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Dissociation of the aggregating effect and the inhibitory effect upon cyclic adenosine monophosphate accumulation by adrenaline and adenosine diphosphate in human platelets
THROMBOSIS RESEARCH 45; 363-370, 1987 0049-3848/87 $3.00 + .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
DISSOCIATION OF THE AGGREGATING EFFECT AND THE INHIBITORY EFFECT UPON CYCLIC ADENOSINE MONOPHOSPHATE ACCUMULATION BY ADRENALINE AND ADENOSINE DIPHOSPHATE IN HUMAN PLATELETS H. Stormorkenl,
T. Lyberg2, L. Hakvaagl and El.Nakstadl
Research Institute for Internal Medicine and Medical Department A, Rikshospitalet,' and Department of Oral Surgery, Ulleval Hospital,2 Oslo, Norway (Received 23.6.1986; Accepted in revised form 28.10.1986
by Editor H. Holmsen)
ABSTRACT Recent evidence indicates that adrenaline and adenosine diphosphate each have separate stimulusresponse pathways for induction of aggregation and inhibition of CAMP accumulation. We have used a natural model to test the validity of this evidence, i.e. patients from two kindreds with an inherited bleeding disorder due to absent aggregation to adrenaline and no secondary wave response to large concentrations of adenosine diphosphate. Our studies showed that the inhibitory effect of adrenaline and adenosine diphosphate on PGEI-induced increase of CAMP in the patients was not different from that of the controls both for adrenaline and adenosine diphosphate. These results therefore support the present evidence that both adrenaline and adenosine diphosphate apply separate pathways in these two platelet functions. INTRODUCTION Adrenaline (ADR) and adenosine diphosphate (ADP) have at least two common features in their capacity as agonists in platelet function: 1) Induction of shape change and aggregation, and 2) inhibition of cyclic adenosine monophosphate (CAMP) accumulation, probably through inhibition of adenylate
Key words:
Platelet aggregation, adrenoceptors, adrenaline, adenosine diphosphate, cyclic AMP
363
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ADP DISSOCIATION & ADRENALINE EFFECTS
Vol. 45, No. 4
cyclase (1,2,3 and ref. therein). ADR has been considered not to induce shape change because no initial decrease in light transmission is observed in aggregometer tracings when ADR is the agonist in contrast to most other agonists. Recent studies (4) using other techniques show, however, that shape change occurs, albeit to a lesser degree than with e.g. ADP. Based on the effect of various adrenoceptor blockers, the alpha2-subtype has been regarded as the mediator of the effects of adrenaline on platelets, although a small effect on beta-receptors may also be traced (1). In 1979, Jakobs (5) brought some disturbance into the picture in providing evidence that a unique alpha-subtype was the responsible mediator. This notion was challenged by Grant and Scrutton (6) who verified Jakops' data, but through additional experiments came to the conclusion that an alpha2-type is the mediator for the natural agonist. Recent studies, comparing the alpha2-selective ligand yohimbine (YOH) and the nonselective dihydroergocryptine (DHE) have again brought this problem on the stage (7,6,9). Thus, Barnett and his group (9) have provided evidence that the DHE identifies an alpha-subtype of more functional relevance than that labelled with YOH. DHE labels some 50% more sites on the platelets than YOH. In cases of myeloproliferative disease (MPD) with absent ADR response, these extra sites were lacking (9). Furthermore, they showed that DHE was considerably more potent in inhibiting ADR aggregation than YOH, both findings indicating that the DHE binding sites are most important for ADR aggregation. Those sites are not of the alpha1 type because DHE is not displaced by the alphal-specific ligand prazosin (6). There is reason to assume, however, that the final word has not been said