©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteolytic Processing of Alzheimers Disease A4 Amyloid Precursor Protein in Human Platelets (*)

Qiao-Xin Li , Geneviève Evin , David H. Small , Gerd Multhaup (1), Konrad Beyreuther (1)(§), Colin L. Masters (¶)

From the (1) Department of Pathology, The University of Melbourne, Parkville, Victoria, 3052 Australia, and The Mental Health Research Institute of Victoria, Royal Park Hospital, Parkville, Victoria, 3052 Australia, Center for Molecular Biology, The University of Heidelberg, Heidelberg, D6900 Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The processing of amyloid precursor protein (APP) and production of A4 amyloid are events likely to influence the development and progression of Alzheimer's disease, since A4 is the major constituent of amyloid deposited in this disorder. Our previous studies showed that human platelets contain full-length APP (APP) and are a suitable substrate to study normal APP processing. In the present study, we show that a 22-kDa A4-containing carboxyl-terminal fragment (22-CTF) of APP is present in unstimulated platelets. Both APP and 22-CTF are proteolytically degraded when platelets are activated with thrombin, collagen, or calcium ionophore A23187. Complete cleavage of APP and 22-CTF require the presence of extracellular calcium. Following stimulation in the presence of calcium, a new CTF of 17 kDa is generated, and the NH-terminal epitope of A4 amyloid is lost. Preincubation of platelets with the cell-permeable cysteine protease inhibitors calpeptin, (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl ester (E64d), Na-p-tosyl-L-lysine chloromethyl ketone, or calcium chelator EGTA before platelet stimulation inhibits the degradation of both APP and 22-CTF. Divalent metal ions including zinc, copper, and cobalt inhibit the degradation of APP and 22-CTF. This study suggests that a calcium-dependent neutral cysteine protease is involved in the proteolytic processing of an amyloidogenic species of APP in human platelets.


INTRODUCTION

The major constituent protein (A4) of amyloid plaques, which are a pathologic hallmark of Alzheimer's disease, is proteolytically derived from a larger glycoprotein precursor, the amyloid precursor protein (APP).() The A4 amyloidogenic region spans 39-44 residues, the carboxyl-terminal third of which is part of the transmembrane domain of APP (Kang et al., 1987). A closely related family of membrane proteins (APLP1 and APLP2) (Sprecher et al., 1993; Wasco et al., 1992) do not contain this amyloidogenic A4 sequence. The family of APP glycoproteins now extends to at least 10 isoforms generated by alternate splicing of the APP gene, principally of exons 7, 8, and 15. APP, the prototypic isoform (Kang et al., 1987) is predominantly expressed in the brain, and lacks a Kunitz-type protease inhibitor domain encoded by exon 7. The Kunitz-type protease inhibitor domain (Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et al., 1988) is of particular interest since it might function in an autoregulatory manner by controlling proteolytic events near the cell membrane. The exclusion of exon 15 through alternate mRNA splicing generates a series of APP molecules (L-APP), which were originally discovered in cells of the lymphocyte/monocyte lineage (König et al., 1992). Subsequent studies have shown that the L-APP isoforms are predominantly expressed in non-neuronal cells, where the absence of the exon 15 domain might also be expected to influence events relating to juxta-membranous domains of APP (Sandbrink et al., 1994).

The mechanism of proteolytic processing of APP is an important problem in the study of Alzheimer's disease, since mutations in the APP gene are etiologically linked to some familial forms of the disease (Ashall and Goate, 1994). The pathogenic mutations occur at or near the cleavage sites involved in the release of the A4 protein. Mutations near the NH terminus (-secretase site) and near the -secretase site of A4 cause increased secretion of A4 in transfected cells (Cai et al., 1993; Citron et al., 1992; Haass et al., 1994). Mutations just outside the COOH-terminal -secretase site of A4 (codon 717) cause an increase in the ratio of A4 to A4 (Suzuki et al., 1994). Longer A4 (A4) shows a greater tendency to aggregate. However, the molecular mechanisms of A4 deposition and accumulation in the more common sporadic forms of the disease are not well understood.

