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Expression of Cyclic ADP-Ribose- Synthetizing CD38 Molecule on Human
Platelet Membrane
By Giuseppe Rarnaschi, Mauro Torti, Enrico Tolnai Festetics, Fabiola Sinigaglia, Fabio Malavasi, and Cesare Balduini
CD38 is a cell surface molecule widely used as a marker for
immature and activated lymphocytes. It has been recently
shown that CD38 displays three enzymatic activities: hydrolysis of NAD' to ADP-ribose, synthesis of cyclic ADP-ribose
from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic
ADP-ribose, acalcium-mobilizing compound. We investigate
here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results
showed that CD38 isexpressed by human platelet mem-
branes. Moreover, we show that platelet CD38 possesses
NAD glycohydrolase,ADP-ribosecyclase, and cyclicADPribose hydrolase activities. This finding indicates that the
calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the
possible role of CD38 expression and enzymatic activities in
the signal transduction pathways leading to platelet activation.
0 1996 by The American Societyof Hematology.
H
nal transduction pathways and undergo, upon activation, profound structural and functional changes." This report shows
that CD38 is present on the membrane of human platelets
and that both intact cells and isolated membranes display
ADP-ribosyl cyclase, cADPR hydrolase, and NADase activities. We also show that surface CD38 is responsible for all
these enzymatic activities observed on platelets. Because
platelets undergo profound structural and functional changes
upon activation, they could serve as a model for studying
the involvement of CD38 in the different signalling pathways
which lead to cell activation. These results also raise stimulating questions on the role of CD38 in platelet functions.
UMAN CD38 IS A type I1 transmembrane glycoprotein
expressed by discrete populations of human leukocytes,',2in which it appears to be involved in relevant cellular
events such as activation, differentiation, and adhe~ion.'.~
Human, murine, and rat CD38 show great similarity both in
amino acid sequence and function with the ADPribosyl
cyclase purified from Aplysia californica5" and fromAplysia
kuroday,Ran enzyme that converts p-NAD+into cyclic ADPribose (cADPR). The catalytic properties shared by these
molecules from such phylogenetically distant organisms
were confirmed by recombinant human and murine CD38,
which are able to catalyze the synthesis of cADPR from pNAD'.9.'" cADPR has been reported to be a second messenger operating in different cell systems and promoting inositol l ,4,5-trisphosphate (IP,)-independent Ca2' mobilization
from discrete intracellular stores.""' CD38 was recently
found on the membrane of human erythrocytes, where it
catalyzes the formation of cADPR from NAD' and the hydrolysis to ADP-ribose (ADPR).14.'5The sum of these two
activities results in the conversion of NAD' to ADPR. Thus,
CD38 appears to be a multicatalytic enzyme possessing
NAD glycohydrolase (NADase), ADP-ribosyl cyclase, and
cADPR hydrolase activities.
Although CD38 is expressed by different blood and bone
marrow cells, until now no evidence has been reported concerning its occurence on platelet membranes. Platelets are
very particular cell fragments that exhibit highly active sigFrom the Department of Biochemistry, University of Pavia, Pavia,
Italy: the hboratory of Cell Biology, University of Turin, Turin,
Italy; the Institute of Biology and Genetics, University of Ancona,
Ancona, Italy: and the Institute of Biological Chemistry, University
of Genoa, Genoa, Italy.
Submitted July 27, 1995; accepted October 23, 1995.
Supported by grants from Ministero dell'universita e della Ricerca Scientijica e Tecnologica-Italy (MURST), Consiglio Nuzionale
delle Ricerche Italy (CNR), AIRC, Telethon (Italy), and Regione
hmbardia.