as to which molecule, or epitope, mediates the aggregating effect of adrenaline. It is well known that several potent aggregation inhibitors, like PGE1, PGD2, PGI2, act by increasing the CAMP-level, and an attractive hypothesis would be that aggregation is induced by lowering the basal CAMP level. Neither ADP nor AOR do this, however, and it now seems accepted (3) that the conclusion made by Haslam long ago (1) that there is no connection between initiation of aggregation and changes in the basal CAMP level is valid (3). On the other hand. it is also fully accepted that ADR, as. well as ADP, inhibit accumulation of platelet CAMP, e.g. as induced by PGEl or PG12 (1,10,11,12, 13). This effect may be of importance in modulating platelet aggregation. Since this latter effect of ADR and ADP and initiation of platelet aggregation seem to be independent phenomena, it would be logical that the pathways are different, either at And the receptor level or further down the reaction sequence. indeed, evidence for this notion has accumulated. Studying MPD patients Swart et al. (9) f ound a normal inhibitory response to adrenaline on PGEl -stimulated CAMP increase, despite absence of response to ADR as aggregating agent. Without commenting on it, Scrutton et al. (14) observed the
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365
same phenomenon in normal blood donors with depressed responsiveness to ADA. Furthermore, Rao et al (15) found normal inhibition of PGI2-stimulated CAMP levels in a kindred with impaired ADR-aggregation. As to the ADP receptor very little is known about its nature. This situation may soon change since it has been found that fluorosulphonylbenzoyladenosine (FSBA) inhibits primary ADPaggregation and shape change, and binds covalently to a ID0 kD platelet membrane protein (for ref. see IS). This opens the possibility to isolate and characterize the protein. The fact that it binds to 40,000 sites per platelet (2) makes its specificity questionable, however. The ability of ADP to inhibit CAMP accumulation is in all probability, like ADR, not involved in induction of aggregation (17). Therefore, the idea of two different pathways for ADP, like for ADR, is not farfetched. And actually, evidence for this exists. Thus MacFarlane et al (3) found that mercuribenzene sulphonate blocks the inhibition of adenylate cyclase by ADP, but not its aggregating ability. Furthermore, Mills et al (2) found that FSBA, which inhibits aggregation by ADP, did not block the inhibitory effect of ADP on CAMP accumulation to PG12. Both findings strongly indicate independence of the two phenomena. Since we have access to patients with inherited extinct response to ADR and absent secondary wave to high concentrations of ADP (lB,19) we have applied this natural model to test the validity of the above evidence for duplicate receptor-effector pathways for both ADR and ADP. METHODS AND MATERIALS To prepare gelfiltered platelets (GFP) blood was collected in one tenth volume ACD-anticoagulant (Na3Citrate BS mmol/L, citric acid 71 mmol/L, glucose 111 mmol/L, dest water to 1000 mL, pH 4.5) and centrifuged at 180 g for 15 min at room temperature to obtain platelet-rich plasma (PRP). The PRP was concentrated by another centrifugation at 900 g for 15 min and 3/4 of the supernatant plasma removed. The pellet was resuspended, and the concentrated PRP subjected to gel filtration principally according to (20), collecting the platelets in Thyrode's solution without added Ca++ containing 0.2% bovine serum albumin. The platelet count was adjusted to 400 x 109/L and used immediately for CAMP studies. The effect of ADR and ADP on PGEI-stimulated was studied in the following system:
CAMP increase
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ADP DISSOCIATION & ADRENALINE EFFECTS
Vol. 45, No. 4
600 L 10 tt
GFP (400 x log/L) CaC12 (11.5 mol/L) Incubation 3 min, 370C
10