APP is modified post-translationally by different proteolytic pathways. Most studies of APP metabolism have been carried out in cell lines transfected with APP or APP cDNA and show that about 15-30% of the newly synthesized full-length APP (APP) passes through a constitutive secretory pathway. This generates the release of a large NH-terminal extracellular fragment of 100-130 kDa into the extracellular compartment and an approximately 10-kDa membrane-associated COOH-terminal fragment (CTF) (Knops et al., 1992; Kuentzel et al., 1993; Weidemann et al., 1989). This process involves cleavage in the middle of the A4 sequence by a putative -secretase activity and therefore precludes formation of intact A4 peptide (Esch et al., 1990; Sisodia et al., 1990). Some of the remaining APP may be reinternalized from the cell surface and degraded in a lysosome/endosome compartment to generate a family of 8-22-kDa CTFs (Golde et al., 1992; Haass et al., 1992; Knops et al., 1992). In addition, low levels of intact A4 or A4 are normally secreted by cells in culture (Cai et al., 1993; Haass et al., 1993; Seubert et al., 1993; Suzuki et al., 1994). This processing may occur either at an early step of the endosomal-lysosomal pathway following endocytosis (Koo and Squazzo, 1994; Shoji et al., 1992), or in a late acidic compartment of the secretory pathway (Busciglio et al., 1993). Proteases termed - and -secretases are required to produce the NH- and COOH termini of A4, respectively. -Secretase activity is either integrated into the cell membrane or operates after release of the transmembrane domain of APP from the membrane. Numerous proteases have been implicated in APP processing (reviewed by Evin et al.(1994)). The proteases which cleave APP in vivo have not been identified. On the basis of specificity and amino acid sequence alone, it is difficult to predict which proteases are important for APP cleavage in vivo because some proteases may require a specific secondary or tertiary conformation of the substrate (Sisodia, 1992). Alternatively, the subcellular compartmentation of many proteases may exclude them from action on APP. For this reason, it is important to study APP processing in a cellular system where normal compartmentation is preserved.

We have demonstrated that human platelets contain APP (Li et al., 1994). Therefore platelets may be a useful model to study normal APP processing. Unlike transfected cell lines in which the processing of APP may not totally reflect processing in vivo (Zhong et al., 1994), purified platelets provide a model system which may be closer to that which occurs in vivo. Platelets are the main source of hematogenous APP and platelet APP is readily released upon physiological stimulation (Bush et al., 1990; Cole et al., 1990; Smith and Broze, 1992; Van Nostrand et al., 1991). The released APP potently inhibits coagulation factor XIa (Smith et al., 1990), suggesting a role for APP in hemostasis.

In the present study, we investigated the processing of APP in stimulated platelets. We describe the identification and degradation of a A4-containing 22-kDa CTF of APP following platelet activation. Our data suggest that a calcium-dependent neutral cysteine protease is involved in the degradation of the amyloidogenic species of APP in platelets.


EXPERIMENTAL PROCEDURES

Materials

Calpeptin was obtained from Calbiochem-Novabiochem Pty. (Alexandria, Australia) and dissolved in dimethyl sulfoxide. Rainbow protein molecular weight markers were purchased from Amersham Corp. (Aylesbury, United Kingdom). Electrophoresis-grade acrylamide, N,N`-methylene bisacrylamide, N,N,N`,N`-tetramethylethylenediamine, and ammonium persulfate were obtained from Bio-Rad. Purified calpain I and II were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Leupeptin was from Auspep (West Melbourne, Australia). PGE, collagen, calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E64), (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl ester (E64d), Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), diisopropyl fluorophosphate (DFP), 1,10-phenanthroline, phosphoramidon, phenylmethylsulfonyl fluoride (PMSF), iodoacetamide, and EGTA were from Sigma. Thrombin was from Boehringer Mannheim (Mannheim, Germany). Protein A-Sepharose CL-4B was purchased from Pharmacia LKB (Uppsala, Sweden). Protein concentrations were determined by the BCA method (Pierce, Rockford, IL). Immobilon P (PVDF) was from Millipore Corp. (Bedford, MA). All other chemicals were of analytical reagent grade.

Antibodies

Monoclonal antibody (mAb) 22C11, which recognizes an epitope near the amino terminus of APP (Weidemann et al., 1989; Hilbich et al., 1993), was from Boehringer Mannheim GmbH (Germany). Polyclonal rabbit antibody 92/9 was raised against purified human APP (Moir et al., 1992). Affinity-purified rabbit antibody to the cytoplasmic domain (residues 645-694) of APP (Ab 369A) (Buxbaum et al., 1990; Nordstedt et al., 1991) was kindly provided by Dr. Sam Gandy (Cornell University Medical College, New York). An anti-CT antibody (raised to 704-730 of APP) was a gift from Dr. B. Cordell (Scios Nova Inc., Mountain View, CA). Monoclonal 6E10 which recognizes residues 1-17 of A4 (Kim et al., 1990) was purchased from the New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY. Polyclonal antibody ``730'' was raised against residues 1-40 of A4. Monoclonal antibodies to talin and thrombospondin were purchased from Sigma. Antibody to GPIb (WM23) has been previously characterized (Berndt et al., 1985; Li et al., 1994). Affinity purified anti-mouse and anti-rabbit alkaline phosphatase-conjugated secondary antibodies were obtained from Promega Corp. (Madison, WI).