Address reprint requests to Cesare Balduini, MD, Department of
Biochemistry, University of Pavia, via Bassi, 21, 27100 Pavia, Italy.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-497//96/8706-0039$3.00/0
2308
MATERIALS AND METHODS
Materials. @-Nicotinamideadenine dinucleotide (P-NAD+', nicotinamide guanine dinucleotide (NGD+), ADP-ribose, ATP, ADP,
AMP, ADP-ribosyl cyclase from Aplysia californica, bovine serum
albumin (BSA), aprotinin, leupeptin, phenylmethylsulfonylfluoride
(PMSF), Nonidet P40, N-hydroxysuccinimidobiotin, fluorescein
(F1TC)-conjugatedantimouse goat IgG were from Sigma (St Louis,
MO). cADPR was a kind gift from A. De Flora (Institute of Biological Chemistry, University of Genoa, Genoa, Italy). The characteristics of the monoclonal antibody (MoAb) IB, against CD38 are
reported elsewhere." MoAb against platelet glycoprotein (GP) IIbIIIa was from AMAC Immunotech (Westbrook, ME), and peroxidase-conjugated avidinwas obtained from Dako (Glostrup, Denmark), Nitrocellulose membrane was purchased from Schleicher &
Schuell (Dassel, Germany). Sepharose CL-2B and protein A-Sepharose were from Pharmacia Biotech (Uppsala, Sweden). Enhanced
chemiluminescence (ECL) reagents and Hyperfilm ECL were from
Amersham (Little Chalfont, UK). The bicinchoninic acid (BCA)
protein determination kit was from Pierce (Rockford, IL). All other
reagents were of analytical grade.
Plateletpreparution. Bloodwas taken from healthy volunteers
from the antecubital vein using citric acid-citrate-dextrose as anticoagulant. Platelets were isolated by gel filtration on a Sepharose CL2B column equilibrated with HEPES buffer ( I O mmol/L HEPES,
137 mmol/L NaCI, 2.9 mmol/L KCI, 12 mmol/L NaHCO9,pH 7.4).
as described." The platelet count was finally adjusted to 10' cells/
mL.
Flow cytornetry. Blood obtained from normal volunteers was
anticoagulated with I O mmolR. EDTA. Platelet-rich plasma was
obtained by centrifuging the whole blood at 12Og for 10 minutes at
room temperature and treated as reported." Briefly, platelets were
fixed in plasma atroom temperature for 5 minutes with2% (wt/
vol) paraformaldehyde, centrifuged at 400g for I O minutes, and
resuspended in phosphate-buffered saline (PBS; IO mmoln
NaH2P04, 140 mmol/L NaCI, pH 7.4). containing 3 mmol/L EDTA
Blood, Vol 87,No 6 (March 15), 1996:pp 2308-2313
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2309
EXPRESSION OF CD38 ON HUMAN PLATELET MEMBRANE
and 5% (wt/vol) BSA. Cells were then centrifuged at 400g for 10
minutes and resuspended in PBS, 0.3 mmoVL EDTA, 5% BSA, pH
7.4. Platelet samples (150 pL, 5 X 10’ cellslmL) were incubated
with IB4 MoAb (IO pg) or control antibodies for 30 minutes at room
temperature. Cells were then washed and resuspended in 200 pL
PBS, 0.3 mmoVL EDTA, 5% BSA, pH 7.4; FITC-conjugated antimouse IgG was then added at a final dilution of 1:100 (voUvol).
After 30 minutes of incubation in the dark at room temperature,
cells were washed and resuspended in 500 to 600 pL of filtered
PBS, 0.3 mmoVL EDTA, 0.1% BSA, pH 7.4. Samples were analyzed
by FACStar flow cytometer (Becton Dickinson, Mountain View,
CA) with argon laser excitation.
Biotinylation of intact platelets. Gel-filtered platelets were incubated with 100 pg/mL of N-hydroxysuccinimidobiotinfor 1 hour at
room temperature. Cells were then gel-filtered again onthe same
column used for platelet preparation. N-hydroxysuccinimidobiotin
was dissolved at 10 mg/mL in dimethylsulfoxide (DMSO); the final
concentration of DMSO in platelet samples did not exceed 1% (VOU
vol).
Membrane preparation. Platelet concentrates were obtained
from the local blood bank (Servizio di Immunoematologia e Trasfusione, IRCCS Policlinico S. Matteo, Pavia, Italy). Platelets were centrifuged at 1.800g for 20 minutes at room temperature and resuspended in a buffer containing 135 mmoUL NaCI, 2.7 mmoVL KCI,
12 mmol/L NaHCO,, 0.36 mmoVL NaH2P04,2 mmoVL MgCI2,0.2
mmoVL EGTA, 5.5 mmoVL glucose, 0.35% (wt/vol) BSA, pH 6.3.
Cells were then washed in the same buffer without EGTA, glucose,
or BSA and finally resuspended at a final count of 2 X IO9 cells/
mL in hypotonic buffer (10 mmoVL HEPES, 100 pmoUL PMSF,
10 pglmL leupeptin, IO pg/mL aprotinin, pH 7.5). Platelets were
cooled at4°Cand disrupted with 5 X 20 seconds strokes with a
Labsonic 2000 sonicator (Terzano, Milano, Italy) at maximal output.
