J. Phy8iol. (1975), 251, pp. 803-816
803
With 7 text-ftgure8 Printed in Great Britain
BINDING OF ADENOSINE DIPHOSPHATE TO INTACT HUMAN PLATELETS
BY G. V. R. BORN* AND H. FEINBERGt From the Department of Pharmacology, Royal College of Surgeons, Lincolns Inn Fields, London W02
(Received 12 March 1975) SUMMARY
1. Human platelet-rich plasma was incubated at 370 C with [8-14C]ADP and with human serum albumin labelled with 125I. The platelets were rapidly separated by centrifugation through silicone oil. From radioactivity determinations of plasma and platelet pellets the uptake of ADP, without or with break-down products, by the platelets was calculated on the assumption that the 1251 radioactivity in the pellet represented trapped plasma. 2. ADP radioactivity was taken up by platelets within 10 sec and increased with time of incubation. The uptake of other nucleotide diphosphates was less initially and increased much more slowly. 3. Radioactivity added as ADP was recovered as ATP to the extent of 60%; as ADP of 30 %; and as AMP of 10%. 4. Prostaglandin E1 which inhibited platelet aggregation had no effect on the initial or subsequent uptake or on this distribution of radioactivity. 5. The rate of rise in uptake was much slower when platelets were resuspended in plasma heated to 56° C for 30 min. 6. Unlabelled adenosine inhibited the later, but not the initial, uptake while unlabelled ADP inhibited both. Dipyridamole, which blocks adenosine uptake, prevented the later but not the initial uptake. 7. [oc-32P]ADP radioactivity was taken up at the earliest sampling time and the extent of uptake did not further increase. 8. Guinea-pig platelets, which do not take up adenosine, took up [8-14C]ADP radioactivity from purine-labelled ADP initially. 9. It was concluded that the initial uptake represented binding of ADP and that the later uptake represented labelled adenosine originating as a break-down product of ADP. *
Present address: Department of Pharmacology, Medical School, Hills Road,
Cambridge, CB2 2QD.
t Present address: Department of Pharmacology, University of Illinois, College of Medicine, Chicago, Illinois, U.S.A.
804 8. V. R. BORN AND H. FEINBERG 10. A Scatchard plot of ADP uptake indicated more than one type of binding site. There were approximately 88,000 high affinity sites per platelet which had an affinity constant of 5-41 x 105 M-1. INTRODUCTION
Mammalian platelets are strongly and specifically aggregated by adenosine diphosphate (ADP), and aggregation is preceded by a rapid and striking morphological reaction (Macmillan & Oliver, 1965; Born, 1970). The only other related nucleotides so far known to cause both shape change and aggregation are deoxy-ADP which is less potent, and 2-chloroadenosine and 2-methylthio-adenosine diphosphates which are more potent than ADP (Maguire & Michal, 1968; Gough, Maguire & Penglis, 1972). Other agents which cause platelet aggregation including thrombin, collagen, adrenaline and 5-hydroxytryptamine, release ADP from platelets; and there is evidence that aggregation by at least thrombin and collagen does not occur without this release. The rapidity and specificity with which ADP brings about these effects suggests that they are initiated by the complexing of ADP with receptors specific for it on or just within the platelet surface membrane because ADP, as a highly ionized molecule at physiological pH, is unlikely to pass through the platelet membrane. It has, therefore, been proposed (Born, 1965) that the mode of action of ADP can be best accounted for by assuming that it forms a short-lived complex with a specific receptor of the platelet membrane in a manner similar to that assumed to account for the action of other pharmacological agonists, e.g. adrenaline or acetylcholine, on smooth muscle membranes; in these reactions, the agonists themselves are not altered. In relation to this proposal, arguments that platelets do not 'bind' ADP (Salzman, Ashford, Chambers & Neri, 1969) are invalid as they depend on the meaning of binding in terms of time (Burgen, 1966). Several attempts have been made to demonstrate binding of ADP to platelets (Born, 1964; Salzman, Chambers & Neri, 1966). When radioactive ADP was added to platelet-rich plasma and the platelets were separated by centrifugation, the radioactivity in the platelet pellet suggested an immediate uptake of about 105 molecules of ADP per platelet (Born, 1965). The uptake of radioactivity increased slowly for some minutes and then more rapidly, presumably due to conversion of ADP to adenosine which is known to be incorporated into platelets. Some uncertainty about these results was caused by the necessity to correct the pellet radioactivity for ADP present in the plasma which remains in pellets of platelets after centrifugation. This ADP has to be
805 BINDING OF ADP TO PLATELETS either removed by washing, which may also remove bound ADP, or determined independently by estimating the volume of trapped plasma with another isotope. This paper describes results obtained with an experimental technique designed to minimize these uncertainties. The uptake of ADP radioactivity by platelets was measured after separating them rapidly from plasma by centrifuging through silicone oil; the small amounts of plasma trapped in the pellets was determined separately. METHODS Non-isotopic nucleotides were obtained from Sigma Co., London.
Inosine-[8-3H]5'-
diphosphate, uridine-[5-3H]diphosphate, guanosine-[8-3H]5'-diphosphate, adenosine[2-3H]5'-diphosphate, adenosine-[8-14C]5'-diphosphate and adenosine-"C(U) were obtained from the Radiochemical Centre in Amersham. Labelled ADP was reisolated on DEAE cellulose using NH4CO3 as the eluant after thorough washing with water to eliminate labelled adenosine as a possible contaminant. [a-32P]ADP was prepared by incubating [oc_32P]ATP (Amersham/Searle) with glucose and hexokinase according to the procedure of Lamprecht & Trautschold (1965); the labelled ADP was isolated from an acid-soluble extract (6%, HCl04 v/v) of the incubation mixture by chromatography on DEAE-cellulose. Human serum albumin labelled with 125I (1251I-HSA) was obtained from Amersham/Searle, London. Prostaglandin E1 was obtained from the Upjohn Company and human fibrinogen from the Kabi Co., Stockholm. Platelet-rich plasma was prepared from human blood obtained from healthy adults of both sexes by the procedures of Born & Hume (1967). Platelets suspended in homologous heated plasma (Spaet & Lejnieks, 1966) were prepared by centrifuging platelet-rich plasma for 10 min at 2000 g and resuspending the platelets in platelet-free plasma that had been incubated at 560 C for 30 min, then centrifuged to sediment denatured protein and brought to pH 7-5 with solid Trizma base (Sigma). It was necessary to add human fibrinogen (30 mg/ml.) and Ca2+ (10-3 M) to restore the aggregating effect of ADP (10-5 M). Aggregability of all platelet suspensions to ADP (10-5-10-6 M) was determined before addition of labelled substances; preparations which showed no aggregation were discarded. Platelets in plasma or in heated plasma were incubated at 370 C with constant stirring. Labelled nucleotides and 125I-HSA were added in 2 % or less of the total volume by rapid injection. Samples of 1 ml. were centrifuged at 12,000 g for 30-60 sec in an Eppendorf Microfuge in tubes containing 0-2 ml. silicone oil (Dow Corning 702 and 200, 9:1 v/v); a modified rotor was used to allow the tubes to swing out horizontally (Feinberg, Michel & Born, 1974). Samples (100,l.) of the platelet-free plasma were transferred to counting vials containing 0 5 ml. Soluene (Packard Instruments) and isopropanol (1: 1 v/v). 125I radioactivity was measured in a gamma counter. Ten ml of 0.5 M-HCl-Instagel (1: 9 v/v, Packard Instruments) scintillation fluid was added. 3H or 14C radioactivity was measured and the values were corrected for 1251 radioactivity. The remaining platelet-free plasma lying above the silicone oil was removed by suction. The platelet pellet under the oil was frozen by immersing the tip of the centrifuge tube in powdered CO2 or liquid N2. The part of the tube that had been occupied by the platelet-free plasma was washed repeatedly with isotonic saline. Most of the silicone oil was removed by draining from the inverted centrifuge tube. The platelet pellet was transferred to a counting vial by brief centrifugation of the inverted centrifuge tube held in the
G. V. R. BORN AND H. FEINBERG
806
mouth of the counting vial. The minute amount of silicone oil transferred with the pellet did not affect the counting rates. The platelet pellet and its trapped plasma were solubilized with the soluene-isopropanol mixture and the 126J and 3H or 125I and 14C radioactivities were determined as above. From the 125I-HSA radioactivity of the pellet, the isotopic nucleotide in the trapped plasma and the 3H or 14C radioactivity of the platelets were calculated. The distribution of 3H amongst the nucleotides in the pellet, after varying periods of incubation, was determined by layering 60 % HC104 beneath the silicone oil; thus, during centrifugation the platelets penetrated the silicone oil and entered the perchloric acid layer. Acid-soluble nucleotides in the neutralized extract were separated by high voltage electrophoresis for 1 hr on Whatman 3 MM paper in 0-05 M citrate buffer pH 4-5. The nucleotide spots were identified under an U.V. lamp, eluted and their radioactivity was counted in 0.5 m-HCl-Instagel mixture.
