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Effect of cyclic amp on cytoplasmic free calcium in human platelets stimulated by thrombin: Direct measurement with quiǹ2
THROMBOSIS RESEARCH 32; 183-188, 1983 0049-3848183 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
EFFECT OF CYCLIC AMP ON CYTOPLASMICFREE CALCIUM IN HUMAN PLATELETS STIMULATEDBY THROMBIN:DIRECT MEASUREMENTWITH QUIN2
J. YAMANISHI,Y. KAWAHARAand H. FUKUZAKI Departmentof InternalMedicine,Division I, Kobe UniversitySchool of Medicine,Kobe 650, Japan
(Received 14.7.1983; Accepted in revised form 8.8.1983 by Editor I. Yamashina) ABSTRACT With the fluorescentCa2+ indicatorquin2, we measured directly cytoplasmicfree Ca2+ ([ Ca2+ Ii) in washed human platelets stimulated by thrombinand examinedthe effect of cyclic AMP on [ Ca2+ ]i levels and 14C-serotoninrelease. Thrombin (0.2 U/ml) evoked a rise in [ Ca2+ ]i from the basal level of about 100 nM to ~3 uM which was fast enough to trigger serotoninrelease. This rise was inhibitedin a dose-dependentmanner by preincubationwith prostaglandinEl (PGEl) (0.1-10uM) or dibutyrylcyclic AMP (0.01 -1 mM). Parallelto this, serotoninreleasewas also inhibited by these drugs. When added to plateletsafter stimulationby thrombin,PGEl caused the rapid decreaseof elevated [ Ca2+ Ii. These resultsprovide direct evidencethat [ Ca2+ ]i levels in plateletsare regulatedby cyclic AMP.
INTRODUCTION It has been su gested that an increasein plateletcyclic AMP lowers cytoplasmicfree Ca5+ ([ Ca2+ Ii) and this action of cyclic AMP is thought to be underlyingmechanismby which certainprostaglandinsi.e. PGEl or PG12, which stimulateplateletadenylatecyclase,inhibitplateletactivation (1). However to date, there is lack of direct evidencethat cyclic AMP actually lowers [ Ca2+ ]i in intact plateletssince direct measurementsof [ Ca2+ ]i have not been previouslyachievedin such small cells. Recently,Tsienet al. (2,3)have synthesizeda new fluorescentCa2+ indicatorquin2 that can be trapped in the cytoplasmof intact cells of any size and have successfully measured [ Ca2+ ]i in lymphocytes. With this method, Rink et al. (4) have shown a rapid rise in [ Ca2+ ]i in intact plateletswhich are stimulated Key words: human platelet,cytoplasmicfree calcium,cyclic AMP, serotonin release,thrombin 183
184
CYCLIC AMP AND Ca*+ IN PLATELET
Vo1.32, No.2
[Ca2+]i -
-
3 NM 1 MM
-500
nM
-100
nM
FIG. 1 Time courses of serotonin release and quin2 fluorescence response in stimulated by thrombin. Samples of platelets which were loaded platelets with both [lklserotonin and quin2 were stimulated by thrombin (0.2 U/ml). Serotonin release was quantified and quin2 fluorescence was recorded as traces described under MATERIALSAND METHODS. The gaps in the fluorescence here and in subsequent figures show the times for addition of agents. A, serotonin release; B, quin2 fluorescence response.
In the present study we examined the effect of cyclic AMP on by thrombin. [ Ca2+ ]i levels in intact platelets stimulated by thrombin by means of this new fluorescent dye. MATERIALSAND METHODS Quin2 acetoxymethyl ester and PGEl were products of Dojindo LaboraBovine thrombin and tories and Ono Pharmaceutical Co., respectively. DBcAMPwere purchased from Mochida Pharmaceutical Co. and Sigma, respectively. [l‘klserotonin (58 mCi/mmol) was obtained from New England Nuclear. The abbreviations used were: PGEl, prostaglandin El; 12; DBcAMP, dibutyryl cyclic AMP; PGD2, prostaglandin
PGI2, prostaglandin D2
CYCLICAMP AND Ca2+ IN PLATELET
Vo1.32, No.2
B
A
-4
Thrombin
-4 Thrombin
185
c)
r
-4 Tbrombin
, 2 min, FIG. 2 Effect of various concentrationsof PGEl on thrombin-induced quin2 fluorescenceresponse. Samples of plateletswhich were loadedwith both [14C]serotonin and quin2 were preincubatedwith various concentrationsof PGEl for 5 min and then thrombin (0.2 U/ml) was added. Quin2 fluorescence was recordedas describedunder MATERIALSAND METHODS. A, with saline; B, with PGEl (0.1 uM); C, with PGEl (1 PM); D, with PGEl (10 @l).
