Coritributed by Robert C. Shulman, February 23, 1979. ABSTRACT. Suspensions ..... Burt, T. C., Glonek, T. & Barany, M. (1976) J. Biol.Chem. 251,. 2584-2591.
Proc. Natl. Acad. Sci. USA
\'ol. 76, No. 5, pp. 2227-2231, NIay 1979 Biochemist ry
Adenine nucleotide storage and secretion in platelets as studied by 31p nuclear magnetic resonance (cytoplasmic nucleotides/granular nucleotides)
K. UGURBIIB*, H. HOLMSENt, AND R. G. SHULMAN* *Bell Laboratories, Murray Hill, New Jersey 07974; and tSpecialized (Center for Thrombosis Research, Temple University, Philadelphia, Pennsylvania 19140 Cori tributed by Robert C. Shulman, February 23, 1979
currently with making the NMR measurements, the cytoplasmic pools of ATP and ADP (which were prelabeled with [14Cjadenine) were monitored radiochemically, and the total (cytoplasmic plus granular) pools of ATP, ADP, PPi, and Pi were analyzed chemically, in cell extracts.
Suspensions of human and pig blood platelets ABSTRACT have been studied by 31p NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with 1j4CJadenine, was selectively reduced by addition of H202/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di- and triphosphate resonances at temperatures examined (4-50'C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that, in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4VC but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.
Blood platelets are free-floating anucleate cells that play a fundamental role in hemostasis and the maintenance of vascular integrity. These functions are mediated, in part, by adenine nucleotides, which are segregated into two distinct compartments (1). One of the two compartments is the membraneenclosed dense granules, in which as much as 65% of the total cellular ATP and ADP is sequestered together with serotonin, pyrophosphate, and Ca2+ in human platelets (2) and with serotonin and Mg2+ in pig (3) platelets. The cytoplasmic pool contains the remaining adenylates with an ATP/ADP ratio of 7-10; this pool participates in the energy metabolism of the cell and, unlike the granular pool, is affected by metabolic inhibitors (4, 5). During platelet secretion at 370C the granular pool is extruded to the extracellular volume, whereas the cytoplasmic ATP is retained intracellularly, and is partially converted into hypoxanthine (1). When platelets are incubated with radioactive nucleotide precursors such as l14C]adenine, the cytoplasmic pool is labeled rapidly (half time ~40 sec); in contrast, the granules incorporate the label slowly (half time 18 hr) (6). High-resolution 31P nuclear magnetic resonance (NMR) provides a rapid, accurate, and noninvasive method for the study of phosphorylated metabolites in vivo (7-9). Furthermore, 31P NMR measurements possess the capability to distinguish among nucleoside phosphate resonances arising from different subcellular compartments when these compartments maintain differences in pH, divalent cation concentration, or both. With this in mind, we have examined the 31P NMR spectra of resting platelets, of cells challenged with thrombin to induce secretion, and of cells metabolically perturbed with H202 plus NaN3 or 2-deoxyglucose plus antimycin A. Con-
EXPERIMENTAL Platelet Suspensions. Human and pig platelet-rich plasma were prepared as described (5, 10). The platelet-rich plasma was incubated for 2-3 hr at 250C with 0.1 mM adenine and 0.2 [MIU-4C]adenine (code CFA from Amersham/Searle, 250-300 Ci/mol; 1 Ci = 3.7 X 1010 becquerels). This mixture was then diluted 1:1 with ice-cold 0.13 M NaCI/0.02 M TrisHCI/0.003 M EDTA/15 mM glucose, pH 7.4 (suspension medium) and centrifuged at 3000 X g for 15 min at 4 0C. The cells were resuspended in 30-40 ml of cold suspension medium, sedimented as above, and finally resuspended with suspension medium to a final volume of 1.5-2 ml, yielding 36-48 mg of platelet protein per ml. Extractions. For NMR measurements, extracts were prepared as described in the figure captions. For chemical and radiochemical analyses, the extracts were prepared by diluting the platelet suspensions 20- to 30-fold with ice-cold suspension medium and subsequently adding one volume of ice-cold 6.6 M HC1O4 to 10 vol of this dilute suspension. Extracts of the extracellular volume of the platelet suspensions were prepared similarly after the cells had been removed from the diluted platelet suspension by centrifugation at 12,000 X g for 2 min. The HC104 extracts were neutralized with K2CO and stored at -60'C. The levels of total and '4C-labeled ATP and ADP were determined separately (5). 31P NMR Studies. 31P NMR spectra were obtained at 145.7 MHz by using 10-mm sample tubes in a Bruker HX-360 spectrometer operating in the Fourier transform mode. Chemical shifts are reported relative to 85% (wt/wt) phosphoric acid. They were obtained by using a concentric capillary (3 mm in outer diameter) containing 0. 1% phosphoric acid in 0.1 M HCl that had been calibrated within a platelet suspension using internally added glycerophosphocholine.