8
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Fig. 1. Apparent uptake (in p-mole/108 platelets) of nucleotide diphosphates, calculated from the uptake of radioactivity, by human platelets incubated at 370 C in citrated plasma. Samples were taken at the intervals shown and the platelets were sedimented through silicone oil. Trapped plasma was determined using 125J-human serum albumin as marker, and the values shown represent the uptake of purine label in excess of that in the trapped plasma. The upper curves represent apparent uptake of ADP (10- M) in the absence (0) and presence ( x ) of prostaglandin E1 (10-6 M) (mean and s.E. of five experiments). The apparent uptakes of GDP (-), IDP (EO) and UDP (0) under the same conditions are shown in the lower curves.
BINDING OF ADP TO PLATELETS
807
RESULTS
Uptake by platelets of radioactivity added as [8-'4C]ADP The uptake of radioactivity by platelets in human platelet-rich plasma, incubated at 370 C, after the addition of [8-14C]ADP (10- M) is shown in Fig. 1. Human serum albumin labelled with 1251 (125I-HSA) was added simultaneously with the ADP to determine the volume of plasma trapped in the platelet pellet; the ADP uptake shown in Fig. 1 is corrected 0.8 C.
0~
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El 1o x ._
o 0
02
._
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2 3 Time (min) Fig. 2. Effect of temperature on uptake by platelets of radioactivity added and expressed as ADP. Human platelet-rich plasma was incubated with 1251-HSA and [3H]ADP (10 4M) at 370 C (-), 270 C (A), or 18' C (0). Samples were taken after the intervals shown and the platelets were sedimented through silicone oil. The ordinate represents the uptake of radioactivity, calculated as ADP (n-mole/108 platelets) from uptake of 3H in excess of that present in the trapped plasma. The vertical bars represent the s.E. of the mean of six determinations. The dashed portion of the lines represent extrapolated parts of the linear regressions calculated from the mean of the observation at 30, 90 and 180 sec at each temperature.
1
for this. Even at the earliest sampling time, i.e. 15 sec, ADP radioactivity was taken up in excess of that present in the trapped plasma. The uptake increased for at least 8 min. Prostaglandin E1 (PGE,), which inhibits platelet aggregation by ADP, has been claimed to inhibit the
808 0. V. R. BORN AND H. FEINBER0 uptake of ADP radioactivity by platelets (Boullin, Green & Price, 1972). In our experiments PGE, added in concentrations sufficient to inhibit aggregation completely had no significant effect on the uptake of ADP radioactivity (Fig. 1). The uptake of radioactivity from ADP greatly exceeded that of other nucleoside diphosphates, namely GDP, IDP, or UDP, all labelled with tritium (Fig. 1). This difference supports other evidence (Gaardner, Jonsen, Laland, Hellem & Owren, 1961) for the specificity of the interaction of ADP with platelets. An attempt was made to resolve further the time course of the initial uptake of ADP. Incubation with 125I-HSA and [-3H]ADP (106 M) for 10 see plus 5 see for centrifugation resulted in an excess of ADP (0.62 + 0-21 p-mole/108 platelets) although centrifugation for 5 see was not long enough to sediment all the platelets.
Effect of temperature on ADP uptake Platelet-rich plasma was incubated with [2-3H]ADP (1.8 x 104 M) and 1251-HSA at three different temperatures. At 270 C, the apparent uptake was slower and at 180 C still slower than at 370 C; however, extrapolation of the linear regressions calculated from the curves at the three temperatures led to closely similar intercepts at zero time (Fig. 2). The simplest explanation of these results (see also Born, 1965) is that the intercept represents an estimate of the initial binding of ADP, which is almost unaffected by temperature in this range; whereas the subsequent increase in the rate of apparent uptake represents an increase in the rate of uptake of break-down product of ADP, presumably adenosine. The fate of isotopic ADP added to platelets Platelet-rich plasma was incubated at 370 C with [2-3H]ADP added at 1 x 10-6 M and the platelets were centrifuged through silicone oil directly into HC104. The distribution of radioactivity present as ATP. ADP and AMP was determined as a percentage of total radioactivity in the acid extract. At the earliest sampling time, i.e. 2 min, ATP accounted for 60 % of the radioactivity, ADP for 30 % and AMP for 10 % (Fig. 3). This distribution remained unchanged for at least 10 min although the absolute amount of radioactivity increased during this time in the manner shown earlier (Fig. 1). Thus, less than 50 % of the radioactivity recovered from pelleted platelets remained as [3H]ADP. The addition of PGE1 (106 M) 1 min before that of [3H]ADP completely inhibited aggregation of the platelets; however, PGE1 had no effect on the distribution of radioactivity between the adenine nucleotides (Fig. 3).
BINDING OF ADP TO PLATELETS
809
80
60 0
40 0 C
0
020
0
2
6
4
8
10
Time (min)
Fig. 3. Distribution of radioactivity taken up by platelets in human platelet-rich plasma incubated at 370 C with 10-6 M [3H]ADP. Samples were taken at the intervals shown and sedimented through silicone oil into 0*6 N-HClO4 containing sufficient sucrose to make the HCl04-sucrose mixture more dense than the silicone oil. The neutralized acid extracts from three assays were pooled and the lyophilized material was taken up in 0.05 M citrate buffer, pH 4-5. Samples of these solutions were subjected to high-voltage electrophoresis together with authentic ATP, ADP and AMP as carriers. The nucleotide spots were made visible under UN. radiation, cut out and oxidized completely to C02 and 3H20. The 3H20 radioactivity was determined by liquid scintillation. The plot represents percent of total radioactivity found in ATP (A A), ADP (U L1) and AMP (@0). The closed symbols represent the radioactivity distribution when labelled ADP only was added, and the open symbols when prostaglandin E1 (106 M) was added 1 min before the radioactive ADP.
Effects of adenosine and dipyridamole on the uptake of ADP radioactivity by platelets ADP added to platelet-rich plasma is broken down by plasma enzymes, viz. adenylate kinase and an ADPase (Haslam & Mills, 1967; Mills, 1966). To determine ADP uptake by platelets under conditions in which this break-down was minimized, platelets were resuspended in homologous plasma which had been heated to 560 C for 30 min. Fibrinogen and Ca2+ were added to restore the aggregation response to ADP.