Sonicate was centrifuged at 1,800g for 20 minutes to eliminate intact
cells and then ultracentrifuged at 100,OOOg at 4°C for 45 minutes.
Cytosol was collected and frozen, and membranes were washed once
with hypotonic buffer. The membrane protein concentration was
determined by BCA assay” and adjusted to 3 to 7 mg/mL . Membranes were stored at -70°C until used.
Purity of platelet
preparations.
Platelet preparations were
checked for contamination by other CD38’ cells both by direct
microscope evaluation of May-Grunwald-Giemsa-stained smears of
the samples and by cytofluorimetric determination of erythrocytes
and B lymphocytes. Both approaches showed that the contamination
of platelet samples by other cells was absent or negligible (data not
shown).
Immunoprecipitation. Platelets or membranes were lysed by
adding 1 v01of ice-cold immunoprecipitation buffer ( I O mmol/L
HEPES, 137 mmoVL NaCl, 2.9 mmoVL KCI, 12 mmoVL NaHC03,
2% [voUvol] Nonidet P40, 1% [wt/vol] sodium deoxycolate, 0.2%
[wtlvol] sodium dodecyl sulfate [SDS], 1 mmol/L PMSF, 20 pg/mL
aprotinin, 20 pglmL leupeptin, pH 7.4).
Lysates were kept in ice for 15 minutes and then centrifuged at
13,000g for 5 minutes. Cleared lysate (500 pL) was incubated with
8 pg/mL IB4 MoAb or control MoAbs for 2 hours at 4°C. Samples
were then added with 100 pL of protein A-Sepharose dissolved in
deionized water at 50 mg/mL and incubated for 30 minutes at 4°C.
The protein A-Sepharose pellets were collected by brief centrifugation and washed five times with ice-cold immunoprecipitation buffer
diluted 1:l with HEPES buffer. Immunoprecipitates were then directly dissociated with SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer, loaded on 10%PAGE gels, electrotransferred to nitrocellulose, and then probed with avidin-peroxidase.
Reactive bands were evidentiated by a chemiluminescent reaction
(Amersham). Immunoprecipitates were resuspended in 10 mmol/L
HEPES, pH 7.5, for enzymatic analysis.
Enzymatic analysis. For enzymatic analysis, intact platelets were
suspended in HEPES buffer (10 mmoVL HEPES, 137 mmoVL NaCI,
2.9 mmoVL KCI, 12 mmoVL NaHC03, pH 7.4) and platelet membranes and immunoprecipitates were suspended in 10 mmol/L
HEPES, pH 7.5. Samples were prewarmed at 37°C for 5 minutes
and then rendered 1 mmoVL NAD+ for NADase and ADP-ribosyl
cyclase assays, 100 pmoVLcADPR for cADPR hydrolase assay,
and 100 pmol/L NGD’ for guanosine 5’-diphosphoribosyl cyclase
(GDP-ribosyl cyclase) assay. Aliquots (100 pL) were taken at different times of incubations (ranging from 0 to 60 minutes) at 37°C and
centrifuged at 13,000g for 5 minutes. supernatants were collected
on Amicon Microcon-3 membrane (molecular
andultrafiltered
weight cut off, 3,000 Daltons). Filtered samples were then analyzed
by reverse-phase high pressure liquid chromatography (HPLC).
HPLC analysis. The reactions of ADP-ribosyl cyclase, cADPR
hydrolase, NAD glycohydrolase, and GDP-ribosyl cyclase activities
of intact platelets, membranes, and immunoprecipitates were monitored by HPLC and quantified with calibration curves of standard
NAD’, NGD’, cADPR, and ADPR. Cyclic guanosine diphosphateribose (cGDPR) was obtained by incubating known amounts of
NGD’ with Aplysia ADP-ribosyl cyclase, as described.” The nucleotides were separated on a 25 X 0.46 cm Supelcosil LC-IST reverse
phase column (Supelco, Bellefonte, PA). The flow rate was maintained at 1.3 mUmin and nucleotides were eluted with a gradient
from 0% to 10% methanol, obtained by mixing buffer A (0.1 moll
L KH2P04, pH 6.00) and buffer B (0.1 moVL KHzP04, 10% VOU
v01 methanol, pH 6.00), as described.” Briefly, buffer B was held
to 0% for 9 minutes, increased linearly to25% by 15 minutes,
increased linearly to 90% from 15 to 17.5 minutes, stepped to 100%
by 19.5 minutes, and held at 100% until 25.5 minutes. Nucleotides
of interest were eluted before 22 minutes. The column was calibrated
with0-NAD’,
AMP, ADP, ATP, cADPR, ADPR, NGD’, and
cGDPR. The peaks were detected at 254 nm.