ADP (1.7 mol/L) or ADP-ADR equal parts or saline
‘1
50 't
The figures in brackets represent final concentrations
Incubation 30 set PGEl (230 nmol/L)
100
tt
100
”
Incubation 30 set Perchloric
acid (PEA) 50%
The PCA-platelet mixture was sonicated 20 set at 20 KHz-50 W and centrifuged for 15 min at 12,000 rpm in an Eppendorf centrifuge. The supernatant was transferred to another Eppendorf tube and the pH adjusted to 7.0 with 5 mol/L K2CO3 using appropriate indicator paper and centrifuged 4 min in the Eppendorf centrifuge. The supernatant was frozen in measured aliquots at -700C until CAMP was assayed using the Amersham RIA-kit (Amersham, Buckinghamshire, England). After considerable training it was possible to start new samples every other minute. Thus 6 samples from one gel filtration procedure could be processed within 15 min. The procedure was performed in a water bath at 370C. Platelet aggregation and release was studied in a Chronolog dual channel aggregometer (Chronolog Corp., Havertown, Pa., USA) using their chrono-lume luciferase luciferine reagent for ATP-monitoring. ADP, PGEl and the phosphodiesterase inhibitor NIX (3-isobutylyl-I-methyl-xanthine) were from Sigma Chemical Company, St. Louis, MO., USA; adrenaline bitartrate from Nycomed A.S., Oslo, Norway. RESULTS
AND
DISCUSSION
Because the platelets from these patients regain their ability to respond to adrenaline with time, a considerable improvement being noted at 2 h (18), it was necessary to finish the experiments as fast as possible. For that reason, we could not apply the most common method used in such experiments, i.e. labelling CAMP with 14C-adenine which requires 1 l/2 h incubation. With the set-up chosen it was possible to finish the procedure down to addition of the PCA for 6 samples within 15 min. Many investigators have added the reagents simultaneously, but since our results conform well with those
Vol. 45, No. 4
ADP DISSOCIATION & ADRENALINE EFFECTS
published by others, this does not seem to be of great importance. The concentrations of ADR and ADP applied usually give both a primary and secondary wave in normals, but not in the patients studied. The reason for chasing 30 see incubation time after addition of PGEl is that the CAMP increase is linear within this time (1). Also, because of the large background level of CAMP in PRP it was necessary to use GFP. Such a procedure may induce some changes in absolute CAMP levels, but this should not invalidate comparison of the responses. FIG. 1 Aggregation
and Release of ATP in Patient AP and Control.
Figure 1 shows the aggregometer and ATP release tracings with ADR (right panel) and ADP (left panel) as agonists from a control and one of the patients studied. It is seen that the patient platelets do not respond to ADR even at very high concentrations, as opposed to those of the control with conventional agonist concentration, although this control is definitely a medium responder. Similarly, ADP 15 mol/L gives only a primary wave with no release in the patient, whereas the control at 4 mol/L shows both secondary aggregation and release. Thus, these platelets should be suitable for studying whether ADR and ADP apply different pathways for the aggregating and adenylate cyclase-inhibiting effect by measuring their response to PGEq-stimulated CAMP increase. Table 1 shows the results of these studies. It can be seen that (line 2) PGET increases the basal level (line 1) by a factor of 5-6, and that adrenaline inhibits this increase by about 50% (line 3) and that there is no
367
368
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TABLE
1
Effect on Adrenaline (ADR) and Adenosine Diphosphate (ADP) on Prostaglandin El (PGE,) Stimulated Cyclic AMP (CAMP) Increase in Gel-Filtered Platelets (GFP) Controls 1 2 3 4 5 6
GFP+saline+saline GFP+PGEq+saline GFP+PGEl+ADR GFP+saline+ADR GFP+PGEl+ADP GFP+PGE,+ADP/ADR
The phosphodiesterase Results are given in in brackets.