8. V. R. BORN AND H. FEINBERG Addition of unlabelled adenosine at 2-5 x 10-5 M at the same time as labelled ADP at 10-6 M considerably diminished the increase in uptake of ADP radioactivity over a 20 min period (Fig. 4); however, adenosine had no effect on the uptake at the earliest sampling time, i.e. at about 10 sec, nor during the first 5 min. Addition of unlabelled ADP instead of adenosine had a similar effect on the uptake of ADP radioactivity except that the earliest uptake was also decreased a little (Fig. 4). 810
12.
0 _I
00
00C
0
10 1520 Time (min) Fig. 4. Effect of non-labelled adenosine or ADP on the uptake of radioactivity expressed as ADP (calculated from the radioactivity taken up in excess of that in the plasma trapped with the platelet pellet) by human platelets incubated at 370 C in pre-heated homologous plasma with [14C]ADP at 1 X 10-6 M. Samples were taken at the intervals shown. The upper curve (@) represents the apparent uptake when [14C]ADP (10-6 M) was added; the middle curve (U) when [14C]ADP and adenosine (2.5 x 10-6 m) were added together; and the lower curve (0), when [14C]AfDP and non-labelled ADP (2-5 x 10-5 M) were added -together. The plot represents the mean of two experiments. 5
Dipyridamole (I10-4m) added to platelet-rich plasma 1 min before the addition of radioactive adenosine almost completely blocked uptake of adenosine radioactivity even in the earliest sample, i.e. 10 sec (Fig. SA). Addition of dipyridamole before [3H]ADP did not alter the rapid initial uptake of radioactivity, but blocked the subsequent increasing uptake of radioactivity over a 20 min period (Fig. 5B). These results are consistent with the proposition that the continuously increasing uptake of ADP radioactivity by platelets in untreated plasma and the delayed increase in pre-heated plasma represented uptake of
BINDING OF ADP TO PLATELETS 0-
8
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~~~~~1-2
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o
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0 1 6 20 4 8 12 20 Time (min) Fig. 5. The effect of dipyridamole (1 x 10-4 M) on the uptake of radioactivity from [8-14C]adenosine or from [8-14C]ADP, both added at 1 X 10-6 M, by human platelets incubated at 370 C in pre-heated homologous plasma. Samples were taken at the intervals shown and the platelets were sedimented through silicone oil. Plotted on the ordinate is the uptake expressed as the added substance, in p-mole/108 platelets. Panel A represents the uptake of adenosine and panel B the uptake of ADP in the absence (0 O) of dipyridamole. The plots *) and presence (0 are the mean of two experiments. 0
0.
8
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16
12
04
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I-
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630 .,
0
12 6 5 4 3 Time (min) Fig. 6. Uptake of [e32P]ADP radioactivity, expressed as ADP, by human platelets incubated at 370 C in pre-heated plasma (0-0); and the uptake of [8-14C]ADP radioactivity, also expressed as ADP, by guineapig platelets incubated in homologous plasma (0-*). ADP radioactivity in the trapped plasma was determined with 125I-HSA as the plasma marker and subtracted from the total uptake of radioactivity. The plot represents the mean of two experiments. 0
1
2
break-down products, most likely adenosine. However, the uptake of ADP by platelets in pre-heated plasma at the earliest time was unaffected by adenosine or by dipyridamole and, therefore, presumably represented binding of unaltered ADP to platelets.
812
G. V. R. BORN AND H. FEINBERG
Uptake of radioactivity from ADP labelled with ax-32P by human platelets and of radioactivity from [14C]ADP by guinea-pig platelets It has been claimed (Salzman et al. 1969) that the uptake of radioactivity from ADP labelled with 32p in the a-position does not increase 5
4
1>-1 U soI6
2
1
0
O
_
o
2
4 6 V (p-mole/108 platelets)
20
22
Fig. 7. The binding of ADP, determined from extrapolation to zero time (samples taken at 30, 90 and 180 see) of the amount of radioactivity from ADP found in platelets sedimented through silicone oil and corrected for ADP radioactivity in the trapped plasma (determined with 1251-HSA). Results from three experiments with ADP concentrations of 107 - 10- M and from eight experiments with single, different ADP concentrations were plotted according to Scatchard. The ordinate represents the ratio of the apparent amount bound to the unbound concentration and the abscissa the apparent amount of bound ADP (p-mole/108 platelets). The interrupted line represents an estimate of the tangent of the first-order binding reaction.
continuously, as with ADP labelled in the purine or ribose moieties; this was taken as evidence that ADP per se is not bound to platelets. Incubation of [a-32P]ADP and 125I-HSA in human platelet-rich plasma confirmed
BINDING OF ADP TO PLATELETS
813 that the increase in 32p uptake by platelets was slight during 6 min. However, the radioactivity in the platelet pellets was always greater than could be accounted for by 32p in the trapped plasma (Fig. 6). Guinea-pig platelets do not take up adenosine (Michel, 1972) and should not, therefore, take up radioactivity from purine-labelled ADP if that uptake is dependent on the break-down of ADP to adenosine. Incubation of guinea-pig platelets with [14C]ADP showed no increase in uptake over 5 min, but, as with human platelets incubated with [a32P]ADP, the radioactivity in the platelet pellets always exceeded the amount that could be attributed to trapped plasma (Fig. 6).
