Expression of Cyclic ADP-Ribose- Synthetizing CD38 - Blood

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, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet mem-

UMAN CD38 IS A type I1 transmembrane glycoprotein expressed by discrete populations of human leuko-

cytes,',2 in 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 ADP ribosyl cyclase purified from Aplysia californica5" and from Aplysia kuroday,R an enzyme that converts p-NAD+ into cyclic ADP- ribose (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 p- NAD'.9.'" cADPR has been reported to be a second messenger operating in different cell systems and promoting inosi- tol 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.'5 The 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 sig-

H

From 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 Ric-

erca 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 costs of this article were defrayed in part by page charge payment. 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

branes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP- ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthe- tized 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 Society of Hematology.

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.

MATERIALS AND METHODS

Materials. @-Nicotinamide adenine 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)-conjugated antimouse goat IgG were from Sigma (St Louis, MO). cADPR was a kind gift from A. De Flora (Institute of Biologi- cal Chemistry, University of Genoa, Genoa, Italy). The characteris- tics of the monoclonal antibody (MoAb) IB, against CD38 are reported elsewhere." MoAb against platelet glycoprotein (GP) IIb- IIIa was from AMAC Immunotech (Westbrook, ME), and peroxidase-conjugated avidin was obtained from Dako (Glostrup, Den- mark), Nitrocellulose membrane was purchased from Schleicher & Schuell (Dassel, Germany). Sepharose CL-2B and protein A-Sepha- rose 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.

Platelet preparution. Blood was taken from healthy volunteers from the antecubital vein using citric acid-citrate-dextrose as antico- agulant. Platelets were isolated by gel filtration on a Sepharose CL- 2B 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 at room temperature for 5 minutes with 2% (wt/ vol) paraformaldehyde, centrifuged at 400g for I O minutes, and resuspended in phosphate-buffered saline (PBS; I O mmoln NaH2P04, 140 mmol/L NaCI, pH 7.4). containing 3 mmol/L EDTA

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EXPRESSION OF CD38 ON HUMAN PLATELET MEMBRANE 2309

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 ( I O 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-hydroxysuccinimidobiotin for 1 hour at room temperature. Cells were then gel-filtered again on the 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 Trasfus- ione, 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 at 4°C and 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 . Mem- branes 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 v01 of 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 centrifuga- tion 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, electrotrans- ferred 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 pmoVL cADPR 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 and ultrafiltered on Amicon Microcon-3 membrane (molecular 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 moni- tored by HPLC and quantified with calibration curves of standard NAD’, NGD’, cADPR, and ADPR. Cyclic guanosine diphosphate- ribose (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 main- tained 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 to 25% 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 with 0-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 anti- GPIIb-IIIa MoAb gave, as expected, a strong fluorescence signal (data not shown).

Immunoprecipitation of biotinylated intact platelets. Platelets were biotinylated with 100 pg/mL of N-hydroxysuccinimidobiotin for 1 hour at room temperature and then lysed with immunoprecipitation buffer as described in the Materials and Methods. 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. Immunoprecipi- tates were then subjected to SDS-PAGE, Western blotting, and avidin-peroxidase staining. 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’ B- lymphoma cell line (data not shown). The band was not




2310 RAMASCHI ET AL

preparations incubated with NGD'; however, we were not able to detect cGDPR production (data not shown). This indicates that cyclase activity is located exclusively on platelet membranes. To further investigate the cellular to- pology 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 hydrolase are detectable in intact platelets and are therefore exposed at the outer surface of the membrane.

Platelet CD38 accounts for NAD glycohydrolase, ADP- ribosyl 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 immunoprecipitation buffer and incubated with the IB4 MoAb. The immunoprecipitate was resuspended in 100 pL of 10 mmol/L HEPES, pH 7.5,

FLUORESCENCE

Fig 1. Flow cytometry analysis of the expression of CD38 on human platelet. Fixed platelets were incubated with 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.