-
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RESULTS 31P NMR spectra of intact 1 145.7-MHz illustrates typical Fig. human platelets and their perchloric acid extract at 40C. The levels of total intra- and extracellular adenine nucleotides and of the '4C-labeled metabolites that were determined in parallel with the NMR measurements are shown in Table 1. The low extracellular nucleotide levels showed that the cells were intact, and the intracellular constituents had not leaked out. The well-resolved nucleoside di- and triphosphate resonances (7) observed in the platelet extract spectra (Fig. lb) have been assigned to ATP and ADP, because the adenine nucleotides constitute ;90% of the total nucleoside phosphates in human
platelets (1). Pyrophosphate (PP1) and inorganic phosphate (P)
2227
Proc. Natl. Acad. Sci. 17SA 76 (1979)
Biochemistry: Ugurbil et al.
2228
lal)le 2. Adenine nucleotides in human platelets treated with NaN:JH2O,2 for the NMR measurements shown in Fig. 2 lotal ATP and ADP, "/ of total nmol/mgprotein radioactivitv. Time.* AI)P ADP ATP} ATP' mill
ATPy
ATPO (b)~~~~~~~~~
ATPa
ATP-y
R
A
7 7.()
18.8
7}9.4 18.8
14.8 20.7
2:3.0 22.4 24.4
34.7 33.9 11.2
IThe cell suspension contained 681 mgn of protein and 140,000 cpm/ml. All extracts were of total suspension. ). Before NMR measurements; 75. after NMR measurement at 4%' ail(l before addition of 1 mM NaN:i followed by 1.4 mM H9O,9; 190), after NMR measurement of the platelet/NaNj/HO, mixture at
B PiTx T7
(a)
0) 7!, 190
-IOU.
0
-5
5
10
15
20
ppm
obtained for cytoplasmic, '4C-labeled, and total (cytoplasmic and granular) adenylates (Table 1) suggests that :31P NMR at 145.7 MHz detects, in intact cells, only the cytoplasmic nucleotides and not the nucleotides in the granules. In order to test this possibility, the platelets were treated with H202 in the presence of azide to decrease the levels of cytoplasmic nucleotides while not affecting the granular contents (4). The chemical and radiochemical analysis of cell extracts (Table 2) shows that, in response to H202 addition, the 14C-labeled cytoplasmic ATP decreased to 24% of its original value, the total ATP to 34%, and the total (ATP + ADP) to 63%. The integrated intensities of the detectable NMR peaks at 5.2 and 19.0 ppm were reduced to 15 + 10% of their initial values by H202 (Fig. 2 a and b)K which, within the errors, agrees only with the observed reduction in cytoplasmic, '4CIabeled ATP. Large signals from ADP and ATP were subsequently observed in the NMR spectrum when HC104 was added to the cell suspension (Fig. 2c). Fig. 2d shows the spectrum obtained from this sample after the cellular debris and divalent cations were removed, and the pH was adjusted to 7.5. The NMR spectra of a different but identically prepared H202/NaN3-treated human platelet sample was examined as a function of temperature from 40C to 500C; no differences from Fig. 2b were observed even when spectra were accumulated with an 8-fold longer repetition time and the same pulse angle as in Fig. 2. The granular constituents of human platelets were detectable by 31P NMR after their thrombin-induced secretion into the extracellular volume (Fig. 3). The NMR spectra before and after thrombin addition were recorded at 40C>C and the sample was incubated at 370C, for -30 sec in between to stimulate secretion, which does not occur at 4°C (12). Thrombin treatment at 370C reduced the cytoplasmic ATP. and ATPjj peaks to 550% of