Binding of labelled ADP to platelets In three experiments, [14C]ADP or [3H]ADP in concentrations between 1 X 10-7 and 10-M was incubated at 370 C with human platelets resuspended in pre-heated plasma. The apparent uptakes of ADP radioactivity were determined after 30, 90 and 180 sec. The early, flat part of the uptake curves were extrapolated to zero time. The values for ADP binding in these experiments, as well as in ten experiments with single concentrations of ADP, were used to construct a Scatchard plot (Fig. 7). The plot indicated that platelets have more than one type of binding site for ADP. The total number of sites could not be determined as it was uncertain where the abscissa was crossed. A plot of the log of bound ADP versus log of unbound ADP (Klotz, 1973) as a basis for calculating the number of sites yielded an estimate of about 88,000 sites per platelet. Because, according to the Scatchard plot, the number of different sites was uncertain, the binding constant was also uncertain. However, for the binding of ADP at low concentrations an affinity constant could be estimated from the Scatchard plot as 5'41 x 105 M-1. DISCUSSION
From experiments in which isolated platelet membranes were incubated with ADP for 1 hr (Nachman & Ferris, 1974), it was estimated that each human platelet can bind about 1 x 105 ADP molecules. An earlier estimate (Born, 1965), based on the uptake of purine-labelled ADP by intact platelets, was very similar. The similarity of the estimates despite different methods may be fortuitous, because a membrane fraction isolated from platelets is unlikely to represent only the outer plasma membrane and because uptake of radioactivity from purine-labelled ADP may represent uptake of metabolites as well as binding of ADP. The present results show that human platelets incubated at 370 C in their citrated plasma took up radioactivity from purine-labelled ADP at
814 G. V. R. BORN AND H. FEINBERG the earliest sampling time, i.e. about 10 sec. This rapid uptake was almost independent of temperature. Thereafter radioactivity continued to be taken up more slowly. This slower uptake was highly dependent on temperature over the range 18-37' C. The rapid uptake is explained most easily on the assumption that it represents binding of intact ADP to the platelets, presumably to receptor sites specific for it on the platelet surface (Born, 1965). The subsequent slower uptake is best explained by assuming that it represented incorporation into the platelets of labelled nucleosides, probably mainly adenosine, formed by the enzymic breakdown of ADP. Thus the difference in the rate of increase of radioactivity at different temperatures indicated a rate of both ADP metabolism and uptake of the metabolites whereas extrapolation to zero time, which yielded a common value, represented ADP binding to platelets. The uptake of radioactivity from other ring-labelled nucleoside diphosphates, i.e. GDP, IDP and UDP, was much less initially and increased at a very much slower rate than that from ADP. This indicates that there was specificity for ADP both in its binding (see also Nachman & Ferris, 1974) and in its metabolism (Salzman et al. 1969). We have shown also that 2 min after the addition of purine-labelled ADP at least 60 % of the radioactivity taken up by the platelets was present as ATP. This is consistent with the assumption that ADP was metabolized to adenosine which was rapidly incorporated and converted to ATP (see Holmsen & Rozenberg, 1968). Prostaglandin E1, which inhibits aggregation by ADP, had no effect on the initial or the later uptake of radioactivity from ADP or on the distribution of intraplatelet radioactivity amongst the adenine nucleotides. Therefore, the claim (Boullin et al. 1972) that aggregation is proportional to the binding of intact ADP was not confirmed, Further evidence for our interpretation came from experiments with plasma heated to 560 C for 30 min which had less than one tenth of the ADPase activity of normal plasma (Spaet & Lejnieks, 1966). From such heated plasma, platelets again took up radioactivity from added [14C]ADP at the earliest sampling time, i.e. about 10 sec; and thereafter the radioactivity of the platelets remained constant for at least 5 min, as would be expected if the break-down of ADP to products capable of being incorporated into platelets had been stopped. Added unlabelled ADP diminished the rapid initial uptake of radioactivity from [1C]ADP as well as the slower subsequent uptake whereas, conversely, added adenosine had no effect on the initial uptake but diminished the slower subsequent uptake. Furthermore dipyridamole, which blocks the uptake of adenosine, inhibited the slow continuous uptake of radioactivity but not the initial uptake. These observations
815 BINDING OF ADP TO PLATELETS also support the conclusion that the initial uptake represents binding of ADP and the later uptake the incorporation of adenosine. The experiment of Salzman et al. (1969) with [a-32P]ADP was repeated with platelets resuspended in heated plasma. An initial rapid uptake of 32p did not increase subsequently during 6 min of incubation. Furthermore, guinea-pig platelets which do not take up adenosine (Michel, 1972) took up radioactivity from [14C]ADP. These observations constitute further evidence that ADP is rapidly bound to platelets. By extrapolating the flat portion of the uptake curve obtained with heated plasma to zero time we estimated initial binding for the range of ADP concentrations from 104 to 10-7 M. The Scatchard plot was non-linear and did not cross the abscissa, indicating more than one class of binding site. We used a log plot method (Klotz, 1973) for calculating that about 88,000 molecules of ADP can be bound per platelet. The initial linear portion of the Scatchard curve led to an affinity constant of 5.4 x 105 M-1 for the ligands formed at low ADP concentration. Nachman & Ferris (1974) found a single class of ADP binding sites on isolated human platelet membranes numbering about 100,000 per platelet; and an affinity constant of 6-5 x 106 M-1. The difference in the number of classes of ADP binding sites between isolated membranes and intact platelets may be due to the treatment necessary for isolating a membrane fraction and the long incubation at 370 C by Nachman & Ferris (1974), which would provide an estimate of steady-state equilibrium values rather than the kinetic parameters appropriate to initial binding and which may have also influenced estimation the affinity constant. Although the estimates of the total number of binding sites available to ADP are quite similar, it is likely that the lower estimate calculated from our results is more correct because the procedures used for isolating membranes are likely to expose binding sites for ADP other than those which mediate its physiological effects on intact platelets. REFERENCES
BORN, G. V. R. (1964). Strong inhibition by 2-chloroadenosine of the aggregation of blood platelets by adenosine diphosphate. Nature, Lond. 202, 95-96. BORN, G. V. R. (1965). Uptake of adenosine and of adenosine diphosphate by human blood platelets. Nature, Lond. 206, 1121-1122. BORN, G. V. R. (1970). Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J. Physiol. 209, 487-511. BORN, G. V. R. & HumE, M. (1967). Effects of the numbers and sizes of platelet aggregates on the optical density of plasma. Nature, Lond. 215, 1027-1029. BOULLIN, D. J., GREEN, A. R. & PRICE, K. S. (1972). The mechanism of adenosine diphosphate induced platelet aggregation: binding to platelet receptors and inhibition of binding and aggregation by prostaglandin E1. J. Physiol. 221, 415-426.
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G. V. B. BORN AND H. FEINBERG
BURGEN, A. S. V. (1966). The drug-receptor complex. J. Pharm. Pharmac. 18, 137-149. FEINBERG, H., MICHEL, H. & BORN, G. V. R. (1974). Determination of the fluid volume of platelets by their separation through silicone oil. J. Lab. clin. Med. 84, 926-934. GAARDER, A., JoNsEN, J., LALAND, S., HELLEM, A. J. & OWREN, P. A. (1961). Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature, Lond. 192, 531-532. GOUGH, G., MAGUIRE, H. M. & PENGLIS, F. (1972). Analogues of adenosine 5'diphosphate - new platelet aggregators. Molec. Pharmacol. 8, 170-177. HAsLAM, R. J. & MiLLs, D. C. B. (1967). The adenylate kinase of human plasma, erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma. Biochem. J. 103, 773-784. HOLMSEN, H. & ROZENBERG, M. C. (1968). Adenine nucleotide metabolism of blood platelets. I. Adenosine kinase and nucleotide formation from exogenous adenosine and AMP. Biochim. biophys. Acta 155, 326-341. KLOTZ, I. M. (1973). Physicochemical aspects of drug-protein interactions: a general perspective. Ann. N.Y. Acad. Sci. 226, 18-35. LAMPRECHT, W. & TRAUTSCHOLD, I. (1965). In Methods of Enzyme Analyais, pp. 543-545. New York: Academic Press. MACMTILLAN, D. C. & OLIVER, M. F. (1965). The initial changes in platelet morphology following the addition of adenosine diphosphate. J. AtheroWler. Re8. 5, 440444. MAGUIRE, M. H. & MICEA, F. (1968). Powerful new aggregator of blood platelets 2-chloroadenosine-5'-diphosphate. Nature, Lond. 217, 571-573. MICHEL, H. (1972). Etude comparative de l'inhibition de I'aggr6gation plaquette par l'ad6nosine et son incorporation dans les plaquettes sanguine humaines et animals. Ph.D. Thesis, University of Paris. MmLTs, D. C. B. (1966). The breakdown of adenosine diphosphate and of adenosine triphosphate in plasma. Biochem. J. 98, 32P. NACHMAN, L. R. & FERRIS, B. (1974). Binding of adenosine diphosphate by isolated membranes from human platelets. J. biol. Chem. 249, 704-710. SALZMAN, E. W., ASHFORD, T. P., CHAMBERS, D. A. & NERI, L. L. (1969). Platelet incorporation of labelled adenosine and adenosine diphosphate. Thromb. Diath.
haemorrh. 22, 304-315. SALzMAN, E. W., CHiAMBERS, D. A. & NERI, L. L. (1966). Incorporation of labelled nucleotides and aggregation of human blood platelets. Thromb. Diath. haemorrh. 15, 52-68. SPAET, T. H. & LEJNIEKS, I. (1966). Studies on the mechanism whereby platelets are clumped by adenosine diphosphate. Thromb. Diath. haemorrh. 15, 36-51.