Platelet Preparation and Activation

Human platelets were prepared from fresh blood as described previously (Li et al., 1994). In brief, platelets were isolated by centrifugation and washed twice in buffer (0.38% sodium citrate, 0.6% glucose, 0.72% NaCl, pH 7.0) in the presence of 3 µM PGE. Platelets were then resuspended (1 10/ml) in Tyrode's buffer (136.9 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO, 0.42 mM NaHPO with 2 mM CaCl, 1 mM MgCl, 5.5 mM glucose, pH 7.4) (calcium-containing medium). In some cases, platelets were resuspended in EHS buffer (10 mM HEPES, 150 mM NaCl, and 1 mM EDTA, pH 7.4) (calcium-free medium). The platelets were then incubated with 1 µM A23187, or 1 µM PMA, or a combination of 1 unit/ml thrombin and 20 µg/ml collagen for 15 min at room temperature with stirring in the presence of calcium (Tyrode's buffer), unless indicated. After the incubation, EGTA (200 mM, pH 9.0) was added to a final concentration of 5 mM just before centrifugation to separate the platelets from the incubation medium. The platelet pellets were lysed in a modified EHS buffer containing 5 mM EDTA, 1% (v/v) Triton X-100, 0.2 mg/ml leupeptin, and 1 mM PMSF followed by brief centrifugation at 9000 g in a microcentrifuge to remove cytoskeletal elements.

Preparation of Platelet Lysate for Calpain Digestion

To obtain platelet lysate for calpain digestion, platelets were lysed in Tyrode's buffer (with 1% Triton X-100, lacking calcium and any protease inhibitors). Digestion of the CTF of APP with calpain was performed as follows. Calcium chloride (final concentration, 2 mM) and calpain I or II (final concentration, 20 µg/ml) were added to 20 µl of lysate (equivalent to 2 10 platelets) and incubated for 30 min at 30 °C. The reaction was terminated by adding sample buffer (42 mM Tris-HCl, 1% (w/v) glycine, 2% (w/v) SDS, and 5% (v/v) -mercaptoethanol). In some cases, platelet lysates were preincubated with the indicated reagents for 5 min before addition of calpain.

SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting

Lysate equivalent to 2 10 platelets were dissolved in sample buffer, reduced, boiled, and separated on 15 or 8.5% discontinuous polyacrylamide gels in the presence of SDS. The proteins were then transferred electrophoretically to Immobilon P membranes (PVDF, Millipore) as described previously (Li et al., 1994). Nonspecific binding of primary antibody was blocked with 3% (w/v) bovine serum albumin and the PVDF membranes were incubated with primary antibody overnight at 4 °C. Affinity-purified secondary anti-mouse or anti-rabbit IgG conjugated to alkaline phosphatase (1:10,000) were used for immunodetection of the bound primary antibody. The alkaline phosphatase-conjugated secondary antibody was visualized by chromogenic reaction using Fast-Red/Naphthol AS-MX phosphate as substrates for the alkaline phosphatase. Where indicated, chemiluminescence was used to detect the primary antibody (369A) on PVDF membrane: a horseradish peroxidase-linked secondary antibody was used and visualized by enhanced chemiluminescence (Boehringer Mannheim) according to the instructions provided by the manufacturer.

Reproducibility of Experiments

The figures and tables present representative results of experiments which were performed at least three times.


RESULTS

Processing of Full-length APP in Whole Platelets

Previous studies have identified 140-150-kDa APP in platelets (Li et al., 1994; Gardella et al., 1992; Schlossmacher et al., 1992). Processing of this species may generate amyloid-containing fragments. Proteolytic cleavage of APP was therefore examined in whole activated platelets. Platelets were incubated with different agonists in the presence or absence of calcium, lysed, and APP immunoreactivities were analyzed by Western blotting with an affinity-purified antibody to the cytoplasmic domain of APP (antibody 369A). This antibody has been shown to label the 140-150-kDa APP and smaller fragments of APP, and does not detect COOH-terminal truncated soluble APP (Buxbaum et al., 1990; Li et al., 1994; Nordstedt et al., 1991). The present study showed that resting platelets (PGE-treated) contained APP (Fig. 1A, lanes 1 and 5), in agreement with our previous report (Li et al., 1994). The APP was also detected by mAb 6E10 which recognizes an epitope of A4 (data not shown). Activation of platelets by physiological stimulants (a combination of thrombin and collagen) or by nonphysiological agonists (PMA or calcium ionophore A23187) in either calcium-free (EHS buffer containing 1 mM EDTA) or calcium-containing medium (Tyrode buffer containing 2 mM CaCl, 1 mM MgCl) resulted in decreased immunostaining of a APP band (Fig. 1A, lanes 2-4 and 6-8). A23187 together with calcium was the most potent treatment as it induced complete cleavage of APP (Fig. 1A, lane 8, and Fig. 2A, lane 4). Platelet activation and degranulation progressed under these conditions with or without calcium, as indicated by release of APP into the medium (Fig. 1B).