FIl;. 1. :P1' NMR sl)e(tra at 145.7 MHz of intact human platelets (a). and their extract (h), at 4O(7. Spectrum a is the sum 400(1 scans of scans (4()0 (40)10 )pulses. (0.34-sec repetitioi time) and h consists (2500 pulses. 6-sec repet it ion time). The cell extract was obtained by HClO.1 addition (final concentration - I M) to the NMR sample. The extract cell debris, passed through was neutralized subsequent to removal (Chelex-lt)t) column (Rio-Had), lyophilized,. and redissolved in H GO at a volume equal to the original sample volume. The pH was adjusted to 7.5 for the spectrum shown. The difference in the chemical shift of' A'I'l', between the extract and cell s)ect~ra reflects ATP binding to divalent cations (e.g.. Mg2+) in the intact cell (7, 183). A and B, unidentified resonances; C is tentatively assigned to granular ATP,, and AI)lP,, on the basis ofFigs. 5 and 6. R is the reference (0. I% phosphoric acid in ).1 M HCI) contained in a concentric capillary. a
assigned on the basis of their known chemical shifts and by addition of the pure compounds into the extract. Peak C is assigned to (V-phosphates of granular ATP as discussed later. The phosphomonoester peaks A and B were not identified. The nucleoside phosphate resonances observed in the intact cells (Fig. la) stem predominantly from ATP. The ADP contribution can be calculated by comparing the intensities of the two resonances located at 5.2 and 19.0 ppm (Fig. la). Using the intensities of these two resonances, we calculate an ATP/(ATP + ADP) ratio of 0.95 for the cell spectrum shown in Fig. la. On a different but similarly prepared platelet suspension the same ratio was 1.0 0.1 when the spectrum was measured as in Fig. la (at both 40C, and 20'C), and 0.91 + 0.1 (40C) when the repetition time was increased 8-fold to avoid possible saturation of the resonances. In contrast to the cell spectra, the ATP/(ATP + ADP) ratio in the extract spectrum (Fig. lb) is 0.59. The comparison of the ATP/(ATP + ADP) ratios obtained from intact cell and extract NMR spectra (Fig. 1) with those were
Table 1.
1l4ClAdenine nucleotides and t~ot~al levels of ATP and ADP in the suspension of human platelets
Sample Platelet suspension
Sulpernatant Secretablet *
used for the NMR measurements shown in Fig. I Total ATP and APP.t Radioactivity. oft* nmol/mg protein AI)P ATP AMP ADP ATP
74.8 0.6 1.7
15.0 0.0
0.5
0.5 0.0 0.7
29.0 0.4 9. 7
16.9 0.2 12.2
ATP/(ATP + A1)) 14C Tot~al 0.6:3 0.83 -
-
-
0.44
The cell suspension contained 71 mg of protein and 190,000 cpm/ml.
2.3% and 7.8%, respectively, in t~he total platelet suspension. Hadioact~ivities found in IMP and (inosine + hypoxanthine) were Only were ATP, AI)P, of the
IMP) cytoplasmic ext~racellular. Approximately all the inosine and hypoxanthine (and none AMP, IMP', inosine, and hypoxanthine are labeled by incubat~ion of platelets with l'4Cladenine (5), and the sum of their radioactivities was constant under our experimental conditions. The radioactivity of each metabolite is therefore expressed as a %YO of the total radioactivity. t Total ATP and ADP represents 14C-labeled cytoplasmic ATP and ADP plus granular ATP and ADP, which are not radioactively labeled. The platelet suspension was diluted 1:31, incubated with 5 units of t~hrombin per ml for 5 min at 370C, and then centrifuged at. 17,550 X g. The values are from t~he supernatant.