803
With 7 text-ftgure8 Printed in Great Britain
BINDING OF ADENOSINE DIPHOSPHATE TO INTACT HUMAN PLATELETS
BY G. V. R. BORN* AND H. FEINBERGt From the Department of Pharmacology, Royal College of Surgeons, Lincolns Inn Fields, London W02
(Received 12 March 1975) SUMMARY
1. Human platelet-rich plasma was incubated at 370 C with [8-14C]ADP and with human serum albumin labelled with 125I. The platelets were rapidly separated by centrifugation through silicone oil. From radioactivity determinations of plasma and platelet pellets the uptake of ADP, without or with break-down products, by the platelets was calculated on the assumption that the 1251 radioactivity in the pellet represented trapped plasma. 2. ADP radioactivity was taken up by platelets within 10 sec and increased with time of incubation. The uptake of other nucleotide diphosphates was less initially and increased much more slowly. 3. Radioactivity added as ADP was recovered as ATP to the extent of 60%; as ADP of 30 %; and as AMP of 10%. 4. Prostaglandin E1 which inhibited platelet aggregation had no effect on the initial or subsequent uptake or on this distribution of radioactivity. 5. The rate of rise in uptake was much slower when platelets were resuspended in plasma heated to 56° C for 30 min. 6. Unlabelled adenosine inhibited the later, but not the initial, uptake while unlabelled ADP inhibited both. Dipyridamole, which blocks adenosine uptake, prevented the later but not the initial uptake. 7. [oc-32P]ADP radioactivity was taken up at the earliest sampling time and the extent of uptake did not further increase. 8. Guinea-pig platelets, which do not take up adenosine, took up [8-14C]ADP radioactivity from purine-labelled ADP initially. 9. It was concluded that the initial uptake represented binding of ADP and that the later uptake represented labelled adenosine originating as a break-down product of ADP. *
Present address: Department of Pharmacology, Medical School, Hills Road,
Cambridge, CB2 2QD.
t Present address: Department of Pharmacology, University of Illinois, College of Medicine, Chicago, Illinois, U.S.A.
804 8. V. R. BORN AND H. FEINBERG 10. A Scatchard plot of ADP uptake indicated more than one type of binding site. There were approximately 88,000 high affinity sites per platelet which had an affinity constant of 5-41 x 105 M-1. INTRODUCTION
Mammalian platelets are strongly and specifically aggregated by adenosine diphosphate (ADP), and aggregation is preceded by a rapid and striking morphological reaction (Macmillan & Oliver, 1965; Born, 1970). The only other related nucleotides so far known to cause both shape change and aggregation are deoxy-ADP which is less potent, and 2-chloroadenosine and 2-methylthio-adenosine diphosphates which are more potent than ADP (Maguire & Michal, 1968; Gough, Maguire & Penglis, 1972). Other agents which cause platelet aggregation including thrombin, collagen, adrenaline and 5-hydroxytryptamine, release ADP from platelets; and there is evidence that aggregation by at least thrombin and collagen does not occur without this release. The rapidity and specificity with which ADP brings about these effects suggests that they are initiated by the complexing of ADP with receptors specific for it on or just within the platelet surface membrane because ADP, as a highly ionized molecule at physiological pH, is unlikely to pass through the platelet membrane. It has, therefore, been proposed (Born, 1965) that the mode of action of ADP can be best accounted for by assuming that it forms a short-lived complex with a specific receptor of the platelet membrane in a manner similar to that assumed to account for the action of other pharmacological agonists, e.g. adrenaline or acetylcholine, on smooth muscle membranes; in these reactions, the agonists themselves are not altered. In relation to this proposal, arguments that platelets do not 'bind' ADP (Salzman, Ashford, Chambers & Neri, 1969) are invalid as they depend on the meaning of binding in terms of time (Burgen, 1966). Several attempts have been made to demonstrate binding of ADP to platelets (Born, 1964; Salzman, Chambers & Neri, 1966). When radioactive ADP was added to platelet-rich plasma and the platelets were separated by centrifugation, the radioactivity in the platelet pellet suggested an immediate uptake of about 105 molecules of ADP per platelet (Born, 1965). The uptake of radioactivity increased slowly for some minutes and then more rapidly, presumably due to conversion of ADP to adenosine which is known to be incorporated into platelets. Some uncertainty about these results was caused by the necessity to correct the pellet radioactivity for ADP present in the plasma which remains in pellets of platelets after centrifugation. This ADP has to be
805 BINDING OF ADP TO PLATELETS either removed by washing, which may also remove bound ADP, or determined independently by estimating the volume of trapped plasma with another isotope. This paper describes results obtained with an experimental technique designed to minimize these uncertainties. The uptake of ADP radioactivity by platelets was measured after separating them rapidly from plasma by centrifuging through silicone oil; the small amounts of plasma trapped in the pellets was determined separately. METHODS Non-isotopic nucleotides were obtained from Sigma Co., London.
Inosine-[8-3H]5'-
diphosphate, uridine-[5-3H]diphosphate, guanosine-[8-3H]5'-diphosphate, adenosine[2-3H]5'-diphosphate, adenosine-[8-14C]5'-diphosphate and adenosine-"C(U) were obtained from the Radiochemical Centre in Amersham. Labelled ADP was reisolated on DEAE cellulose using NH4CO3 as the eluant after thorough washing with water to eliminate labelled adenosine as a possible contaminant. [a-32P]ADP was prepared by incubating [oc_32P]ATP (Amersham/Searle) with glucose and hexokinase according to the procedure of Lamprecht & Trautschold (1965); the labelled ADP was isolated from an acid-soluble extract (6%, HCl04 v/v) of the incubation mixture by chromatography on DEAE-cellulose. Human serum albumin labelled with 125I (1251I-HSA) was obtained from Amersham/Searle, London. Prostaglandin E1 was obtained from the Upjohn Company and human fibrinogen from the Kabi Co., Stockholm. Platelet-rich plasma was prepared from human blood obtained from healthy adults of both sexes by the procedures of Born & Hume (1967). Platelets suspended in homologous heated plasma (Spaet & Lejnieks, 1966) were prepared by centrifuging platelet-rich plasma for 10 min at 2000 g and resuspending the platelets in platelet-free plasma that had been incubated at 560 C for 30 min, then centrifuged to sediment denatured protein and brought to pH 7-5 with solid Trizma base (Sigma). It was necessary to add human fibrinogen (30 mg/ml.) and Ca2+ (10-3 M) to restore the aggregating effect of ADP (10-5 M). Aggregability of all platelet suspensions to ADP (10-5-10-6 M) was determined before addition of labelled substances; preparations which showed no aggregation were discarded. Platelets in plasma or in heated plasma were incubated at 370 C with constant stirring. Labelled nucleotides and 125I-HSA were added in 2 % or less of the total volume by rapid injection. Samples of 1 ml. were centrifuged at 12,000 g for 30-60 sec in an Eppendorf Microfuge in tubes containing 0-2 ml. silicone oil (Dow Corning 702 and 200, 9:1 v/v); a modified rotor was used to allow the tubes to swing out horizontally (Feinberg, Michel & Born, 1974). Samples (100,l.) of the platelet-free plasma were transferred to counting vials containing 0 5 ml. Soluene (Packard Instruments) and isopropanol (1: 1 v/v). 125I radioactivity was measured in a gamma counter. Ten ml of 0.5 M-HCl-Instagel (1: 9 v/v, Packard Instruments) scintillation fluid was added. 3H or 14C radioactivity was measured and the values were corrected for 1251 radioactivity. The remaining platelet-free plasma lying above the silicone oil was removed by suction. The platelet pellet under the oil was frozen by immersing the tip of the centrifuge tube in powdered CO2 or liquid N2. The part of the tube that had been occupied by the platelet-free plasma was washed repeatedly with isotonic saline. Most of the silicone oil was removed by draining from the inverted centrifuge tube. The platelet pellet was transferred to a counting vial by brief centrifugation of the inverted centrifuge tube held in the
G. V. R. BORN AND H. FEINBERG
806
mouth of the counting vial. The minute amount of silicone oil transferred with the pellet did not affect the counting rates. The platelet pellet and its trapped plasma were solubilized with the soluene-isopropanol mixture and the 126J and 3H or 125I and 14C radioactivities were determined as above. From the 125I-HSA radioactivity of the pellet, the isotopic nucleotide in the trapped plasma and the 3H or 14C radioactivity of the platelets were calculated. The distribution of 3H amongst the nucleotides in the pellet, after varying periods of incubation, was determined by layering 60 % HC104 beneath the silicone oil; thus, during centrifugation the platelets penetrated the silicone oil and entered the perchloric acid layer. Acid-soluble nucleotides in the neutralized extract were separated by high voltage electrophoresis for 1 hr on Whatman 3 MM paper in 0-05 M citrate buffer pH 4-5. The nucleotide spots were identified under an U.V. lamp, eluted and their radioactivity was counted in 0.5 m-HCl-Instagel mixture.