Figure 1: Western blotting analysis of cleavage of APP in intact platelets and appearance of APP and GPIb in the medium, indicating that the presence of A23187 plus calcium induces maximum APP degradation and GPIb release. Washed platelets were activated with the agonists indicated in the presence of 2 mM CaCl (+Ca) or 1 mM EDTA (-Ca) and platelet lysates and medium were prepared. Samples were separated by a 8.5% SDS-PAGE gel, transferred to PVDF membranes, and then probed with indicated antibodies. The primary antibody was detected with alkaline phosphatase-conjugated secondary antibody by chromogenic reaction. Panel A, lysates derived from 2 10 platelets and analyzed with an antibody to the COOH terminus of APP (369A). Panel B, platelet incubation medium derived from 2 10 platelets were analyzed with an antibody to the N terminus of APP (mAb 22C11). Panel C, platelet incubation medium derived from 1 10 platelets were analyzed with an antibody to GPIb (WM23). The relative mass of standard protein markers is shown on the left. The localization of APP and GC immunoreactivities are indicated on the right. T/C, thrombin plus collagen; A, calcium ionophore A23187.




Figure 2: Western blot analysis of APP degradation within platelets and GPIb appearance in the medium in the presence of protease inhibitors and metal ions, showing that APP degradation and GPIb release are partially inhibited by calpeptin and EGTA. A, fluorogram to show the inhibition of APP degradation. Washed platelets were treated with the agonists in the presence of the indicated agents. Platelet lysates were prepared and analyzed by Western blotting as described in the legend to Fig. 1 except that the primary antibody 369A was detected by chemiluminescence. B, inhibition profile of GPIb release. Platelet incubation media were analyzed with an antibody to GPIb (WM23). T/C, thrombin plus collagen; A, A23187; Cal, calpeptin.



Platelet APP could also be detected by Western blotting using the chemiluminescence visualization technique, which is more sensitive than chromogenic visualization (Fig. 2A). Following activation, APP disappeared and two faint bands of 90-95 kDa labeled by 369A appeared, these bands were not present in the PGE-treated platelets (Fig. 2A, lanes 1 and 4). By semi-quantitation using densitometric analysis, only about 60-70% of APP was cleaved when platelets were stimulated by thrombin plus collagen or by PMA, in contrast to nearly 100% of APP being degraded in A23187-treated platelets.

In order to determine whether the disappearance of the APP was due to a proteolytic process, washed platelets were preincubated with various protease inhibitors for 15 min before addition of A23187. Cleavage of APP in A23187-treated platelets was inhibited by calpeptin, TLCK, E64d, and EGTA as well as metal ions, Zn, Cu, and Co (Fig. 2A and ). Pretreatment of platelets with calpeptin, EGTA, or Zn protected 40-60% of APP from degradation. Cu and Co blocked almost all of the APP degradation (90%). A lower concentration of calpeptin (2 µg/ml) also partially inhibited APP degradation (data not shown). APP degradation induced by thrombin or PMA was also inhibited by calpeptin and EGTA, similar to that found by A23187 induction (data not shown).

Cleavage of APPIs Not Inhibited by Agents That Affect the Acidic Compartments

There is considerable evidence suggesting a role for lysosomal and acidic compartments in the processing of APP (Busciglio et al., 1993; Caporaso et al., 1992; Haass et al., 1992; Knops et al., 1992). Involvement of a lysosomal or acidic compartment in the degradation of APP was examined by preincubating whole platelets with NHCl, chloroquine, or monensin before addition of A23187. The APP immunoreactivity in the platelet lysate was analyzed by Western blotting. None of these agents inhibited the cleavage of APP (), suggesting that the enzymes of lysosomal or acidic compartments were not involved in the degradation of APP of platelets.

Proteolysis and release of platelet membrane glycoproteins GPIb to the medium as a result of A23187 plus calcium treatment have been reported (McGowan et al., 1983). To confirm this result, we separated the platelets from the incubation medium and analyzed the appearance of glycocalicin (GC, an extracellular region of GPIb) in the medium by Western blotting. Increased amounts of GC were detected when platelets were treated with thrombin plus collagen, PMA, or A23187, and maximum release of GC was found with A23187 plus calcium treatment (Fig. 1C). The release of GC to the medium was partially inhibited by pretreatment of platelets with calpeptin, TLCK, E64d, and EGTA, but not by other protease inhibitors including PMSF, DFP, pepstatin (Fig. 2B).

Identification of APP CTF Containing A4

Initially, we examined the A4-containing CTF of APP by Western blotting of total platelet lysates. A lysate from PGE-treated platelets was found to contain a 22-kDa COOH-terminal fragment of APP which was recognized by antibody 369A (Fig. 3A, lane 1). Two different approaches then confirmed that this 22-kDa band was a CTF of APP. First, when the antibody was preincubated with the peptide homologue used to raise antibody 369A, the staining was abolished (Fig. 3B). Second, other antibodies including the polyclonal Ab 730 (anti-A4) (Fig. 3A, lane 2), anti-CT (raised to 704-730 of APP) (Fig. 3A, lane 3), 92/9 (anti-APP) (Fig. 3A, lane 4), and mAb 6E10 (anti-A4) (Fig. 4A) recognized this 22-kDa fragment. Since the epitope for 6E10 is within the sequence 1-17 of A4 (Kim et al., 1990), this result indicated that the 22-kDa fragment also contains most of the A4 sequence, in addition to the COOH-terminal sequence of APP. The 22-kDa fragment was reproducibly detected in platelets from several normal subjects. The 369A antibody also recognized other CTFs with molecular mass of 15, 16, and 18 kDa (Fig. 3). Since these bands were present in both lysates from PGE- and A23187-treated platelets in similar amounts and therefore were unrelated to the activation process, they were not examined further.