89 513 240 82 350 112
(6)
(79-115) (251-672) (143-375) (77-96) (260-345) (76-203)
inhibitor pmol/lOg
Patients
583: 267 65 315 123
(3)
71-97) 274-656) 196-329) 75-99) 242-391) 75-206)
MIX was added in all samples. platelets as mean with ranges
difference between controls and the patients. This, therefore, substantiates the existing evidence that the pathway leading to aggregation is different from that governing CAMP regulation. Considering line 5, it is seen that the same applies to ADP, its effect being somewhat less than that of ADR, however. By combining ADP and ADR (line 6) each in half the concentration applied in line 3 and 4, the results could indicate a synergistic inhibition, contrary to an earlier observation (21), but further experiments are needed to settle this problem. Comparison of line 1 and line 4 shows that ADR does not influence basal CAMP levels, confirming earlier observations. It should be noted that the basic abnormality of aggregation in these patients' platelets is not yet clear. The fact that primary aggregation by ADP is present indicates normal ADP receptor function. As to ADR, the same may apply as inferred both from presence of ADR proaggregatory effect and apparently normal binding of yohimbine The abnormality (16). therefore seems to be located beyond the receptor level. Whether the aggregatory pathway goes through CAMP with a deficient step beyond this stage, cannot be entirely excluded. It is unlikely, however, according to findings referred to in the introductory remarks. With this reservation in mind, our findings support the notion that both ADR and ADP apply separate routes in stimulating aggregation and in inhibiting agonist-induced CAMP increase. Since the abnormalities in our patients were evident with the optical method, we do not believe that the single platelet disappearance method (22,23) would add critical information relative to the problem focused.
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ADP DISSOCIATION & ADRENALINE EFFECTS
ACKNOWLEDGEMENTS The authors are grateful to Drs. T. Skomedal and J.B. Osnes for valuable advice in preparing the manuscript, and to Tori11 Halvorsen for skillful secretarial assistance. REFERENCES 1.
HASLAN, R.J. Interactions of the pharmacological receptors of blood platelets with adenylate cyclase. Ser. Haemat VI, 3, 333-350, 1973.
2.
MILLS, D.C.B., FIGURES, W.R., SCEARCE, L.N., STEWART, for G.J., COLMAN R.F. and COLMAN, R.W. Two mechanisms inhibition of ADP-induced platelet shape change by 5'-pfluorosulfonylbenzyladenosine. J. Biol. Chem. 260, 8D780003, 1905.
3.
NACFARLANE D.E., SRIVASTAVA, P.C. and MILLS D.C.B. 2methylthioadenosine(beta-32P)diphosphate: an agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets. J. Clin. Invest. 71, 420-428, 1983.
4.
MILTON, J.G. and FROJMOVIC, M.M. Adrenaline and adenosine diphosphate-induced platelet aggregation require shape change. Importance of pseudopods. J. Lab. Clin. Med. 104, 805-815, 1984.
5.
JAKOBS, K.H. Synthetic a-adrenergic agonists are potent a-adrenergic blockers in human platelets. Nature 274, 819-820, 1978.
6.
GRANT, J.A. and SCRUTTON, M.C. Novel alpha2-adrenoceptors primarily responsible for inducing human platelet Nature 277 i, 659-661, 1979. aggregation.
7.
BOON, N.A., ELLIOT, J.M., GRAHAME-SMITH, D.G. and ST. JOHN-GREEN, T. A comparison of alpha-2 adrenoceptor binding characteristics of intact human platelets identiand 3H-dihydroergocryptin. J fied by 3H Yohimbine 2 Auton. Pharmacol. 3, 89-95, 1983.
8.
MOTULSKY, H.J. and INSEL, P.A. (3H)-dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. Biochem. Pharmacol. 31, 2591-2597, 1982.
9.
SWART, S.S., KEYTHWOOT, J. and BARNETT D.B. Differential labelling of platelet alpha2 adrenoceptors by 3H-dihydroergocryptin and 3H-yohimbine in patients with myeloproliferative disorders. Thromb. Res. 40, 623-629, 1985.
10.
COLE, B., ROBINSON, G.A. and HARTMANN, R.C. Studies on the role of cyclic AMP in platelet function. Ann. NY Acad. Sci. 185, 477-487, 1971.
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11.
of platelet MILLS, D.C.B. and SMITH, J.6. The control responsiveness by agents that influence cyclic AMP metabolism. Ann. NY Acad. Sci. 201, 391-399, 1972.
12.
NELLWIG, adenylate
13.