Biochemistry: Ugurbil et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
their original values and led to the appearance of new resonances (Fig. 3b). These new resonances were shown to be extracellular because they (and Pi) were quantitatively detectable in the supernatant after the cells were removed by centrifugation. They were specifically assigned to ATP, ADP, and PPi after the supernatant was treated as described in the Fig. 3 legend to obtain spectra with narrower linewidths (Fig. 3 inset). Chemical analysis of supernatants from similar experiments confirmed the presence of ATP, ADP, and PP1 and further showed that the nucleotides were not 14C labeled. The extraand intracellular resonances were resolved from each other (Fig. 3b) because the suspension medium contained EDTA; therefore, the extracellular ATP, ADP, and PP1 resonances displayed chemical shifts that are typical of these compounds in the absence of divalent cations at the extracellular pH (t6.6) of this sample. In contrast, ~s85% of the cytoplasmic ATP is bound to divalent cations as determined from the ATP, and ATP11 chemical shift difference (13) and consequently appears downfield of the extracellular ATP. The a-phosphate resonance of ATP and ADP is not very sensitive to pH or metal ion binding, and therefore is not resolved into intra- and extracellular components in Fig. 3b. Pi
ADPg ADPcv
ATPf
ATPy AADPf
ATPa ADPa I
2229 ATPfl
_
5
7
9
11 21
23
(ATPao + ADPa) (Cy + ex) A
(b) R
ATPCY
In
(a -5
0
5
--I
ppm
10
15
20
FIC(. 3. 31p NMR spectra at 145.7 MHz and 40C of resting human platelets before thrombin addition (a), and after thrombin addition (11.7 units/ml) and incubation in a 370C bath for -3(0 sec (b). The suspension was cooled to 4VC in an ice bath subsequent to the incubation at 370C. Both spectra consist of 4000 scans accumulated using 400 pulses and 0.34-sec repetition time. The cells suspension was prepared as described in the experimental section except that the suspension medium contained 1 mM Pi. R is the reference capillary. Superscripts cy and ex stand for cytosolic and extracellular, respectively. (Inset) Spectrum (5000 scans, 450 pulses, and 2.8-sec repetition time) of the supernatant after removal of the thrombin-treated cells. This supernatant was passed through a Chelex-100 column, and its pH was readjusted to 7.5.
Pig platelets have also been examined with SIP NMR and chemical analysis because the granular composition of pig platelets is different [they contain Mg2+ instead of Ca2+, and store predominantly ATP (3)] and because a 31P NMR spectrum of pig platelets, showing resonances assigned to granular ATP, has been reported (11). The spectrum of resting pig platelets at 4VC (Fig. 4a) was very similar to that of human platelets. As in human platelets, the prominent nucleoside phosphate resonances observed in pig platelets at 4VC were assigned to cyto-
c
(C) ATPCj
ATPp
A B
pi
(b)
(a) -5
0
5
10
ppm
15
20
Fi(;. 2. :4p NMR spectra at 145.7 MHz of human platelets at 4-C; (a) beftore HO,/NaN:i addition (6000 scans); (b) after addition of 1 mM NaN:, and 1.4 mM H209 and incubation at 250C for 3 min (6000 scans); (c) after addition of 20% by volume of 60% HCl04 (wt/wt) to the H)O,/NaN:I-treated sample (4000 scans); and (d) after the cell debris was removed from the sample (from which spectrum c was obtained), its pH was adjusted to 7.5, and the divalent cations were removed with Chelex-100 (1800 scans). All spectra were measured using 400 pulses. The repetition time was 0.34 sec for a, b, and c and 2.8 sec for d. The pH of the sample after HCI04 addition was -0. The phosphomonoester peak located at approximately -3.7 ppm in spectrum b is a composite resonance as shown in spectrum d and includes contributions from AMP and IMP (which are not resolved from each other). The two resonances at 10.5 and 11.6 ppm in spectrum c stem from a and terminal phosphates of ATP, ADP, and PPi.
ppm
FIG. 4. :3p NMR spectra at 145.7 MHz of pig platelets at 40C. (a) Resting cells; (b) after addition of 3 mM NaN3/3 mM H202; (C) after addition of 20% by volume of 60% HCl04. Each spectrum consists of 4000 scans accumulated with 400 pulses repeated every 0.34 sec. All experimental conditions are the same as for Fig. 2. R is the reference capillary described in Fig. 1.
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Ugurbil et al.
2230
Temp., C 30
(c) 25
20
5TPCY
+,
10
5
10
20
15
ppm
Fic,.