Figure 3: Characterization of a 22-kDa A4 containing fragment in lysates of PGE-treated platelets by Western blot. Platelets isolated in the presence of PGE were lysed and lyastes derived from 2 10 platelets were loaded onto a 15% SDS-PAGE gel and analyzed as described in the legend to Fig. 1. A, APP CTFs were analyzed with different antibodies. Lane 1, antibody to the COOH terminus of APP (369A); lane 2, antibody to the 1-40 sequence of A4 (730); lane 3, antibody to the 704-730 sequence of APP (anti-CT); and lane 4, antibody to purified human brain APP (92/9). B, the immunoreactivity to the 22-kDa band was abolished in the presence of the peptide used to raise the 369A antibody (lane 1). The relative mass of standard protein markers is shown on the left. The localization of APP COOH-terminal immunoreactivities are indicated on the right. Only the 22-kDa band was recognized by all four antibodies.




Figure 4: Western blot showing that cleavage of 22-CTF of APP in activated platelets required external calcium. Washed platelets were stimulated with the indicated agonists in the presence of 2 mM CaCl (+Ca) or 1 mM EDTA (-Ca) and analyzed as described in the legend to Fig. 1. Panel A, platelet lysates were analyzed with antibody 369A or 6E10; panel B, platelet lysates were analyzed with anti-talin. ``*'' indicates the new 17-kDa fragment generated upon platelet activation. T/C, thrombin plus collagen; A, A23187.



Cleavage of the 22-kDa CTF of APP in Whole Platelets is a Calcium-dependent Process

We next examined the degradation of the APP 22-CTF associated with platelet activation. Platelets incubated with various agonists were lysed and the APP immunoreactive polypeptides in the lysate were analyzed by Western blotting using antibody 369A. When platelets were incubated with the calcium ionophore A23187 (1 µM) in a calcium-containing medium, the 22-kDa band (Fig. 4A) disappeared and a 17-CTF was observed (Fig. 4A, lane 8, +Ca). The 17-CTF was not present in the PGE-treated platelets (Fig. 4A, lane 5, +Ca), and was not recognized by antibody 6E10 (Fig. 4A, lane 4, +Ca). This process was dependent on the presence of external calcium, since platelets treated with A23187 in a calcium-free medium did not lead to the degradation of the 22-kDa CTF, nor to the generation of the 17-kDa CTF (Fig. 4A, lanes 4 and 8, -Ca), even though platelets were stirred in both situations to maximize platelet activation.

The cleavage of the 22-CTF in whole platelets was also examined after stimulation with other agonists. When platelets were treated with thrombin (1 unit/ml, or even 10 units/ml) and collagen (20 µg/ml) or PMA (1 µM) in the presence or absence of calcium, degradation of the 22-CTF was not observed (Fig. 4A, lanes 2 and 3; 6 and 7, +Ca and -Ca). Under these conditions, however, the platelets were activated, as indicated by increased APP secretion following degranulation (Fig. 1B, lanes 2, 3, 6, and 7).

Effect of Protease Inhibitors and Metal Ions on the Proteolytic Cleavage of the 22-CTF of APP

The effect of different protease inhibitors was examined to identify the classes of proteases which may be involved in the cleavage of the A4-containing 22-CTF. Platelets were preincubated with protease inhibitors or metal ions for 15 min, then the platelets were treated with A23187. Platelet lysates were prepared and the APP immunoreactivities were observed by Western blotting using antibody 369A. When platelets were preincubated with calpeptin, TLCK, E64d, or EGTA, degradation of the 22-CTF in A23187-treated platelets was inhibited (Fig. 5A). Inhibition of the 22-CTF cleavage was also detected with lower concentration of calpeptin at 2 µg/ml (data not shown). Cleavage of the 22-kDa CTF was not inhibited by the serine protease inhibitors PMSF or DFP, or the metalloprotease inhibitors 1,10-phenanthroline or phosphoramidon, or the aspartic protease inhibitor pepstatin A (). Cleavage of the 22-CTF still occurred in the presence of the cysteine protease inhibitors iodoacetamide, or the chymotrypsin-like serine protease inhibitor TPCK, but the intensity of the 17-kDa band was diminished compared to the A23187-treated platelets (). Preincubation of platelets with Zn, Cu, or Co inhibited the cleavage of 22-CTF, while Fe did not have any inhibitory effect ().