JAKOBS, H.K., SAUR, W. and SCHULTZ, G. Reduction of adenylate cyclase activity in lysates of human platelets by the alpha-adrenergic component of epinephrine. J 2 Cycl. Nucl. Res. 2, 381-392, 1976.
14.
SCRUTTON, M.C., CLARE. K.A., HUTTON, R.A. and BROCKresponsiveness to adrenaline in DORFER, K.R. Depressed platelets from apparently normal human donors: A familial trait. Elrit. J. Haemat. 49, 303-314, 1981.
15.
RAO, A.K., WILLIS, J., WACHTFOGEL, Y. and BECKETTE, C. Normal inhibition of PGI2-stimulated cyclic ANP levels despite impaired platelet aggregation response to epinephrine. Thrombos. Haemostas. 54, 41, 1985 (Abstr.)
16.
COLMAN, R.W. Platelet activation: tor. Sem. Hematol. 23, 2, 119-128,
17.
HASLAN, R.J. and CUSACK, N.J. Blood platelet receptors for ADP and adenosine. In: Purinergic Receptors. Burnstock (Ed.), Chapman & Hall Ltd. London 1981 p.221-286.
18.
STORNORKEN, H., GOGSTAD, G. and SDLUN, N-0. A new bleeding disorder: Lack of platelet aggregatory response to adrenaline and lack of secondary aggregation to ADP and platelet activating factor (PAF). Thromb. Res. 29, 391402, 1982.
19.
STORMORKEN, H., NAKSTAD, B., LYBERG, T., TESTART, N.-C. and THORSRUD, A.K. Adrenalin thrombocytopathia. Thrombos. Haemostas. 54, 74, 1985. (Abstr.)
20.
TANGEN, O., BERMAN, H.J. and NARFEY, P. Gel filtration. A new technique for separation of blood platelets from plasma. Thromb. Oiath. Haemorrh. 25, 268-278, 1971.
21.
OSNANI, A.H., CLARE, K.A., SCRUTTON, interaction and platelet inhibitory Res. 31, 665-674, 1983.
22.
GEAR, A.R.L. In vitro platelet responses: Aggregation. in Platelet Responses and Metabolism I. Ed. H. Holmsen, CRC Press, Boca Raton, Fa., in press.
23.
THOMPSON, N.T., SCRUTTON, N.C. and WALLIS, R.8. Particle volume changes associated with light transmittance changes in the platelet aggregometer: Dependence upon aggregating agent and effectiveness of stimulus. Thromb. Res. 41, 615-626, 1986.
K.P. and JAKOBS, K.H. cyclase by ADP. Thromb.
Inhibition of platelet Res. 18, 7-17, 1980.
Role of an ADP 1986.
recep-
N.C. Synergistic agonists. Thromb.
