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Cytosolic ionized calcium in human platelets: The influence of collagen and a novel antiplatelet agent
Vol.
177,
June
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
14, 1991
CYTOSOLIC INFLUENCE
IONIZED CALCIUM IN HUMAN PLATELETS: OF COLLAGEN AND A NOVEL ANTIPLATELET
888-893
TEE AGENT
Ranmchander Gollautudi, Elwood 0. Dillingham, Stephen E. Bond and Beverly A. Lyman Department of Medicinal Chemistry, College of Pharmacy, The University of Tennessee, Memphis, 26 South Dunlap, Memphis, Tennessee 38163
Received May 9, 1991 Two phases of calcium mobilization were observed when aequorin-loaded humanplatelets, suspended in a nominally calcium-free medium containing 0.1 mMEGTA, were stimulated with collagen. The first phase coincided with platelet shape change, and the second phase corresponded to aggregation. On the other hand, only one [Ca2+]. peak was found in systems containing 1.0 m.M compoundu,u'-bis Ca, or 1.0 or 2.0 mM EGTA: A novel antiplatelet [3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, inhibited both [C$+]. peaks. It is suggested that inhibition of the mobilization of intrapiatelet calcium stores as well as the blocking of transmembrane calcium flux may be responsible for the platelet aggregation-inhibitory action of this compound. 0 1991Academic Press,Inc.
Stimulus-response coupling in platelets is regulated by a complex signal transduction mechanism, in which calcium plays a key role (1-3). A 2+ steep concentration gradient exists between the free Ca levels in plasma (1 mu) and the platelet cytosol. The cytosolic ionized calcium ([Ca 2+]i) in resting humanplatelets varies with the calcium indicator used, being 100 nh when measured with quin-2 (4) and 2-4 uh when measured with aequorin (5). Agonist binding to platelet receptors is accompanied by a sharp rise in [Ca2+li which originates from one or more dischargeable intracellular stores as well as from the extracellular fluid (2,6). Receptor binding by a number of platelet agonists results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C to form the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) (7,8). The primary role of IP3 is to release Ca2+ from nonmitochondrial intracellular stores (9) whereas DG activates protein kinase C and causes phosphorylation of a 40-47 kDa protein (10). On the other hand, the mechanism controlling Ca2+-influx is more complex and appears to be mediated by at least three pathways (3). Other phosphoinositides, inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate have also been implied as possible Ca2+-mobilizing agents (11). Any interference with this chain of events is anticipated to inhibit the [CaZcli levels.
Vol.
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One of the most promising inhibitors of human blood platelet aggregation developed in this laboratory isop'-bis[3-(N,N-diethylcarbamoyl)piperidino]-pxylene dihydrobromide (I). This compound was effective in inhibiting human blood platelet aggregation in vitro (12), platelet clustering induced by polymer surfaces in whole human blood in vitro (13), platelet aggregation in dogs in vivo (14), and platelet deposition in baboons in vivo (15), while showing relatively low toxicity in mice and rats In experiments with monolayer films, compound I showed substantial (16). interaction with anionic phospholipids (phosphatidylinositol and phosphatidylserine) but not with phosphatidylethanolamine or phosphatidylcholine A concentration of I (10 uM) affecting substantial inhibition of (17). ADP-induced aggregation in vitro, failed to retain that effect when 5.0 mM Ca2+ was added to the test medium. Considerably higher concentrations of I 2+ were required to inhibit aggregation in the presence of 5.0 mM Ca or when the Ca2+-ionophore A23187 was used as the agonist (18). Based on these observations, it was postulated that this compound may restrain [Ca2+li mobilization (18). We report for the first time, a biphasic aequorin-indicated [Ca 2+1 i response to stimulation of human platelets with collagen and blockade of this by compound I.