Washed platelets,which were prepared as describedby Baenzigerand Majerus (!?I), were incubatedfor 25 min at 37'C with Hepes buffered saline (145 mM NaCl, 5 mM KCl, 1 mM MgS04, 0.5 mM Na2HP04, 10 mM Hepes, 5 mM glucose,pH 7.4 at 37°C) containing5 uM quin2 acetoxymethylester. The suspensionwas washed 2 times with Hepes buffered saline to remove any extraneousdye and finallyresuspendedto make a concentrationof 1 x 108 cells/mlin Hepes bufferedsaline containing1 mM CaC12. Fluorescencewas recordedat 37'C in Hitachi fluorescencespectrophotometer650-60. Excitationwas at 339 nm and emissionat 500 nm. [ Ca2+ ]i was calculatedfrom fluorescencesignal as describedby Rink et al. (4). Serotoninreleasewas quantifiedsimultaneouslywith quin2 fluorescenceby measuringradioactivematerial released from the plateletswhich were preloadedwith [14C]serotonin as describedby Haslam et al. (6). RESULTS Fig. 1 shows time courses of serotoninrelease and fluorescence responsein plateletswhich were stimulatedby thrombin. In comfirmationof the reviousobservationby Rink et al. (4), thrombinevoked a rapid rise in 1 Ca'+ ]i from the basal level of about 100 nM to s3 pM. This risewas fast enough to trigger serotoninrelease. Since the reactionCa2+ + quin2++ Ca2+-quin2is in equilibrium,it seems reasonableto speculatethat,inplatelets which are not loadedwith quin2, thrombin-inducedrise in [ Ca2+ ]i may be faster than that shown in Fig. 1. In fact, serotoninrelease from unloaded plateletswas faster than that from loaded cells (datanot shown). PGEl is known to stimulateplatelet adenylatecyclase activityresultingin marked elevationof platelet cyclic AMP levels. Preincubationof plateletswith this prostaglandininhibitedthrombin-inducedincreaseof [ Ca2* ]i in a dose-
186
CYCLIC AMPAND Ca*+ IN PLATELET
Vol.32, No.2
2.0
21.5 =I .C Kl.0 tu u Y 0.5
I 10-7
0
I 10-6 PGEl
(MI
I
1
10-5 0
I 10-5
I
10-a
DBcAMP
I 10-3
(MI
FIG. 3 Parallelinhibitionby PGEl or DBcAMP of thrombin-inducedincreaseof [ Ca2+ ]i and serotoninrelease. Samples of plateletswhich were loaded with both [14C]serotonin and quin2 were preincubatedwith various concentrationsof PGEl or DBcAMP for 5 min and then thrombin (0.2 U/ml) was added. [ Ca2+ ]i was calculatedfrom fluorescencesignal and serotonin releasewas quantifiedas describedunder MATERIALSAND METHODS. A, with PGEl; 8, with DBcAMP. o-, [ Ca2+ 1ii , serotoninrelease.
100
r
FIG. 4 Reversalof thrombin-induced increaseof [ Ca2+ ]i by PGEl. Samples of platelets which were loaded with both [14C]serotonin and quin2 were stimulated by thrombin (0.1 U/ml). After 100 set, PGEl (10 ~JM)or saline was added. Quin2 fluorescence was recorded as described under MATERIALS AND METHODS. A, with PGEl; B, with saline.