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 cADPR at 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 completely absent. The specific activities expressed as nanomoles of products formed per minute by 1 mg of membrane proteins (Table 1) indicate a NADase/ cADPR hydrolase/ADP-ribosyl cyclase ratio of about 100:6:1. In our experimental conditions, 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 of NAD' 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

kDa

206- 137-

8 3 4

44.4-

3 2 . 5 4 1 2

Fig 2. Immunoprecipitation of human CD38 molecule from biotinylated intact platelets. Gel-filtered platelets were biotinylated with N-hydroxysuccinimidobiotin, gel-filtered, and then lysed with immunoprecipitation buffer. The lysate was immunoprecipitated with IB4 MoAb (anti-CD381 (lane 2) or with CB13 MoAb (anti-HLA class 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.




EXPRESSION OF CD38 ON HUMAN PLATELET MEMBRANE

0 10 20 30 40 50 60 TIME (min)

0 10 20 30 40 50 60 TIME (min)

Fig 3. NADaw, ADP-ribosyl cyclase, and cyclic ADPR hydrolase 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 HPLC analysis. Cyclic ADP-ribose and ADP-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 by 1 mg of membrane proteins. Data of a typical experiment of 6 different preparations that gave similar results are reported.

and enzyme activities were tested (Fig 5). Despite the presence of the IB4 MoAb, the enzyme activities could be detected in the samples, confirming previous observations concerning the independence of the CD38 catalytic do- main from the IB4 ep i t~pe . ' . '~ 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

231 1

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

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. Be- cause biotin in these experiments can react only with proteins 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, demon- strated 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 IB4 MoAb 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

(mU/mg of protein) Specific Activities

%

NADase 2.16 100 cADPR hydrolase 0.132 6 ADP-ribosyl cyclase 0.024 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.




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. At present it is only possible to speculate, but it is interesting that platelets undergo many of the processes in which CD38 and its enzymatic products have already been reported to be involved in several cell types. Platelets exibit many signal transduction pathways, including Ca2+movement, are regulated in their function by different mechanisms, and adhere to each other and to other cell types. These cells therefore represent a very

- CGDPR

I I I I I I 1 I l f

0 6 12 18

TlME(min) Fig 4. Platelet membranes produce 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'. The peaks corresponding to NOD+ and cGDPR are shown. cGDPR was identified by coelution with standard cGDPR obtained by incubation of 100 pmol/L NGD+ with 3 pg of Ap1p.e califor- nice ADP-ribosyl cyclase for 60 minutes at room temperature (B). The peak marked by an asterisk is the putative GDPR?'

A

600 - 500-

h

1 8 3 400-

E

3

9.

- B 300- 0

- 200-

100

e ADPR e cADPR p 1:: P -14 %

V

-12 3 -10 ;

2

:4 E

L

al -8 = - 6 -

E

- 2

0 10 20 30 40 50 60 TIME (min)

B

2o 1 c

P f 3 U L 0 9.

0 10 20 30 40 50 60 TIME (min)

Fig 5. NADase, ADP-ribosyl cyclase, and cADPR hydrolase activities in IB.-immunoprecipitates. The immunoprecipiiatcH, 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. The rerrults are axpressed as nanomoles of product obtained per milliliter of incubation suspension.

stimulating model for future research on the biologic role of CD38 and may provide valuable information for the identi- fication of its receptor.

ACKNOWLEDGMENT

We thank Prof A. DeFlora (University of Genoa, Genoa, Italy) and Prof M.E. Cosulich (University of Pavia, Pavia, Italy and Istituto Scien- tific~ 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.




EXPRESSION OF CD38 ON HUMAN PLATELET MEMBRANE 2313

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1996 87: 2308-2313

G Ramaschi, M Torti, ET Festetics, F Sinigaglia, F Malavasi and C Balduini human platelet membraneExpression of cyclic ADP-ribose-synthetizing CD38 molecule on

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Expression of Cyclic ADP-Ribose- Synthetizing CD38 - Blood