Temperature dependence of the granular ADP and ATP pig platelets. Each spectrum is the sum of 1000 scans obtained with 450 pulses and 0.68-sec repetition time. Platelets were incubated with antimycin A and 2-deoxyglucose in plasma for ;:30 mmn before the preparation of the NMR sample. The NMR sampl6 also contained ;z:50 mM 2-deoxyglucose.
ATPCY 0
----A-,. 1I5
1
10
5
15
6.
resonances
20
of
ppm
a
of
FIG. 5. ADP and ATP resonances of pig platelets at 145.7 MHz. (a) Spectrum at 40C (1000 scans); (b) spectrum at 370C, 0.5-6 min after addition of 12.5 Mg of antimycin A per ml and 100 mM 2-deoxyglucose (500 scans); (c) spectrum at 370C, 9-14.5 min after the addition of 2-deoxyglucose/antimycin A (500 scans). All spectra were obtained with 450 pulses and 0.68-sec repetition time. Superscripts cy and gr denote cytoplasmic and granular, respectively. After spectrum c was obtained, another spectrum was measured at 370C with a 2.7-sec repetition time and 450 pulse; it showed that under the pulsing condition used for the spectra shown, the granular peaks at 6.3 and 19.5 ppm were not saturated, while the granular peaks at ; 10 ppm were affected minimally (:' 10%).
plasmic ATP because they were drastically reduced upon H202/NaNs treatment, whereas the subsequent disruption of the cells with HC104 revealed new and intense nucleoside phosphate resonances (Fig. 4c). Chemical and radiochemical analyses of extracts taken from this sample showed that H202/NaN3 addition reduced 14C-labeled cytoplasmic ATP to t8% of its original value, whereas the total ATP was reduced only to 60% of its original value; ADP was unchanged. Unlike human platelets, however, pig platelet granular contents were detectable in the NMR spectrum at temperatures > 150 C. Fig. 5 illustrates the nucleoside phosphate resonances Table 3.
at 40C (Fig. 5a), and as a function 2-deoxyglucose/antimycin A at 370C
pig platelet suspension
of time after addition of
(Fig.
5 b and
' c). The new
.-
tt_ in _ observed Fig. 5 b and
-
resonances
assigned to granular ATP and ADP because 2-deoxygluplus antimycin A depletes the cytoplasmic ATP and ADP but leaves the granular pool unperturbed (5); in this sample, radiochemical analysis showed that '4C-labeled (cytoplasmic) ATP was reduced to ~~5% of its original value (Table 3), while the cytoplasmic ATP resonance observed at 40C disappeared -~9 min after deoxyglucose/antimycin A addition, at 370 C. The temperature dependence of the granular ATP and ADP peaks of pig platelets after the cytoplasmic ATP and ADP had been removed by incubation with 2-deoxyglucose and antimycin' A
c are cose
is shown in
Fig.
6. As the temperature
was
lowered below 300C,
integrated intensities also a-phosphate peak at 10.6 ppmn was affected the
the linewidths increased, and the decreased. The least
by
the low temperatures and was still visible at C; its to that of peak C observed in the intact cell
position corresponds
spectra at 40C (Fig. 4a). The chemical shifts of the granular ATP and AT P1j peaks indicate that the intragranular volume is acidic relative to the
cytoplasm.
This is consistent with the
on isolated granules made by using methylamine distribution, in which the intragranular pH was reported to be 5.5-5.8 (1 1). For an accurate measurement of the intragranular pH by using the granular ATP res-
results of earlier pH measurements
14CIAdenine nucleotides and total levels of ATP and ADP in the suspension of pig platelets treated with 2-deoxyglucose and antimycin A for the NMR measurements in Fig. 5
Time,* min
Sample
ATP
ADP
Radioactivity, %t IMP AMP
Hypoxanthine
Total ATP and ADP, nmol/mg protein ATP ADP
9.3 26.5 2.8 4.5 63.5 2.6 26.5 2.0 1.3 1.0 0.8 2.9 10.2 14.5 53.7 30 2.2 15.4 26.0 2.9 38.1 0.8 1.5 8.0 0.4 Sup The cell suspension contained 58 mg of protein and 195,000 cpm/ml. PS represents values obtained from the platelet suspension and Sup indicates values from the supernatant of the same suspension. * 0, Before NMR measurements and addition of 2-deoxyglucose and antimycin A; 30, ;20 min after addition of 100 mM 2-deoxyglucose and 12.5 pg of antimycin A per ml (after NMR measurements). Percent radioactivity, see * footnote, Table 1. By using the total radioactivity of the extracts together with the specific radioactivity of cytoplasmic ATP and ADP obtained from the ethanol-insoluble ADP fraction (4), the absolute sizes of the cytoplasmic ATP and ADP were calculated to be 13.0 and 5.4 nmol/mg of protein, respectively. 0
PS Sup PS
Biochemistry: Ugurbil et al. onances, an in situ calibration would be necessary due to the well-known effects of divalent cations on ATP titration (13).