DISSOCIATION OF THE AGGREGATING EFFECT AND THE INHIBITORY EFFECT UPON CYCLIC ADENOSINE MONOPHOSPHATE ACCUMULATION BY ADRENALINE AND ADENOSINE DIPHOSPHATE IN HUMAN PLATELETS H. Stormorkenl,
T. Lyberg2, L. Hakvaagl and El.Nakstadl
Research Institute for Internal Medicine and Medical Department A, Rikshospitalet,' and Department of Oral Surgery, Ulleval Hospital,2 Oslo, Norway (Received 23.6.1986; Accepted in revised form 28.10.1986
by Editor H. Holmsen)
ABSTRACT Recent evidence indicates that adrenaline and adenosine diphosphate each have separate stimulusresponse pathways for induction of aggregation and inhibition of CAMP accumulation. We have used a natural model to test the validity of this evidence, i.e. patients from two kindreds with an inherited bleeding disorder due to absent aggregation to adrenaline and no secondary wave response to large concentrations of adenosine diphosphate. Our studies showed that the inhibitory effect of adrenaline and adenosine diphosphate on PGEI-induced increase of CAMP in the patients was not different from that of the controls both for adrenaline and adenosine diphosphate. These results therefore support the present evidence that both adrenaline and adenosine diphosphate apply separate pathways in these two platelet functions. INTRODUCTION Adrenaline (ADR) and adenosine diphosphate (ADP) have at least two common features in their capacity as agonists in platelet function: 1) Induction of shape change and aggregation, and 2) inhibition of cyclic adenosine monophosphate (CAMP) accumulation, probably through inhibition of adenylate
Key words:
Platelet aggregation, adrenoceptors, adrenaline, adenosine diphosphate, cyclic AMP
363
364
ADP DISSOCIATION & ADRENALINE EFFECTS
Vol. 45, No. 4
cyclase (1,2,3 and ref. therein). ADR has been considered not to induce shape change because no initial decrease in light transmission is observed in aggregometer tracings when ADR is the agonist in contrast to most other agonists. Recent studies (4) using other techniques show, however, that shape change occurs, albeit to a lesser degree than with e.g. ADP. Based on the effect of various adrenoceptor blockers, the alpha2-subtype has been regarded as the mediator of the effects of adrenaline on platelets, although a small effect on beta-receptors may also be traced (1). In 1979, Jakobs (5) brought some disturbance into the picture in providing evidence that a unique alpha-subtype was the responsible mediator. This notion was challenged by Grant and Scrutton (6) who verified Jakops' data, but through additional experiments came to the conclusion that an alpha2-type is the mediator for the natural agonist. Recent studies, comparing the alpha2-selective ligand yohimbine (YOH) and the nonselective dihydroergocryptine (DHE) have again brought this problem on the stage (7,6,9). Thus, Barnett and his group (9) have provided evidence that the DHE identifies an alpha-subtype of more functional relevance than that labelled with YOH. DHE labels some 50% more sites on the platelets than YOH. In cases of myeloproliferative disease (MPD) with absent ADR response, these extra sites were lacking (9). Furthermore, they showed that DHE was considerably more potent in inhibiting ADR aggregation than YOH, both findings indicating that the DHE binding sites are most important for ADR aggregation. Those sites are not of the alpha1 type because DHE is not displaced by the alphal-specific ligand prazosin (6). There is reason to assume, however, that the final word has not been said as to which molecule, or epitope, mediates the aggregating effect of adrenaline. It is well known that several potent aggregation inhibitors, like PGE1, PGD2, PGI2, act by increasing the CAMP-level, and an attractive hypothesis would be that aggregation is induced by lowering the basal CAMP level. Neither ADP nor AOR do this, however, and it now seems accepted (3) that the conclusion made by Haslam long ago (1) that there is no connection between initiation of aggregation and changes in the basal CAMP level is valid (3). On the other hand. it is also fully accepted that ADR, as. well as ADP, inhibit accumulation of platelet CAMP, e.g. as induced by PGEl or PG12 (1,10,11,12, 13). This effect may be of importance in modulating platelet aggregation. Since this latter effect of ADR and ADP and initiation of platelet aggregation seem to be independent phenomena, it would be logical that the pathways are different, either at And the receptor level or further down the reaction sequence. indeed, evidence for this notion has accumulated. Studying MPD patients Swart et al. (9) f ound a normal inhibitory response to adrenaline on PGEl -stimulated CAMP increase, despite absence of response to ADR as aggregating agent. Without commenting on it, Scrutton et al. (14) observed the
Vol. 45, No. 4
ADP DISSOCIATION 81ADRENALINE EFFECTS
365