8
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Fig. 1. Apparent uptake (in p-mole/108 platelets) of nucleotide diphosphates, calculated from the uptake of radioactivity, by human platelets incubated at 370 C in citrated plasma. Samples were taken at the intervals shown and the platelets were sedimented through silicone oil. Trapped plasma was determined using 125J-human serum albumin as marker, and the values shown represent the uptake of purine label in excess of that in the trapped plasma. The upper curves represent apparent uptake of ADP (10- M) in the absence (0) and presence ( x ) of prostaglandin E1 (10-6 M) (mean and s.E. of five experiments). The apparent uptakes of GDP (-), IDP (EO) and UDP (0) under the same conditions are shown in the lower curves.
BINDING OF ADP TO PLATELETS
807
RESULTS
Uptake by platelets of radioactivity added as [8-'4C]ADP The uptake of radioactivity by platelets in human platelet-rich plasma, incubated at 370 C, after the addition of [8-14C]ADP (10- M) is shown in Fig. 1. Human serum albumin labelled with 1251 (125I-HSA) was added simultaneously with the ADP to determine the volume of plasma trapped in the platelet pellet; the ADP uptake shown in Fig. 1 is corrected 0.8 C.
0~
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2 3 Time (min) Fig. 2. Effect of temperature on uptake by platelets of radioactivity added and expressed as ADP. Human platelet-rich plasma was incubated with 1251-HSA and [3H]ADP (10 4M) at 370 C (-), 270 C (A), or 18' C (0). Samples were taken after the intervals shown and the platelets were sedimented through silicone oil. The ordinate represents the uptake of radioactivity, calculated as ADP (n-mole/108 platelets) from uptake of 3H in excess of that present in the trapped plasma. The vertical bars represent the s.E. of the mean of six determinations. The dashed portion of the lines represent extrapolated parts of the linear regressions calculated from the mean of the observation at 30, 90 and 180 sec at each temperature.
1
for this. Even at the earliest sampling time, i.e. 15 sec, ADP radioactivity was taken up in excess of that present in the trapped plasma. The uptake increased for at least 8 min. Prostaglandin E1 (PGE,), which inhibits platelet aggregation by ADP, has been claimed to inhibit the
808 0. V. R. BORN AND H. FEINBER0 uptake of ADP radioactivity by platelets (Boullin, Green & Price, 1972). In our experiments PGE, added in concentrations sufficient to inhibit aggregation completely had no significant effect on the uptake of ADP radioactivity (Fig. 1). The uptake of radioactivity from ADP greatly exceeded that of other nucleoside diphosphates, namely GDP, IDP, or UDP, all labelled with tritium (Fig. 1). This difference supports other evidence (Gaardner, Jonsen, Laland, Hellem & Owren, 1961) for the specificity of the interaction of ADP with platelets. An attempt was made to resolve further the time course of the initial uptake of ADP. Incubation with 125I-HSA and [-3H]ADP (106 M) for 10 see plus 5 see for centrifugation resulted in an excess of ADP (0.62 + 0-21 p-mole/108 platelets) although centrifugation for 5 see was not long enough to sediment all the platelets.
Effect of temperature on ADP uptake Platelet-rich plasma was incubated with [2-3H]ADP (1.8 x 104 M) and 1251-HSA at three different temperatures. At 270 C, the apparent uptake was slower and at 180 C still slower than at 370 C; however, extrapolation of the linear regressions calculated from the curves at the three temperatures led to closely similar intercepts at zero time (Fig. 2). The simplest explanation of these results (see also Born, 1965) is that the intercept represents an estimate of the initial binding of ADP, which is almost unaffected by temperature in this range; whereas the subsequent increase in the rate of apparent uptake represents an increase in the rate of uptake of break-down product of ADP, presumably adenosine. The fate of isotopic ADP added to platelets Platelet-rich plasma was incubated at 370 C with [2-3H]ADP added at 1 x 10-6 M and the platelets were centrifuged through silicone oil directly into HC104. The distribution of radioactivity present as ATP. ADP and AMP was determined as a percentage of total radioactivity in the acid extract. At the earliest sampling time, i.e. 2 min, ATP accounted for 60 % of the radioactivity, ADP for 30 % and AMP for 10 % (Fig. 3). This distribution remained unchanged for at least 10 min although the absolute amount of radioactivity increased during this time in the manner shown earlier (Fig. 1). Thus, less than 50 % of the radioactivity recovered from pelleted platelets remained as [3H]ADP. The addition of PGE1 (106 M) 1 min before that of [3H]ADP completely inhibited aggregation of the platelets; however, PGE1 had no effect on the distribution of radioactivity between the adenine nucleotides (Fig. 3).
BINDING OF ADP TO PLATELETS
809
80
60 0
40 0 C
0
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0
2
6
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10
Time (min)
Fig. 3. Distribution of radioactivity taken up by platelets in human platelet-rich plasma incubated at 370 C with 10-6 M [3H]ADP. Samples were taken at the intervals shown and sedimented through silicone oil into 0*6 N-HClO4 containing sufficient sucrose to make the HCl04-sucrose mixture more dense than the silicone oil. The neutralized acid extracts from three assays were pooled and the lyophilized material was taken up in 0.05 M citrate buffer, pH 4-5. Samples of these solutions were subjected to high-voltage electrophoresis together with authentic ATP, ADP and AMP as carriers. The nucleotide spots were made visible under UN. radiation, cut out and oxidized completely to C02 and 3H20. The 3H20 radioactivity was determined by liquid scintillation. The plot represents percent of total radioactivity found in ATP (A A), ADP (U L1) and AMP (@0). The closed symbols represent the radioactivity distribution when labelled ADP only was added, and the open symbols when prostaglandin E1 (106 M) was added 1 min before the radioactive ADP.
Effects of adenosine and dipyridamole on the uptake of ADP radioactivity by platelets ADP added to platelet-rich plasma is broken down by plasma enzymes, viz. adenylate kinase and an ADPase (Haslam & Mills, 1967; Mills, 1966). To determine ADP uptake by platelets under conditions in which this break-down was minimized, platelets were resuspended in homologous plasma which had been heated to 560 C for 30 min. Fibrinogen and Ca2+ were added to restore the aggregation response to ADP.