Vo1.32, No.2
CYCLIC AMP AND Ca2+ IN PLATELET
187
analysis shown in Fig. dependent manner as shown in Fig. 2. The quantitative 3A indicates that thrombin-induced increase of [ Ca2+ ]i and serotonin release were inhibited parallel to the increasing concentrations of PGEl. Similarly preincubation of platelets with DBcAMPalso inhibited thrombininduced above reactions in a dose-dependent manner (Fig. 3B). In the experiment shown in Fig. 4, platelet suspensions were exposed to thrombin for 100 set by which time the increase of [ Ca2+ ]i was maximal and then PGEl was added. Although [ Ca2+ ]i was slightly decreased in a control experiment (Fig. 4B), PGEl caused the rapid decrease of elevated [ Ca2+ Ii. These results provide direct evidence that cyclic AMP regulates [ Ca2+ ]i levels in platelets. DISCUSSION role in platelet functional change Intracellular Ca2+ plays a crucial and it is believed that [ Ca2+ Ii, which is low in the resting state, rises However to date, conclusions regardrapidly after platelet activation (7). ing the involvement of Ca2+ in responses of intact platelets have dependent Since observation by Kisermostly on indirect and circumstantial evidence. Glanzman et al. (8) that cyclic AMP can increase the rate of uptake of Ca2+ it has also been suggested that an ions by platelet membrane preparation, increase in platelet cyclic AMPmay inhibit platelet function by lowering Recently, Rink et al. (4) have measured successfully [ Ca2+ ]i [ Ca2+ ]j_. in intact platelets by means of the novel fluorescent indicator dye quin2, and they have shown for the first time that thrombin actually raises In the present study, we comfirmed their [ Ca2+ ]i in intact platelets. observation first and then provided direct demonstration that cyclic AMP actually lowers [ Ca2+ ]i and inhibits platelet activation. Although mechanism of this action of cyclic AMP is not clarified in the present study, it is possible that this compound may act by stimulating active transport of Ca2+ ions from the cytoplasm across the dense tubular membranes. During preparation of this manuscript, Feinstein et al. (9), also using quin2, have found that adenylate cyclase stimulators PGD2, PGEl or forskorin The present inhibit thrombin-induced increase of [ Ca2+ ]i in platelets. results and those of theirs are in close agreement with each other as far as [ Ca2+ ]i response to cyclic AMP although they have not monitored serotonin release. ACKNOWLEDGEMENT This investigation was supported in part by research grant from the Mitsuhisa Cancer Research Foundation (1982). Nishizuka and Y. Takai, the The authors are grateful to ProfessorsY. Department of 6iochemistry, Kobe University School of Medicine for valuable discussion and support in this work. REFERENCES 1. FEINSTEIN, M.B., RODAN,G.A. and CUTLER, L.S. Cyclic AMPand calcium in platelet function. In: Platelets in Biology and Pathology J.L. Gordon (Ed.) Vol. 2, Amsterdam:Elsevier/North-Holland Biomedical bress, 1981, pp. 437-472. 2. TSIEN, R.Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 2396-2404, 1980.
188
CYCLIC AMP AND Ca2+
IN PLATELET
Vol.32,
No. 2
3. TSIEN, R.Y., POZZAN,T. and RINK, T.J. Calciumhomeostasisin intact lymphocytes:cytoplasmicfree calciummonitoredwith a new intracellularly trapped fluorescentindicator. _. J Cell. Biol. 94, 325-334,1982. 4. RINK, T.J., SMITH, S.W. and TSIEN R.Y. Cytoplasmicfree Ca2+ in human independentactivationfor shapeplatelets:Ca2' thresholdsand Cai+-* change and secretion. -FEBS Lett. 148, 21-26, 1982. 5. BAENZIGER,N.L. and MAJERUS,P.W. Isolationof human plateletsand platelet surface membranes. Methods Enzymol.3l_,149-155,1974. 6. HASLAM, R.J., DAVIDSON,M.M.L. and MCCLENAGHAN,M.D. CytochalasinB, the blood platelet releasereactionand cyclic GMP. Nature 253, 455457, 1975. 7. GERRARD,J.M., PETERSON,D.A. and WHITE, J.G. Calcium mobilization. In: Plateletsin Biologyand Pathology J.L. Gordon (Ed.) Vol. 2, Amsterdam:Elsevier/North-Holland Biome&.calPress, 1981, pp. 407-436. 8. &ER-GLANZMANN, R., JAKABOVA,M., GEORGE, J.N. and LiiSCHER, E.F. Stimulationof calciumuptake in plateletmembranevesiclesby adenosine 3',5'-cyclicmonophosphateand protein kinase. Biochim.Biophys.Acta 466, 429-440, 1977. 9. FEINSTEIN,M.B., EGAN, J.J., SHA'AFI,R.I. and WHITE, J. The cytoplasmic concentrationof free calcium in plateletsis controlledby stimulators of cyclic AMP production (PGD2,PGEl, forskolin). Biochem. Biophys.Res. Commun. 113, 598-604, 1983.