same phenomenon in normal blood donors with depressed responsiveness to ADA. Furthermore, Rao et al (15) found normal inhibition of PGI2-stimulated CAMP levels in a kindred with impaired ADR-aggregation. As to the ADP receptor very little is known about its nature. This situation may soon change since it has been found that fluorosulphonylbenzoyladenosine (FSBA) inhibits primary ADPaggregation and shape change, and binds covalently to a ID0 kD platelet membrane protein (for ref. see IS). This opens the possibility to isolate and characterize the protein. The fact that it binds to 40,000 sites per platelet (2) makes its specificity questionable, however. The ability of ADP to inhibit CAMP accumulation is in all probability, like ADR, not involved in induction of aggregation (17). Therefore, the idea of two different pathways for ADP, like for ADR, is not farfetched. And actually, evidence for this exists. Thus MacFarlane et al (3) found that mercuribenzene sulphonate blocks the inhibition of adenylate cyclase by ADP, but not its aggregating ability. Furthermore, Mills et al (2) found that FSBA, which inhibits aggregation by ADP, did not block the inhibitory effect of ADP on CAMP accumulation to PG12. Both findings strongly indicate independence of the two phenomena. Since we have access to patients with inherited extinct response to ADR and absent secondary wave to high concentrations of ADP (lB,19) we have applied this natural model to test the validity of the above evidence for duplicate receptor-effector pathways for both ADR and ADP. METHODS AND MATERIALS To prepare gelfiltered platelets (GFP) blood was collected in one tenth volume ACD-anticoagulant (Na3Citrate BS mmol/L, citric acid 71 mmol/L, glucose 111 mmol/L, dest water to 1000 mL, pH 4.5) and centrifuged at 180 g for 15 min at room temperature to obtain platelet-rich plasma (PRP). The PRP was concentrated by another centrifugation at 900 g for 15 min and 3/4 of the supernatant plasma removed. The pellet was resuspended, and the concentrated PRP subjected to gel filtration principally according to (20), collecting the platelets in Thyrode's solution without added Ca++ containing 0.2% bovine serum albumin. The platelet count was adjusted to 400 x 109/L and used immediately for CAMP studies. The effect of ADR and ADP on PGEI-stimulated was studied in the following system:
CAMP increase
366
ADP DISSOCIATION & ADRENALINE EFFECTS
Vol. 45, No. 4
600 L 10 tt
GFP (400 x log/L) CaC12 (11.5 mol/L) Incubation 3 min, 370C
10
ADP (1.7 mol/L) or ADP-ADR equal parts or saline
‘1
50 't
The figures in brackets represent final concentrations
Incubation 30 set PGEl (230 nmol/L)
100
tt
100
”
Incubation 30 set Perchloric
acid (PEA) 50%
The PCA-platelet mixture was sonicated 20 set at 20 KHz-50 W and centrifuged for 15 min at 12,000 rpm in an Eppendorf centrifuge. The supernatant was transferred to another Eppendorf tube and the pH adjusted to 7.0 with 5 mol/L K2CO3 using appropriate indicator paper and centrifuged 4 min in the Eppendorf centrifuge. The supernatant was frozen in measured aliquots at -700C until CAMP was assayed using the Amersham RIA-kit (Amersham, Buckinghamshire, England). After considerable training it was possible to start new samples every other minute. Thus 6 samples from one gel filtration procedure could be processed within 15 min. The procedure was performed in a water bath at 370C. Platelet aggregation and release was studied in a Chronolog dual channel aggregometer (Chronolog Corp., Havertown, Pa., USA) using their chrono-lume luciferase luciferine reagent for ATP-monitoring. ADP, PGEl and the phosphodiesterase inhibitor NIX (3-isobutylyl-I-methyl-xanthine) were from Sigma Chemical Company, St. Louis, MO., USA; adrenaline bitartrate from Nycomed A.S., Oslo, Norway. RESULTS
AND
DISCUSSION
Because the platelets from these patients regain their ability to respond to adrenaline with time, a considerable improvement being noted at 2 h (18), it was necessary to finish the experiments as fast as possible. For that reason, we could not apply the most common method used in such experiments, i.e. labelling CAMP with 14C-adenine which requires 1 l/2 h incubation. With the set-up chosen it was possible to finish the procedure down to addition of the PCA for 6 samples within 15 min. Many investigators have added the reagents simultaneously, but since our results conform well with those
Vol. 45, No. 4
ADP DISSOCIATION & ADRENALINE EFFECTS
published by others, this does not seem to be of great importance. The concentrations of ADR and ADP applied usually give both a primary and secondary wave in normals, but not in the patients studied. The reason for chasing 30 see incubation time after addition of PGEl is that the CAMP increase is linear within this time (1). Also, because of the large background level of CAMP in PRP it was necessary to use GFP. Such a procedure may induce some changes in absolute CAMP levels, but this should not invalidate comparison of the responses. FIG. 1 Aggregation
and Release of ATP in Patient AP and Control.