Figure 5: Western blot of the 22-CTF degradation in the presence of protease inhibitors, showing that calpeptin and EGTA prevent the disappearance of the 22-CTF and the appearance of the 17-CTF, and inhibit talin degradation. Washed platelets were preincubated with the indicated reagents, then treated with A23187. Platelets were separated from incubation medium and the platelet lysates were analyzed by Western blot using antibody 369A (panel A) or anti-talin (panel C); platelet incubation media were analyzed with 22C11 (panel B). ``*'' indicates the new 17-kDa fragment generated upon platelet activation. Cal, calpeptin.



The inhibitors calpeptin and EGTA which inhibited the degradation of 22-CTF of APP did not significantly affect the secretion of -granule proteins including APP (Fig. 5B), indicating that platelets under these conditions were physiologically competent and that the processing of the 22-CTF was specifically associated with the calcium influx. Previous studies have shown that the concentration of calpeptin used does not interfere with platelet aggregation and secretion (Fox et al., 1990). However, the inhibitory effect of metal ions (Zn, Cu, or Co) on the 22-CTF degradation may be due in part to their effects on inhibiting platelet activation, since they inhibited the release of both APP and thrombospondin (data not shown).

The involvement of a lysosomal or acidic compartment in the cleavage of 22-CTF was also examined. When whole platelets were preincubated with NHCl, chloroquine, or monensin before the addition of A23187, cleavage of the 22-CTF was not inhibited (). This suggests that the enzymes of lysosomal or acidic compartments were not involved in the degradation of the 22-CTF.

We also examined cleavage of other platelet proteins as a result of A23187 treatment. Talin is an endogenous substrate of calpain and its degradation is used as an index of calpain activity upon platelet activation (Fox et al., 1985, 1990). Complete cleavage of talin was only observed in the calcium-containing medium, but not in the calcium-free medium (Fig. 4B, lanes 4 and 8), where under the same conditions the disappearance of the 22-CTF of APP and the appearance of the 17-CTF occurred (Fig. 4A). A combination of thrombin and collagen treatment or PMA treatment in the presence or absence of calcium resulted in partial degradation of talin (Fig. 4B, lanes 2, 3, 6, and 7), in agreement with previous reports (Fox et al., 1990; Yano et al., 1993), but the cleavage of 22-CTF was not apparent (Fig. 4). In contrast, platelet plasma membrane glycoprotein IIIa and the cytoskeleton protein actin were not cleaved under any of these conditions (data not shown). The cleavage of talin was also inhibited by calpeptin, TLCK, E64d, or EGTA (Fig. 5C).

Digestion of CTF of APP by Purified Calpain

The previous results suggested that the cleavage of the 22-CTF was mediated by a calcium-dependent neutral cysteine protease, most of which is composed of calpain in platelets (Phillips and Jakabova, 1977). To avoid the problem that some inhibitors may not have been able to penetrate the platelet membrane and interfere with intracellular processing, a platelet lysate was prepared in Tyrode buffer containing 1% Triton X-100 without any added calcium or protease inhibitors. Under these conditions, a 20-CTF of APP was identified in the initial lysate by antibody 369A. From the previous experiments (Fig. 3-5), it was apparent that platelets lysed in the presence of protease inhibitors (leupeptin, PMSF, and EDTA) contained a 22-CTF which disappeared after repeated freeze-thawing of the lysate. This finding suggests that the 22-CTF is a labile intermediate in the APP processing pathway. Interestingly, intact talin was not recovered in the lysate prepared without protease inhibitors, suggesting that activation of endogenous calpain due to the lysis procedure was sufficient to cleave the 22-CTF and talin, but not sufficient to produce a 17-CTF of APP. To investigate the possibility that calpain may be involved in the degradation of APP fragments, platelet lysates prepared without protease inhibitors were incubated with purified calpain I or II in the presence of 2 mM calcium. On analysis of the APP fragments by Western blotting with antibody 369A, the addition of calpain I resulted in the generation of a 17-CTF, while calpain II did not generate a 17-CTF (Fig. 6, lanes 3 and 4). The generation of the 17-CTF by calpain I was inhibited by preincubation of the lysate with calpeptin, E64, TLCK, EGTA, or Cu, partially inhibited by Zn, but not by iodoacetamide, DFP, Co, Fe, or pepstatin A (). The study of APP processing in cell-free lysate was not possible because there was no detectable APP in the platelet lysates prepared without protease inhibitors, indicating that APP was rapidly degraded upon platelet lysis as previously reported (Li et al., 1994; Schlossmacher et al., 1992).


Figure 6: Western blot of calpain digestion of platelet lysates, showing that purified calpain I can produce a 17-CTF. Platelet lysates were preincubated with the indicated reagents followed by the addition of calpain. Samples were separated on a 15% SDS-PAGE gel followed by Western blotting using antibody 369A. ``*'' indicates the 17-kDa fragment generated by exogenous calpain I. C, platelet lysate without incubation; C`: 0.2% MeSO was added to the lysate during incubation. Cal: calpeptin.