EFFECT OF CYCLIC AMP ON CYTOPLASMICFREE CALCIUM IN HUMAN PLATELETS STIMULATEDBY THROMBIN:DIRECT MEASUREMENTWITH QUIN2
J. YAMANISHI,Y. KAWAHARAand H. FUKUZAKI Departmentof InternalMedicine,Division I, Kobe UniversitySchool of Medicine,Kobe 650, Japan
(Received 14.7.1983; Accepted in revised form 8.8.1983 by Editor I. Yamashina) ABSTRACT With the fluorescentCa2+ indicatorquin2, we measured directly cytoplasmicfree Ca2+ ([ Ca2+ Ii) in washed human platelets stimulated by thrombinand examinedthe effect of cyclic AMP on [ Ca2+ ]i levels and 14C-serotoninrelease. Thrombin (0.2 U/ml) evoked a rise in [ Ca2+ ]i from the basal level of about 100 nM to ~3 uM which was fast enough to trigger serotoninrelease. This rise was inhibitedin a dose-dependentmanner by preincubationwith prostaglandinEl (PGEl) (0.1-10uM) or dibutyrylcyclic AMP (0.01 -1 mM). Parallelto this, serotoninreleasewas also inhibited by these drugs. When added to plateletsafter stimulationby thrombin,PGEl caused the rapid decreaseof elevated [ Ca2+ Ii. These resultsprovide direct evidencethat [ Ca2+ ]i levels in plateletsare regulatedby cyclic AMP.
INTRODUCTION It has been su gested that an increasein plateletcyclic AMP lowers cytoplasmicfree Ca5+ ([ Ca2+ Ii) and this action of cyclic AMP is thought to be underlyingmechanismby which certainprostaglandinsi.e. PGEl or PG12, which stimulateplateletadenylatecyclase,inhibitplateletactivation (1). However to date, there is lack of direct evidencethat cyclic AMP actually lowers [ Ca2+ ]i in intact plateletssince direct measurementsof [ Ca2+ ]i have not been previouslyachievedin such small cells. Recently,Tsienet al. (2,3)have synthesizeda new fluorescentCa2+ indicatorquin2 that can be trapped in the cytoplasmof intact cells of any size and have successfully measured [ Ca2+ ]i in lymphocytes. With this method, Rink et al. (4) have shown a rapid rise in [ Ca2+ ]i in intact plateletswhich are stimulated Key words: human platelet,cytoplasmicfree calcium,cyclic AMP, serotonin release,thrombin 183
184
CYCLIC AMP AND Ca*+ IN PLATELET
Vo1.32, No.2
[Ca2+]i -
-
3 NM 1 MM
-500
nM
-100
nM
FIG. 1 Time courses of serotonin release and quin2 fluorescence response in stimulated by thrombin. Samples of platelets which were loaded platelets with both [lklserotonin and quin2 were stimulated by thrombin (0.2 U/ml). Serotonin release was quantified and quin2 fluorescence was recorded as traces described under MATERIALSAND METHODS. The gaps in the fluorescence here and in subsequent figures show the times for addition of agents. A, serotonin release; B, quin2 fluorescence response.
In the present study we examined the effect of cyclic AMP on by thrombin. [ Ca2+ ]i levels in intact platelets stimulated by thrombin by means of this new fluorescent dye. MATERIALSAND METHODS Quin2 acetoxymethyl ester and PGEl were products of Dojindo LaboraBovine thrombin and tories and Ono Pharmaceutical Co., respectively. DBcAMPwere purchased from Mochida Pharmaceutical Co. and Sigma, respectively. [l‘klserotonin (58 mCi/mmol) was obtained from New England Nuclear. The abbreviations used were: PGEl, prostaglandin El; 12; DBcAMP, dibutyryl cyclic AMP; PGD2, prostaglandin
PGI2, prostaglandin D2
CYCLICAMP AND Ca2+ IN PLATELET
Vo1.32, No.2
B
A
-4
Thrombin
-4 Thrombin
185
c)
r
-4 Tbrombin
, 2 min, FIG. 2 Effect of various concentrationsof PGEl on thrombin-induced quin2 fluorescenceresponse. Samples of plateletswhich were loadedwith both [14C]serotonin and quin2 were preincubatedwith various concentrationsof PGEl for 5 min and then thrombin (0.2 U/ml) was added. Quin2 fluorescence was recordedas describedunder MATERIALSAND METHODS. A, with saline; B, with PGEl (0.1 uM); C, with PGEl (1 PM); D, with PGEl (10 @l).