8. V. R. BORN AND H. FEINBERG Addition of unlabelled adenosine at 2-5 x 10-5 M at the same time as labelled ADP at 10-6 M considerably diminished the increase in uptake of ADP radioactivity over a 20 min period (Fig. 4); however, adenosine had no effect on the uptake at the earliest sampling time, i.e. at about 10 sec, nor during the first 5 min. Addition of unlabelled ADP instead of adenosine had a similar effect on the uptake of ADP radioactivity except that the earliest uptake was also decreased a little (Fig. 4). 810
12.
0 _I
00
00C
0
10 1520 Time (min) Fig. 4. Effect of non-labelled adenosine or ADP on the uptake of radioactivity expressed as ADP (calculated from the radioactivity taken up in excess of that in the plasma trapped with the platelet pellet) by human platelets incubated at 370 C in pre-heated homologous plasma with [14C]ADP at 1 X 10-6 M. Samples were taken at the intervals shown. The upper curve (@) represents the apparent uptake when [14C]ADP (10-6 M) was added; the middle curve (U) when [14C]ADP and adenosine (2.5 x 10-6 m) were added together; and the lower curve (0), when [14C]AfDP and non-labelled ADP (2-5 x 10-5 M) were added -together. The plot represents the mean of two experiments. 5
Dipyridamole (I10-4m) added to platelet-rich plasma 1 min before the addition of radioactive adenosine almost completely blocked uptake of adenosine radioactivity even in the earliest sample, i.e. 10 sec (Fig. SA). Addition of dipyridamole before [3H]ADP did not alter the rapid initial uptake of radioactivity, but blocked the subsequent increasing uptake of radioactivity over a 20 min period (Fig. 5B). These results are consistent with the proposition that the continuously increasing uptake of ADP radioactivity by platelets in untreated plasma and the delayed increase in pre-heated plasma represented uptake of
BINDING OF ADP TO PLATELETS 0-
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0 1 6 20 4 8 12 20 Time (min) Fig. 5. The effect of dipyridamole (1 x 10-4 M) on the uptake of radioactivity from [8-14C]adenosine or from [8-14C]ADP, both added at 1 X 10-6 M, by human platelets incubated at 370 C in pre-heated homologous plasma. Samples were taken at the intervals shown and the platelets were sedimented through silicone oil. Plotted on the ordinate is the uptake expressed as the added substance, in p-mole/108 platelets. Panel A represents the uptake of adenosine and panel B the uptake of ADP in the absence (0 O) of dipyridamole. The plots *) and presence (0 are the mean of two experiments. 0
0.
8
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12 6 5 4 3 Time (min) Fig. 6. Uptake of [e32P]ADP radioactivity, expressed as ADP, by human platelets incubated at 370 C in pre-heated plasma (0-0); and the uptake of [8-14C]ADP radioactivity, also expressed as ADP, by guineapig platelets incubated in homologous plasma (0-*). ADP radioactivity in the trapped plasma was determined with 125I-HSA as the plasma marker and subtracted from the total uptake of radioactivity. The plot represents the mean of two experiments. 0
1
2
break-down products, most likely adenosine. However, the uptake of ADP by platelets in pre-heated plasma at the earliest time was unaffected by adenosine or by dipyridamole and, therefore, presumably represented binding of unaltered ADP to platelets.
812
G. V. R. BORN AND H. FEINBERG
Uptake of radioactivity from ADP labelled with ax-32P by human platelets and of radioactivity from [14C]ADP by guinea-pig platelets It has been claimed (Salzman et al. 1969) that the uptake of radioactivity from ADP labelled with 32p in the a-position does not increase 5
4
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2
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O
_
o
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22
Fig. 7. The binding of ADP, determined from extrapolation to zero time (samples taken at 30, 90 and 180 see) of the amount of radioactivity from ADP found in platelets sedimented through silicone oil and corrected for ADP radioactivity in the trapped plasma (determined with 1251-HSA). Results from three experiments with ADP concentrations of 107 - 10- M and from eight experiments with single, different ADP concentrations were plotted according to Scatchard. The ordinate represents the ratio of the apparent amount bound to the unbound concentration and the abscissa the apparent amount of bound ADP (p-mole/108 platelets). The interrupted line represents an estimate of the tangent of the first-order binding reaction.
continuously, as with ADP labelled in the purine or ribose moieties; this was taken as evidence that ADP per se is not bound to platelets. Incubation of [a-32P]ADP and 125I-HSA in human platelet-rich plasma confirmed
BINDING OF ADP TO PLATELETS
813 that the increase in 32p uptake by platelets was slight during 6 min. However, the radioactivity in the platelet pellets was always greater than could be accounted for by 32p in the trapped plasma (Fig. 6). Guinea-pig platelets do not take up adenosine (Michel, 1972) and should not, therefore, take up radioactivity from purine-labelled ADP if that uptake is dependent on the break-down of ADP to adenosine. Incubation of guinea-pig platelets with [14C]ADP showed no increase in uptake over 5 min, but, as with human platelets incubated with [a32P]ADP, the radioactivity in the platelet pellets always exceeded the amount that could be attributed to trapped plasma (Fig. 6).