DISCUSSION

This study provides evidence that A4 containing CTF of APP and APP are present in platelets and are proteolytically processed following platelet activation. APP is cleaved when platelets are stimulated by thrombin, collagen, or a combination of both, or by PMA or A23187. Stimulation of platelets by PMA or by a combination of thrombin and collagen leads to the loss of 60-70% of APP. In contrast, treatment with A23187 in the presence of calcium induces complete degradation of APP, and induces the generation of two new CTFs of 90-95 kDa identified by 369A, which are not present in PGE, thrombin/collagen, or PMA-treated platelets. This indicates that the APP can be cleaved in the NH-terminal extracellular domain. It is not clear whether the degradation of APP produces any large NH-terminal fragments (by cleavage at the COOH-terminal side), since it is difficult to differentiate any newly generated NH-terminal fragments from the existing sAPP species (e.g. APP, APP) in PGE-treated platelets. The cleavage of APP is partially inhibited by cysteine protease inhibitors, calpeptin, TLCK, E64d, or the calcium chelator EGTA, but not inhibited by serine, metallo, or aspartic protease inhibitors (DFP, PMSF, phosphoramidon, 1,10-phenanthroline, or pepstatin) and acidic compartment inhibitors (monensin, chloroquine, or NHCl). Although metal ions (Zn, Cu, and Co) inhibit APP degradation, this may be due to complex effects on the physiological function of platelets, since these ions affect the secretion of platelet -granule contents, including APP and thrombospondin.()

A 22-kDa polypeptide was found in platelets which reacts with a variety of antibodies to the A4 and COOH-terminal region of APP, including 6E10 and 369A. This indicates that the 22-CTF contains the A4 sequence, and is therefore derived from APP, rather than from the related molecules APLP1 and APLP2 (Sprecher et al., 1993; Wasco et al., 1992), and also is potentially amyloidogenic. Because the 22-CTF is present in resting platelets, it is likely to have been produced by a normal processing event. Previous studies have also identified APP CTFs in platelets and lymphoblastoid cells in familial Alzheimer's disease (Ghiso et al., 1994; Matsumoto and Fujiwara, 1993). In both studies, the NH terminus of the CTF was found to begin 30 residues NH-terminal to the A4 sequence. By immunoreactivity with specific antibodies, a 22-kDa APP fragment containing the A4 epitope was also found in transfected 293 cells, brain microvessels, and human umbilical vein endothelial cells (Haass et al., 1992; Knops et al., 1992; Tamaoka et al., 1992). When platelets were treated with A23187 plus calcium, the 22-CTF was not detected. Concomitant with the loss of the 22-CTF, there was the appearance of a 17-CTF. This 17-CTF did not react with mAb 6E10 (which recognizes A4), indicating that the NH terminus of A4 may have been removed with the loss of this epitope. Since the 17-CTF band reacts more strongly with antibody 369A than the 22-CTF band, this suggests that larger fragments (including APP) could also be the precursors of the 17-CTF. Low levels of 22-CTF cleavage and 17-CTF production in thrombin/collagen-treated platelets may occur, but this may be beyond the sensitivity of the Western blotting detection used in this study. The cleavage of 22-CTF is also inhibited by calpeptin, TLCK, E64d, the calcium chelator EGTA, or metal ions (Zn, Cu, Co), but not by serine, metallo, or aspartic protease inhibitors, and not by reagents that affect the acidic compartment and lysosomes.

The protease responsible for the degradation of APP and 22-CTF in the stimulated platelets remains to be identified. The characteristics of inhibition of degradation of APP and 22-CTF are consistent with the involvement of a calcium-dependent neutral cysteine protease, since degradation of APP and 22-CTF occurred at neutral pH and was inhibited by cysteine protease inhibitors (calpeptin, TLCK, E64d), and the calcium chelator EGTA. Calpeptin inhibits a number of cysteine proteases (Tsujinaka et al., 1988; Sasaki et al., 1990) and is widely used for studying the function of platelet calpain which constitutes most of the calcium-dependent protease activity in platelets (Ariyoshi et al., 1991; Fox et al., 1990; Oda et al., 1993). Calpeptin is known to inhibit lysosomal cathepsin B as well as calpain (Sasaki et al., 1990). However, cathepsin B is most active under acidic conditions and does not require calcium. Involvement of cathepsin B in the platelet APP degradation is unlikely, since the platelet APP degradation was not affected by NHCl and chloroquine. Addition of purified calpain to the platelet lysate generated a 17-CTF, similar to that found in the A23187-treated platelets. The generation of the 17-CTF was also inhibited by calpeptin, E64, TLCK, and EGTA. Taken together our data suggest that a calcium-dependent mechanism involving a neutral cysteine protease with characteristics similar to calpain participates in the degradation of APP and 22-CTF. However, the involvement of other proteases in APP degradation seems likely.