RESULTS
Cytojuorimetric
analysis.
A significant fluorescence
signal was obtained after reacting platelets with IB4, an
MoAb specific for human CD38, whereas negligible fluorescence was present on platelet incubated with unrelated
antigens (anti-HLA 11) or with the second antibody alone
(Fig 1). Also, the positive control performed using an antiGPIIb-IIIa MoAb gave, as expected, a strong fluorescence
signal (data not shown).
Immunoprecipitation of biotinylatedintactplatelets.
Platelets were biotinylated with 100pg/mL of N-hydroxysuccinimidobiotin for 1 hour at room temperature and then
lysed with immunoprecipitation buffer as describedin the
MaterialsandMethods.Immunoprecipitation
was performed by adding the IB4 MoAb or isotype-matched control MoAbs directed against surface molecules, eg, HLA
class 11, not expressed by the platelets. Immunoprecipitates were then subjected to SDS-PAGE, Western blotting,
andavidin-peroxidasestaining.
IB,-immunoprecipitates
from intact biotinylated resting platelets showed a single
band with a molecular mass of 46 kD(Fig 2, lane 2).
This band had the same electrophoretic mobility of that
observed in the IB4-immunoprecipitate from a CD38’ Blymphoma cell line (data not shown). The band was not
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RAMASCHI ET AL
2310
FLUORESCENCE
Fig 1. Flow cytometry analysis of the expression of CD38 on human platelet. Fixed platelets were incubatedwith IB. MoAb and with
anti-HLA II negative control and stained as described in the Material
and Methods. Similar results were obtained with three different
platelet preparations.
preparations incubated with NGD'; however, we were not
able to detect cGDPR production (data not shown). This
indicates that cyclaseactivity is located exclusively on
platelet membranes. To further investigate the cellular topology of the enzyme activities, intact platelets were used
to perform the determinations. Using NAD+ as the substrate, both NADase and hydrolase activities could be detected (data not shown).Moreover, platelet incubation
with NGD' resulted in the production of cGDPR in a
manner similar to that shown in Fig 4 (data not shown),
thus confirming that NADase, ADP-ribosyl cyclase, and
cADPR hydrolasearedetectable
in intact platelets and
are therefore exposed at the outer surface of the membrane.
Platelet CD38 accounts for NAD glycohydrolase, ADPribosyl cyclase and cADPR hydrolase activities. To test
whether the CD38 protein was responsible for the formation and hydrolysis of cADPR, platelet membranes were
solubilized with immunoprecipitationbuffer and incubated with the IB4 MoAb. The immunoprecipitate was
resuspended in 100 pL of 10 mmol/L HEPES, pH 7.5,
kDa
present when the negative control MoAb (HLA class 11)
was used (Fig 2, lane 1 ) .
Enzyme activities determination. Plasma membrane
preparations were obtained as described from platelet concentrates and were used to assay NADase, ADP-ribosyl cyclase, and cADPR hydrolase activities. When platelet membranes were incubated with 1 mmol/L NAD' at 37"C, a peak
corresponding to ADPR and progressively increasing with
time appeared in the HPLC chromatogram (Fig 3A). A much
less evident peak corresponding to cADPR could also be
detected (Fig 3A).
