activated platelets. The 37-, 35-, and 34-kDa CAP bands in A23187-stimulated platelets did not decrease upon incubation with thrombin, indicating that these forms of CAP do not form SDS-stable complexes with thrombin and are not further processed by thrombin.


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 (10^7 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 (10^7/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.



Evidence That Cleaved CAP Is Present in an SDS-labile Complex with a Cellular Component in A23187-stimulated Platelets

Molecular sieving chromatography was utilized to investigate whether the cleaved CAP species in A23187-stimulated platelets result from separation of higher molecular weight complexes under the conditions of SDS-PAGE. Fig. 8demonstrates that the majority of 39-kDa CAP antigen in Triton X-100 lysates of both resting and A23187-activated platelets elutes at a volume corresponding to ovalbumin (43 kDa). In contrast, approximately 50% of cleaved CAP in A23187-stimulated platelets (Fig. 8B, open squares) elutes at the position of bovine serum albumin, suggesting that these low molecular weight forms arise by disruption of approximately 68-kDa complexes that are composed of CAP and another cellular protein. Similar chromatographic profiles for CAP antigen in nonstimulated and A23187-activated platelets were obtained from four additional donors (data not shown).


Figure 8: Gel chromatography of platelet lysates on a molecular sieving column. Nonstimulated (A) and A23187-activated (B) platelets (1 ml, 10^9 platelets) were lysed (0.5% Triton X-100) and passed over a Sephacryl S-200 (Pharmacia) column (1.77 cm^2 times 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.




DISCUSSION

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 times 10^6M s), thrombin (K = 5 times 10^5M s), factor Xa (K = 1.3 times 10^5M 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/10^6 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. (^3)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-1beta 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.^3 Experiments in our laboratory are currently directed on the identification of the proteins (e.g. proteases) interacting with CAP in stimulated platelets.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL45954 and HL49563 (to R. R. S.) and M01 RR00833 (to the General Clinical Research Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
Present address: Vertex Pharmaceuticals, 40 Allston St., Cambridge, MA 02139-4211.

§
To whom correspondence should be addressed: Dept. of Vascular Biology (VB-1), The Scripps Research Inst., 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7129; Fax: 619-784-7323.

(^1)
The abbreviations used are: PN-1, protease nexin-1; CAP, cytoplasmic antiproteinase; 6xHis/CAP, recombinant fusion protein of a six consecutive histidine residues containing peptide and CAP; DTT, dithiothreitol; HEL, human erythroleukemia; MBP/CAP, recombinant fusion protein of maltose-binding protein and CAP; PAI-2, plasminogen activator inhibitor-2; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMA, phorbol myristate acetate; PN-1p, platelet-associated protease nexin-1; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Riewald and R. R. Schleef, manuscript in preparation.

(^3)
M. Riewald and R. R. Schleef, unpublished observations.


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