Since APP is membrane-associated (Li et al., 1994), it could be expected that the proteolytic activity (a calcium-dependent neutral cysteine protease) involved in APP processing also co-localizes to membranes. The main calcium-dependent neutral cysteine protease in platelets is calpain and it is usually located intracellularly in the cytoplasm or associated with membrane (Saido et al., 1993, 1994). Our results confirm the presence of a calcium-dependent neutral cysteine protease activity (calpeptin and EGTA sensitive) inside platelets which degrades talin. This activity would correspond to the previously described calpain activity (Fox et al., 1985, 1990; Yano et al., 1993). We could also demonstrate an activity resembling calcium-dependent neutral cysteine protease outside platelets by studying the release of GPIb (the receptor for von Willebrand factor). Increased amounts of GC were detected in the medium as a result of platelet activation, and this process was partially inhibited by calpeptin, TLCK, E64d, or EGTA. This is consistent with the finding that GPIb is cleaved by a cysteine protease (calpain) within the extracellular juxta-membranous segment to release GC (McGowan et al., 1983; Yoshida et al., 1983). The activity which cleaves GPIb therefore shares similar characteristics with the one that processes APP. Whether cleavage of APP and GPIb involves the same or different calcium-activated neutral cysteine proteases remained to be determined.

Other studies support our view that calcium is involved in the regulation of APP processing. Buxbaum et al.(1994) showed that APP processing is regulated by intracellular calcium levels, independent of regulation by protein kinase C activation. Calcium might also be involved in the amyloidogenic processing of APP (Mattson et al., 1992; Peterson et al., 1986; 1991; Saito et al., 1993). Increasing intracellular calcium levels with A23187 mediates a 3-fold increase in production of A4 in human kidney 293 cells transfected with APP (Querfurth and Selkoe, 1994). An increase in the activated form of calpain is found in Alzheimer's disease brain regions where neurons are most affected (Nilsson et al., 1990; Saito et al., 1993). Although we have been unable to demonstrate any qualitative differences between Alzheimer's disease and control platelets in their Ca-activated processing of APP,() the possibility remains that a chronic disturbance of calcium homeostasis in neurons (especially if caused by the toxicity of the A4 protein itself) (Furukawa et al., 1994) might perturb a calpain-like enzyme mediated cleavage of APP. This may modulate a cycle of calcium-mediated APP processing, and thereby affect an amyloidogenic pathway contributing to the evolution of Alzheimer's disease.

  
Table: Effect of various protease inhibitors, acidic compartment inhibitors, and metal ions on the cleavage of 22-CTF and APP in stimulated platelets

The platelets were preincubated with the reagents for 15 min at room temperature and activated with A23187 (1 µM). The samples were analyzed by Western blotting as described under ``Materials and Methods.'' The final concentrations of inhibitors are shown. ``+'' indicates that the cleavage of substrate was inhibited or that product was formed, ``+/-'' indicates partial inhibition, and ``-'' indicates lack of inhibition of substrate cleavage or no product formation. Numerical values are not assigned as these were not quantitative assays.


  
Table: Effects of various protease inhibitors and metal ions on the generation of the 17-CTF by calpain I in platelet lysates

Platelet lysates were preincubated with the indicated reagents followed by addition of calpain I and analyzed by Western blotting as described in the ``Materials and Methods.'' ``+'' indicates the 17-CTF product was detected, and ``-'' indicates lack of product formation, ``+/-'' indicates that a small amount of the 17-CTF product was detected compared to the control with protease alone. Numerical values are not assigned as this was not a quantitative assay.



FOOTNOTES

*
This work was supported in part by grants from the National Health and Medical Research Council of Australia, the Aluminium Development Council of Australia, and the Victorian Health Promotion Foundation. 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.

§
Supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie.

To whom correspondence should be addressed: Dept. of Pathology, The University of Melbourne, Parkville, Victoria, 3052 Australia. Tel.: 61-3-344-5868; Fax: 61-3-344-4004.

The abbreviations used are: APP, amyloid precursor protein; A4, the APP amyloid cleavage product; CTF, COOH-terminal fragment; APP, full-length APP; sAPP, secreted APP; APP, secreted APP product generated by -secretase; APP, secreted APP product generated by -secretase; PGE, prostaglandin E; PMA, phorbol 12-myristate 13-acetate; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane; E64d, (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethylester; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; DFP, diisopropyl fluorophosphate; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; GPIb, glycoprotein Ib; PVDF, polyvinylidine difluoride; mAb, monoclonal antibody; GC, glycocalicin.

Q-X. Li and C. L. Masters, unpublished results.

Q-X. Li and C. L. Masters, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Dr. Sam Gandy (Cornell University Medical College, New York) for the generous supply of the rabbit polyclonal antibody 369A. We thank Dr. Michael Berndt for critical review of this manuscript. We are grateful to Dr. Jon Currie and Valcy Malone of the Mental Health Research Institute of Victoria, Melbourne, Australia, for coordination of volunteers.

Note Added in Proof-A calpain inhibitor (MDL 28170) has been reported to block A4 amyloid production in an APP-transfected cell line (Higaki, J., Quon, D., Zhong, Z., and Cordell, B.(1995) Neuron 14, 651-659).


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