DISCUSSION The data presented above demonstrate that ADP and ATP stored in the dense granules of human platelets are not detectable in the intact cell 31P NMR spectra at the temperatures examined (4-50'C). 19F NMR experiments have shown that resonances from fluorinated serotonin contained in human platelet granules are also undetectable (14). The ATP and ADP contained in pig platelet granules, on the other hand, are undetectable by 31P NMR at 4VC, but they become observable at higher temperatures. Clearly, the granular contents of the two different platelets possess different physical properties. The inability to detect 31P resonances from the granular adenine nucleotides in human platelets indicates that the resonance linewidths must be >300 Hz. Interactions with paramagnetic ions cannot account for this large width because atomic absorption measurements showed that intracellular concentrations of Mn2+ and Cu2+ in human platelets are 300 Hz resonance linewidths, and using 140 ppm for the chemical shift anisotropy of the 31P nucleus (15), the rotational correlation time of the granular ATP and ADP complex is calculated to be > 1 Msec; this would correspond to a molecular weight of >106 for a spherical aggregate rotating in an environment with the viscosity of water. In pig platelet granules, which contain Mg2+ and predominantly ATP, extensive aggregation does not exist at 370C because granular ATP is sufficiently mobile to yield resonances ;70 Hz wide. The linewidths, however, increase with decreasing temperatures below t30'C, indicating an increase in the apparent molecular weight of ATP, again probably due to aggregation. The possibility that the granular contents in pig platelets are undergoing a phase transition is excluded because the linewidths increase gradually as the temperature is decreased. Divalent alkaline earth cations as well as monoamines, such as serotonin, have been known to cause aggregation in aqueous solutions of ATP (16, 17). In Mg2+/ATP mixtures the apparent average molecular weight was shown to increase rapidly with decreasing temperatures, finally resulting in the formation of a second phase (16). In our experiments (to be published) Ca2+ caused precipitate formation in the temperature range of 037°C (pH 5.7) at Ca2+ nucleotide molar ratios of ;2, when the ATP and ADP concentration exceeded z10 mM. In the presence of Mg2+ (Mg2+/nucleotide ;1), on the other hand, both ATP and ADP formed a second, transparent, gel phase as described by Berneis et al. (16), when their concentrations exceeded ;250 mM and I110 mM at 370C and 0WC, respectively. These results demonstrate the-possibility of aggregation of ATP, ADP, Ca2+, and Mg2+ at concentrations found in platelet granules and suggest that the difference between Ca2+ and Mg2+ in inducing this aggregation may explain the 31P NMR results from human and pig platelets.
Proc. Natl. Acad. Sci. USA 76 (1979)
2231
In addition to the granular adenine nucleotides, part of the cytoplasmic ADP may not be detectable in the NMR spectra of htman platelets. The two reasons for suggesting this are the following: First, ATP/(ATP + ADP) ratios calculated from radiochemical data on human platelets were always t0.8 (Tables 1 and 2), whereas the intact cell NMR values for the same parameter ranged from 0.9 to 1. Second, in the H202treated sample (Fig. 2, Table 2), the intensity of the 5.2-ppm peak, which consisted of ATPz and ADP# resonances, decreased to 15 ± 10% of its initial intensity in response to H202, whereas the radiochemical analysis showed that cytoplasmic (ATP + ADP) decreased only to 42% of its original value; in better agreement with NMR data 14C-labeled cytoplasmic ATP was reduced to 24% of its initial value. It is known that