The cADPR hydrolase activity of membrane preparations was measured by incubating samples with 100 pmoll
L cADPRat 37°C for increasing times;in these conditions,
production of ADPR was clearly detected by HPLC analysis (Fig 3B). When the substrates or the membranes were
not present in the incubation mixture, the formation of the
products was completelyabsent. The specific activities
expressed as nanomoles of products formed per minute by
1 mg of membrane proteins (Table 1) indicate a NADase/
cADPRhydrolase/ADP-ribosylcyclaseratio
of about
100:6:1. In ourexperimentalconditions,
the production
of cADPR was clearly detectable, even if small; cADPR
was a substrate for the cADPR hydrolase activity. For this
reason, the presence of the ADP-ribosyl cyclase activity
was also studied using NGD' instead ofNAD' as substrate; this compound is converted by CD38 to cGDPR,
which, in contrast to cADPR, is a poor substrate for the
hydrolase activity.*' This allows accumulation of the cyclic product that can be easily detected by HPLC. Platelet
membranes in the presence of 100 pmol/L NGD' produce
cGDPR, as shown by HPLC analysis(Fig 4). Cyclase
activity assays were also performed with platelet cytosol
206137-
8 3 4
44.4-
32.54
1
2
Fig 2. Immunoprecipitationof human CD38 moleculefrom biotinylated intact platelets. Gel-filtered platelets were biotinylated with
N-hydroxysuccinimidobiotin, gel-filtered, and then lysed with immunoprecipitation buffer. The lysate was immunoprecipitatedwith IB4
MoAb (anti-CD381(lane 2) or with CB13 MoAb (anti-HLAclass II) used
as negative control (lane 1) and the resulting protein-A Sepharose
pellets were loaded onto a 10% SDS-PAGE gel, electroblotted, and
probed with avidin-peroxidase. A 46-kD biotinylated protein corresponding to the apparent molecular weight of CD38 was detectable
only in lane 2. This experiment is representative of three different
platelet preparations.
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231 1
EXPRESSION OF CD38 ON HUMAN PLATELETMEMBRANE
were used as the substrate (Fig 5A and B). The ratio between NADasekADPR hydrolase/ADP-ribosyl cyclase is
similar to that observed using platelet membrane preparations, thus confirming that CD38 is responsible for all the
enzymatic activities under investigation.
DISCUSSION
0
10
30
20
40
50
60
TIME (min)
0
10
20
30
40
50
60
TIME (min)
Fig 3. NADaw, ADP-ribosylcyclase,andcyclicADPRhydrolase
activiies in platelet membranes. Platelet membranes (100 pL, 6 to
7 mglmL of proteins) resuspended in
10 mmollL HEPES, pH 7.5, were
incubated for increasing times at 37°C with 1 mmol/L B-NAD+ (A)
or 100 pmol/L cADPR IB), centrifuged, and uttrafiltered on Amicon
Microcon-3 membrane. Twenty-microliter aliquots were then subjected to HPLCanalysis.CyclicADP-riboseandADP-ribose
were
quantified by using a calibration curve obtained
with known amounts
of the standard nucleotides. The plots show the amounts of
cADPR
and ADPR produced
at different times 1
by
mg of membrane proteins.
Data of a typical experiment of 6 different preparations that gave
similar results are reported.
The purpose of this study was to investigate the presence
of the CD38 molecule on platelet membranes and to examine
its catalytic features, ie, its reported ADP-ribosyl cyclase
and cADPR hydrolase activity in several cell types.
Using the murine MoAb IB4 specific for human CD38,
we showed using cytofluorimetric analysis that the molecule
is significantly expressed by platelets. Confirmation of this
observation was obtained by immunoprecipitation experiments from biotinylated platelets using the same IB4 MoAb;
avidin-peroxidase staining evidentiated the expected single
band of 46 kD after SDS-PAGE and Western blotting. Because biotin in these experiments can react only withproteins
exposed to the extracellular side of the membrane, this result
highlights the surface localization of CD38. Plasma membrane preparations were able to convert NAD' to ADPR;
additionally, in the same incubation mixture, the formation
of cADPR was detected by reverse-phase HPLC. Because
the levels of this compound were very low, the cyclase activity was also assayed using NGD+ as the substrate. NGD+ is
converted by the same cyclase to cGDPR, which can only
be poorly hydrolyzed to GDPR." In these experimental conditions, a significant cyclase activity was detectable in the
membrane preparations. The cADPR hydrolase activity is
present in the membrane preparation, because the formation
of
ADPR
is stoichiometric with the disappearance of
cADPR. All the catalytic activities under investigation were
expressed on the extracellular side of the membrane, demonstrated also by the fact that intact platelets were able to use
NAD+, cADPR, and NGD+. Moreover, the same enzymatic
activities can be recovered in the IB4MoAb immunoprecipitates, confirming that the CD38 molecule is a multifunctional
enzyme also in platelets. It remains unclear whether the production of cADPR is an obligatory step in the conversion
of NAD to ADPR or whether a direct NADase activity is
present together with ADP-ribosyl cyclase and cADPR hydrolase. However, the evidence that the ratio NADase/
cADPR hydrolase/ADP-ribosyl cyclase is similar in mem-
Table 1. NADase, ADP-Ribosyl Cyclase, and cADPR Hydrolase
Specific Activities of Platelet Membranes
Specific Activities
(mU/mg of protein)
and enzymeactivitiesweretested(Fig
5). Despitethe
presence of the IB4 MoAb, the enzyme activities couldbe
detected in the samples,confirming previous observations
concerning the independence of the CD38 catalytic domain from the IB4 e p i t ~ p e . ' . 'Also,
~
in this case, NAD+
could be transformed by the cyclase to the corresponding
cyclic product (Fig 5A). Moreover, the IB4-immunoprecipitate clearly produced ADPR when NAD+ or cADPR
NADase 100
cADPR hydrolase
ADP-ribosyl cyclase
2.16
0.132
0.024
%
6
l
One unit of enzyme activiry is defined as the amount of enzyme
that produces 1 bmol of ADPR (NADase) or cADPR (ADP-ribosyl cyclase) from NAD' and 1 pmol ADPR (cADPR hydrolase) from cADPR
per minute at 37°C. under the conditions described in the Materials
and Methods.