Washed platelets,which were prepared as describedby Baenzigerand Majerus (!?I), were incubatedfor 25 min at 37'C with Hepes buffered saline (145 mM NaCl, 5 mM KCl, 1 mM MgS04, 0.5 mM Na2HP04, 10 mM Hepes, 5 mM glucose,pH 7.4 at 37°C) containing5 uM quin2 acetoxymethylester. The suspensionwas washed 2 times with Hepes buffered saline to remove any extraneousdye and finallyresuspendedto make a concentrationof 1 x 108 cells/mlin Hepes bufferedsaline containing1 mM CaC12. Fluorescencewas recordedat 37'C in Hitachi fluorescencespectrophotometer650-60. Excitationwas at 339 nm and emissionat 500 nm. [ Ca2+ ]i was calculatedfrom fluorescencesignal as describedby Rink et al. (4). Serotoninreleasewas quantifiedsimultaneouslywith quin2 fluorescenceby measuringradioactivematerial released from the plateletswhich were preloadedwith [14C]serotonin as describedby Haslam et al. (6). RESULTS Fig. 1 shows time courses of serotoninrelease and fluorescence responsein plateletswhich were stimulatedby thrombin. In comfirmationof the reviousobservationby Rink et al. (4), thrombinevoked a rapid rise in 1 Ca'+ ]i from the basal level of about 100 nM to s3 pM. This risewas fast enough to trigger serotoninrelease. Since the reactionCa2+ + quin2++ Ca2+-quin2is in equilibrium,it seems reasonableto speculatethat,inplatelets which are not loadedwith quin2, thrombin-inducedrise in [ Ca2+ ]i may be faster than that shown in Fig. 1. In fact, serotoninrelease from unloaded plateletswas faster than that from loaded cells (datanot shown). PGEl is known to stimulateplatelet adenylatecyclase activityresultingin marked elevationof platelet cyclic AMP levels. Preincubationof plateletswith this prostaglandininhibitedthrombin-inducedincreaseof [ Ca2* ]i in a dose-
186
CYCLIC AMPAND Ca*+ IN PLATELET
Vol.32, No.2
2.0
21.5 =I .C Kl.0 tu u Y 0.5
I 10-7
0
I 10-6 PGEl
(MI
I
1
10-5 0
I 10-5
I
10-a
DBcAMP
I 10-3
(MI
FIG. 3 Parallelinhibitionby PGEl or DBcAMP of thrombin-inducedincreaseof [ Ca2+ ]i and serotoninrelease. Samples of plateletswhich were loaded with both [14C]serotonin and quin2 were preincubatedwith various concentrationsof PGEl or DBcAMP for 5 min and then thrombin (0.2 U/ml) was added. [ Ca2+ ]i was calculatedfrom fluorescencesignal and serotonin releasewas quantifiedas describedunder MATERIALSAND METHODS. A, with PGEl; 8, with DBcAMP. o-, [ Ca2+ 1ii , serotoninrelease.
100
r
FIG. 4 Reversalof thrombin-induced increaseof [ Ca2+ ]i by PGEl. Samples of platelets which were loaded with both [14C]serotonin and quin2 were stimulated by thrombin (0.1 U/ml). After 100 set, PGEl (10 ~JM)or saline was added. Quin2 fluorescence was recorded as described under MATERIALS AND METHODS. A, with PGEl; B, with saline.