Figure 1 shows the aggregometer and ATP release tracings with ADR (right panel) and ADP (left panel) as agonists from a control and one of the patients studied. It is seen that the patient platelets do not respond to ADR even at very high concentrations, as opposed to those of the control with conventional agonist concentration, although this control is definitely a medium responder. Similarly, ADP 15 mol/L gives only a primary wave with no release in the patient, whereas the control at 4 mol/L shows both secondary aggregation and release. Thus, these platelets should be suitable for studying whether ADR and ADP apply different pathways for the aggregating and adenylate cyclase-inhibiting effect by measuring their response to PGEq-stimulated CAMP increase. Table 1 shows the results of these studies. It can be seen that (line 2) PGET increases the basal level (line 1) by a factor of 5-6, and that adrenaline inhibits this increase by about 50% (line 3) and that there is no
367
368
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TABLE
1
Effect on Adrenaline (ADR) and Adenosine Diphosphate (ADP) on Prostaglandin El (PGE,) Stimulated Cyclic AMP (CAMP) Increase in Gel-Filtered Platelets (GFP) Controls 1 2 3 4 5 6
GFP+saline+saline GFP+PGEq+saline GFP+PGEl+ADR GFP+saline+ADR GFP+PGEl+ADP GFP+PGE,+ADP/ADR
The phosphodiesterase Results are given in in brackets.
89 513 240 82 350 112
(6)
(79-115) (251-672) (143-375) (77-96) (260-345) (76-203)
inhibitor pmol/lOg
Patients
583: 267 65 315 123
(3)
71-97) 274-656) 196-329) 75-99) 242-391) 75-206)
MIX was added in all samples. platelets as mean with ranges
difference between controls and the patients. This, therefore, substantiates the existing evidence that the pathway leading to aggregation is different from that governing CAMP regulation. Considering line 5, it is seen that the same applies to ADP, its effect being somewhat less than that of ADR, however. By combining ADP and ADR (line 6) each in half the concentration applied in line 3 and 4, the results could indicate a synergistic inhibition, contrary to an earlier observation (21), but further experiments are needed to settle this problem. Comparison of line 1 and line 4 shows that ADR does not influence basal CAMP levels, confirming earlier observations. It should be noted that the basic abnormality of aggregation in these patients' platelets is not yet clear. The fact that primary aggregation by ADP is present indicates normal ADP receptor function. As to ADR, the same may apply as inferred both from presence of ADR proaggregatory effect and apparently normal binding of yohimbine The abnormality (16). therefore seems to be located beyond the receptor level. Whether the aggregatory pathway goes through CAMP with a deficient step beyond this stage, cannot be entirely excluded. It is unlikely, however, according to findings referred to in the introductory remarks. With this reservation in mind, our findings support the notion that both ADR and ADP apply separate routes in stimulating aggregation and in inhibiting agonist-induced CAMP increase. Since the abnormalities in our patients were evident with the optical method, we do not believe that the single platelet disappearance method (22,23) would add critical information relative to the problem focused.
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ADP DISSOCIATION & ADRENALINE EFFECTS
ACKNOWLEDGEMENTS The authors are grateful to Drs. T. Skomedal and J.B. Osnes for valuable advice in preparing the manuscript, and to Tori11 Halvorsen for skillful secretarial assistance. REFERENCES 1.
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