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231 2
RAMASCHI ET AL
brane preparations to that in IB4 MoAb immunoprecipitates
strongly suggests that, in any case, CD38 is responsible for
all the enzymatic activities converting NAD to ADPR. The
finding that CD38 is present on platelet membrane raises the
intriguing question of its function. Atpresent it is only possible to speculate, but it is interesting that platelets undergo
whichCD38 andits enzymatic
many of theprocessesin
products have already been reportedto beinvolved in several
celltypes.Plateletsexibitmanysignaltransductionpathways, including Ca2+movement, areregulated in their function by different mechanisms, and adhere to each other and
to other cell types. These cells thereforerepresent a very
A
600 h
1
83
3
p 1::
e ADPR
e
cADPR
-14
500-
0
-E
3
;
V
-12
400-
L
-10
9.
-B
P
%
=2
al
300-
-8
200-
-6
:4
100
E
E
-2
0
10
20
30
50
60
50
60
40
TIME (min)
B
c
P
2o
1
f
3
U
0
-
L
9.
CGDPR
0
10
20
30
40
TIME (min)
Fig 5. NADase, ADP-ribosyl cyclase, and cADPR hydrolase activities in IB.-immunoprecipitates.TheimmunoprecipiiatcH,obtained
from 250 pL of membranes (6 to 7 mglmL1 and resuspended in 10
mmollL HEPES, pH 7.5, were directly incubated with 1 mmollL NAD'
(A) or 100 pmol/L cADPR (B) (final volume, 100 pL);ADPR and cADPR
were quantified by HPLC analysis. The figure shows a typical time
coursa of ADPR and cADPR production from NAD' and of ADPR production from cADPR. Thererrultsare axpressedas nanomolesof product obtained per milliliter of incubation suspension.
I
0
I
I
I
6
I
I
1
12
I
l
f
18
TlME(min)
Fig 4. Plateletmembranesproduce cGDPR from NGD+.Typical
chromatographic pattern obtained after incubation for 30 seconds
(A) or for 30 minutes (C) of membrane samples at 37°C with 100
pmol/L NGD'.Thepeaks
corresponding to NOD+ and cGDPRare
shown. cGDPR was identified by coelution with standard cGDPR obtained by incubation of 100 pmol/L NGD+with 3 p g of Ap1p.e californice ADP-ribosyl cyclase
for 60 minutes at room temperature (B). The
peak marked by an asteriskis the putative GDPR?'
stimulating model for futureresearch on the biologic role of
CD38 and may provide valuable information for the identification of its receptor.
ACKNOWLEDGMENT
We thank Prof A. DeFlora (University of Genoa, Genoa, Italy) and
Prof M.E. Cosulich (Universityof Pavia, Pavia, Italy and Istituto Scientific~Tumori,Genoa,Italy) for fruitful discussion,Dr G. Mazzini
(CNR-Center for Histochemistry-Centro Grandi Strumenti, Pavia, Italy)
for performing FACS analysis, and Drs G. Nicolella and H. Abbasali
(Servizio di Immunoernatologia e Trasfusione, IRCCS Policlinic0 S.
Matteo, Pavia, Italy) for providing blood samples of healthy donors.
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EXPRESSION OF CD38 ON HUMAN PLATELET MEMBRANE
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1996 87: 2308-2313
Expression of cyclic ADP-ribose-synthetizing CD38 molecule on
human platelet membrane
G Ramaschi, M Torti, ET Festetics, F Sinigaglia, F Malavasi and C Balduini
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