Vo1.32, No.2
CYCLIC AMP AND Ca2+ IN PLATELET
187
analysis shown in Fig. dependent manner as shown in Fig. 2. The quantitative 3A indicates that thrombin-induced increase of [ Ca2+ ]i and serotonin release were inhibited parallel to the increasing concentrations of PGEl. Similarly preincubation of platelets with DBcAMPalso inhibited thrombininduced above reactions in a dose-dependent manner (Fig. 3B). In the experiment shown in Fig. 4, platelet suspensions were exposed to thrombin for 100 set by which time the increase of [ Ca2+ ]i was maximal and then PGEl was added. Although [ Ca2+ ]i was slightly decreased in a control experiment (Fig. 4B), PGEl caused the rapid decrease of elevated [ Ca2+ Ii. These results provide direct evidence that cyclic AMP regulates [ Ca2+ ]i levels in platelets. DISCUSSION role in platelet functional change Intracellular Ca2+ plays a crucial and it is believed that [ Ca2+ Ii, which is low in the resting state, rises However to date, conclusions regardrapidly after platelet activation (7). ing the involvement of Ca2+ in responses of intact platelets have dependent Since observation by Kisermostly on indirect and circumstantial evidence. Glanzman et al. (8) that cyclic AMP can increase the rate of uptake of Ca2+ it has also been suggested that an ions by platelet membrane preparation, increase in platelet cyclic AMPmay inhibit platelet function by lowering Recently, Rink et al. (4) have measured successfully [ Ca2+ ]i [ Ca2+ ]j_. in intact platelets by means of the novel fluorescent indicator dye quin2, and they have shown for the first time that thrombin actually raises In the present study, we comfirmed their [ Ca2+ ]i in intact platelets. observation first and then provided direct demonstration that cyclic AMP actually lowers [ Ca2+ ]i and inhibits platelet activation. Although mechanism of this action of cyclic AMP is not clarified in the present study, it is possible that this compound may act by stimulating active transport of Ca2+ ions from the cytoplasm across the dense tubular membranes. During preparation of this manuscript, Feinstein et al. (9), also using quin2, have found that adenylate cyclase stimulators PGD2, PGEl or forskorin The present inhibit thrombin-induced increase of [ Ca2+ ]i in platelets. results and those of theirs are in close agreement with each other as far as [ Ca2+ ]i response to cyclic AMP although they have not monitored serotonin release. ACKNOWLEDGEMENT This investigation was supported in part by research grant from the Mitsuhisa Cancer Research Foundation (1982). Nishizuka and Y. Takai, the The authors are grateful to ProfessorsY. Department of 6iochemistry, Kobe University School of Medicine for valuable discussion and support in this work. REFERENCES 1. FEINSTEIN, M.B., RODAN,G.A. and CUTLER, L.S. Cyclic AMPand calcium in platelet function. In: Platelets in Biology and Pathology J.L. Gordon (Ed.) Vol. 2, Amsterdam:Elsevier/North-Holland Biomedical bress, 1981, pp. 437-472. 2. TSIEN, R.Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 2396-2404, 1980.
188
CYCLIC AMP AND Ca2+
IN PLATELET
Vol.32,
No. 2
3. TSIEN, R.Y., POZZAN,T. and RINK, T.J. Calciumhomeostasisin intact lymphocytes:cytoplasmicfree calciummonitoredwith a new intracellularly trapped fluorescentindicator. _. J Cell. Biol. 94, 325-334,1982. 4. RINK, T.J., SMITH, S.W. and TSIEN R.Y. Cytoplasmicfree Ca2+ in human independentactivationfor shapeplatelets:Ca2' thresholdsand Cai+-* change and secretion. -FEBS Lett. 148, 21-26, 1982. 5. BAENZIGER,N.L. and MAJERUS,P.W. Isolationof human plateletsand platelet surface membranes. Methods Enzymol.3l_,149-155,1974. 6. HASLAM, R.J., DAVIDSON,M.M.L. and MCCLENAGHAN,M.D. CytochalasinB, the blood platelet releasereactionand cyclic GMP. Nature 253, 455457, 1975. 7. GERRARD,J.M., PETERSON,D.A. and WHITE, J.G. Calcium mobilization. In: Plateletsin Biologyand Pathology J.L. Gordon (Ed.) Vol. 2, Amsterdam:Elsevier/North-Holland Biome&.calPress, 1981, pp. 407-436. 8. &ER-GLANZMANN, R., JAKABOVA,M., GEORGE, J.N. and LiiSCHER, E.F. Stimulationof calciumuptake in plateletmembranevesiclesby adenosine 3',5'-cyclicmonophosphateand protein kinase. Biochim.Biophys.Acta 466, 429-440, 1977. 9. FEINSTEIN,M.B., EGAN, J.J., SHA'AFI,R.I. and WHITE, J. The cytoplasmic concentrationof free calcium in plateletsis controlledby stimulators of cyclic AMP production (PGD2,PGEl, forskolin). Biochem. Biophys.Res. Commun. 113, 598-604, 1983.