Binding of labelled ADP to platelets In three experiments, [14C]ADP or [3H]ADP in concentrations between 1 X 10-7 and 10-M was incubated at 370 C with human platelets resuspended in pre-heated plasma. The apparent uptakes of ADP radioactivity were determined after 30, 90 and 180 sec. The early, flat part of the uptake curves were extrapolated to zero time. The values for ADP binding in these experiments, as well as in ten experiments with single concentrations of ADP, were used to construct a Scatchard plot (Fig. 7). The plot indicated that platelets have more than one type of binding site for ADP. The total number of sites could not be determined as it was uncertain where the abscissa was crossed. A plot of the log of bound ADP versus log of unbound ADP (Klotz, 1973) as a basis for calculating the number of sites yielded an estimate of about 88,000 sites per platelet. Because, according to the Scatchard plot, the number of different sites was uncertain, the binding constant was also uncertain. However, for the binding of ADP at low concentrations an affinity constant could be estimated from the Scatchard plot as 5'41 x 105 M-1. DISCUSSION
From experiments in which isolated platelet membranes were incubated with ADP for 1 hr (Nachman & Ferris, 1974), it was estimated that each human platelet can bind about 1 x 105 ADP molecules. An earlier estimate (Born, 1965), based on the uptake of purine-labelled ADP by intact platelets, was very similar. The similarity of the estimates despite different methods may be fortuitous, because a membrane fraction isolated from platelets is unlikely to represent only the outer plasma membrane and because uptake of radioactivity from purine-labelled ADP may represent uptake of metabolites as well as binding of ADP. The present results show that human platelets incubated at 370 C in their citrated plasma took up radioactivity from purine-labelled ADP at
814 G. V. R. BORN AND H. FEINBERG the earliest sampling time, i.e. about 10 sec. This rapid uptake was almost independent of temperature. Thereafter radioactivity continued to be taken up more slowly. This slower uptake was highly dependent on temperature over the range 18-37' C. The rapid uptake is explained most easily on the assumption that it represents binding of intact ADP to the platelets, presumably to receptor sites specific for it on the platelet surface (Born, 1965). The subsequent slower uptake is best explained by assuming that it represented incorporation into the platelets of labelled nucleosides, probably mainly adenosine, formed by the enzymic breakdown of ADP. Thus the difference in the rate of increase of radioactivity at different temperatures indicated a rate of both ADP metabolism and uptake of the metabolites whereas extrapolation to zero time, which yielded a common value, represented ADP binding to platelets. The uptake of radioactivity from other ring-labelled nucleoside diphosphates, i.e. GDP, IDP and UDP, was much less initially and increased at a very much slower rate than that from ADP. This indicates that there was specificity for ADP both in its binding (see also Nachman & Ferris, 1974) and in its metabolism (Salzman et al. 1969). We have shown also that 2 min after the addition of purine-labelled ADP at least 60 % of the radioactivity taken up by the platelets was present as ATP. This is consistent with the assumption that ADP was metabolized to adenosine which was rapidly incorporated and converted to ATP (see Holmsen & Rozenberg, 1968). Prostaglandin E1, which inhibits aggregation by ADP, had no effect on the initial or the later uptake of radioactivity from ADP or on the distribution of intraplatelet radioactivity amongst the adenine nucleotides. Therefore, the claim (Boullin et al. 1972) that aggregation is proportional to the binding of intact ADP was not confirmed, Further evidence for our interpretation came from experiments with plasma heated to 560 C for 30 min which had less than one tenth of the ADPase activity of normal plasma (Spaet & Lejnieks, 1966). From such heated plasma, platelets again took up radioactivity from added [14C]ADP at the earliest sampling time, i.e. about 10 sec; and thereafter the radioactivity of the platelets remained constant for at least 5 min, as would be expected if the break-down of ADP to products capable of being incorporated into platelets had been stopped. Added unlabelled ADP diminished the rapid initial uptake of radioactivity from [1C]ADP as well as the slower subsequent uptake whereas, conversely, added adenosine had no effect on the initial uptake but diminished the slower subsequent uptake. Furthermore dipyridamole, which blocks the uptake of adenosine, inhibited the slow continuous uptake of radioactivity but not the initial uptake. These observations
815 BINDING OF ADP TO PLATELETS also support the conclusion that the initial uptake represents binding of ADP and the later uptake the incorporation of adenosine. The experiment of Salzman et al. (1969) with [a-32P]ADP was repeated with platelets resuspended in heated plasma. An initial rapid uptake of 32p did not increase subsequently during 6 min of incubation. Furthermore, guinea-pig platelets which do not take up adenosine (Michel, 1972) took up radioactivity from [14C]ADP. These observations constitute further evidence that ADP is rapidly bound to platelets. By extrapolating the flat portion of the uptake curve obtained with heated plasma to zero time we estimated initial binding for the range of ADP concentrations from 104 to 10-7 M. The Scatchard plot was non-linear and did not cross the abscissa, indicating more than one class of binding site. We used a log plot method (Klotz, 1973) for calculating that about 88,000 molecules of ADP can be bound per platelet. The initial linear portion of the Scatchard curve led to an affinity constant of 5.4 x 105 M-1 for the ligands formed at low ADP concentration. Nachman & Ferris (1974) found a single class of ADP binding sites on isolated human platelet membranes numbering about 100,000 per platelet; and an affinity constant of 6-5 x 106 M-1. The difference in the number of classes of ADP binding sites between isolated membranes and intact platelets may be due to the treatment necessary for isolating a membrane fraction and the long incubation at 370 C by Nachman & Ferris (1974), which would provide an estimate of steady-state equilibrium values rather than the kinetic parameters appropriate to initial binding and which may have also influenced estimation the affinity constant. Although the estimates of the total number of binding sites available to ADP are quite similar, it is likely that the lower estimate calculated from our results is more correct because the procedures used for isolating membranes are likely to expose binding sites for ADP other than those which mediate its physiological effects on intact platelets. REFERENCES
BORN, G. V. R. (1964). Strong inhibition by 2-chloroadenosine of the aggregation of blood platelets by adenosine diphosphate. Nature, Lond. 202, 95-96. BORN, G. V. R. (1965). Uptake of adenosine and of adenosine diphosphate by human blood platelets. Nature, Lond. 206, 1121-1122. BORN, G. V. R. (1970). Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J. Physiol. 209, 487-511. BORN, G. V. R. & HumE, M. (1967). Effects of the numbers and sizes of platelet aggregates on the optical density of plasma. Nature, Lond. 215, 1027-1029. BOULLIN, D. J., GREEN, A. R. & PRICE, K. S. (1972). The mechanism of adenosine diphosphate induced platelet aggregation: binding to platelet receptors and inhibition of binding and aggregation by prostaglandin E1. J. Physiol. 221, 415-426.
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G. V. B. BORN AND H. FEINBERG
BURGEN, A. S. V. (1966). The drug-receptor complex. J. Pharm. Pharmac. 18, 137-149. FEINBERG, H., MICHEL, H. & BORN, G. V. R. (1974). Determination of the fluid volume of platelets by their separation through silicone oil. J. Lab. clin. Med. 84, 926-934. GAARDER, A., JoNsEN, J., LALAND, S., HELLEM, A. J. & OWREN, P. A. (1961). Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature, Lond. 192, 531-532. GOUGH, G., MAGUIRE, H. M. & PENGLIS, F. (1972). Analogues of adenosine 5'diphosphate - new platelet aggregators. Molec. Pharmacol. 8, 170-177. HAsLAM, R. J. & MiLLs, D. C. B. (1967). The adenylate kinase of human plasma, erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma. Biochem. J. 103, 773-784. HOLMSEN, H. & ROZENBERG, M. C. (1968). Adenine nucleotide metabolism of blood platelets. I. Adenosine kinase and nucleotide formation from exogenous adenosine and AMP. Biochim. biophys. Acta 155, 326-341. KLOTZ, I. M. (1973). Physicochemical aspects of drug-protein interactions: a general perspective. Ann. N.Y. Acad. Sci. 226, 18-35. LAMPRECHT, W. & TRAUTSCHOLD, I. (1965). In Methods of Enzyme Analyais, pp. 543-545. New York: Academic Press. MACMTILLAN, D. C. & OLIVER, M. F. (1965). The initial changes in platelet morphology following the addition of adenosine diphosphate. J. AtheroWler. Re8. 5, 440444. MAGUIRE, M. H. & MICEA, F. (1968). Powerful new aggregator of blood platelets 2-chloroadenosine-5'-diphosphate. Nature, Lond. 217, 571-573. MICHEL, H. (1972). Etude comparative de l'inhibition de I'aggr6gation plaquette par l'ad6nosine et son incorporation dans les plaquettes sanguine humaines et animals. Ph.D. Thesis, University of Paris. MmLTs, D. C. B. (1966). The breakdown of adenosine diphosphate and of adenosine triphosphate in plasma. Biochem. J. 98, 32P. NACHMAN, L. R. & FERRIS, B. (1974). Binding of adenosine diphosphate by isolated membranes from human platelets. J. biol. Chem. 249, 704-710. SALZMAN, E. W., ASHFORD, T. P., CHAMBERS, D. A. & NERI, L. L. (1969). Platelet incorporation of labelled adenosine and adenosine diphosphate. Thromb. Diath.
haemorrh. 22, 304-315. SALzMAN, E. W., CHiAMBERS, D. A. & NERI, L. L. (1966). Incorporation of labelled nucleotides and aggregation of human blood platelets. Thromb. Diath. haemorrh. 15, 52-68. SPAET, T. H. & LEJNIEKS, I. (1966). Studies on the mechanism whereby platelets are clumped by adenosine diphosphate. Thromb. Diath. haemorrh. 15, 36-51.