Materials. The test compound, a,o'-bis[3-(N,N-diethylcarbamoyl)piperidino]mdihydrobromide (I), was synthesized in this laboratory as described Aequorin was obtained from Friday Harbor Photoproteins, Friday (19). Harbor, WA. Ethylene glycol tetraacetate (EGTA), prostaglandin E (PGE 1, albumin (bovine, fraction V, powder), and dextrose were obtained $ rom Si.b Chemical Co., St. Louis, MO. Collagen was obtained from Hormon Chemie Mtichen, GmbH, Miinchen, Germany. Fibrinogen (grade L lyophilized powder) was purchased from Kabi Vitrum, Franklin, OH. Dimethyl sulfoxide (DMSO) was obtained from Pierce Chemical Co., Rockford, IL. The Platelet Ionized Calcium Aggregometer (PICA) Chrono-Log Corp., Ltd., Havertown, PA was used in all assays. Platelet Preparation. The basic procedure of Yamaguchi et al. (20) was Blood was collected from six blood donors, aged 24-38 years, who followed. had fasted overnight and affirmed abstinence from alcohol, caffeine and medications of any kind for a period of at least one week prior to donation. Platelet rich plasma (PRP) was obtained by centrifugation (12Og, 15 min) of titrated venous blood (3.8% trisodium citrate, final concentration) and 1.0 0.009 of the volume of PRP, was added. Platelets were M citric acid, collected (8OOg, 15 min), resuspended in and washed (8OOg, 15 min) with HEPES-buffered saline (140 mM NaCl, 2.7 mM KCl, 0.1% bovine albumin, 0.5% The glucose, 3.8 mM HEPES, pH 7.6) with added EGTA (5 mM) and PGE (1 uM). platelet pellet was resuspended in 90 ul of the same buffer and added to 20 Six ul of 3 mg/ml aequorin solution in a 1.5 ml Eppendorf centrifuge tube. 1 ul aliquots of DMSO were added at 90 second intervals with brief, gentle 2 min mixing on a Vortex mixer and the platelet suspension was incubated gently One ml of HEPES-buffered saline was added, after the last addition. were pelleted (lOOOg, mixed, and again incubated for 2 min. The platelets and pelleted again. The final resuspended in the same buffer, 30 set), pellet was resuspended in HEPES-buffered saline to which was added either 1 (calcium mM Mg Cl2 (calcium-free buffer) or 1 mM Mg Cl2 and 1 mM Ca Cl The platelet count was idjusted to depending on the experiment. buffer) 3-4 x lb5 platelets/ul. 889
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The PICA was used to obtain simultaneous Experimental Procedure. aggregation and luminescence data under the following experimental (1) platelet suspension, 1 ml, was equilibrated at 37 C with conditions: stirring at 1100 rpm; (2) 100 ul (1.2 ug) of fibrinogen was added; (3) the vehicle (95% ethanol) or the test compound in vehicle was added; (4) when 10 ul of the desired concentration of EGTA was added 30 seconds needed, prior to addition of the agonist; then (5) the agonist (collagen, 5 ug or 20 ug) was added and the time noted. At one minute after addition of agonist in Channel 1, a platelet sample containing fibrinogen in Channel 2 was lysed with 10 ul of a 1:l solution of Triton X-100 (final concentration 0.005%) and 10 mM CaCl (final concentration 0.5 mM). Treated and control fluorescence dat 2 were corrected for time dependent decay of fluorescence using data from the untreated lysed controls. RESULTS
Representative tracings of the collagen-induced [Cazili response in aequorin-loaded human platelets are shown in Figure 1. Platelets (3-4 x 105/u1) were resuspended in HEPES-buffered saline containing 1 mM Ca2+. Luminescence tracings were recorded at the gain settings indicated. The addition of collagen to the suspension medium containing 1 mM Ca 2+ ([Ca2+lo) resulted in the appearance of a single peak within 1 min (Figure IA). The appearance of this peak coincided with shape change and returned to baseline as the aggregation response approached maximum. Treatment of platelet suspensions with compound I (36 uM) inhibited the [Ca2+] i peak by 59% (Figure 18). The influence of 1 and 2 mM EGTA added to the nominally Ca '+-free suspending medium is shown in Figures 2A and B, respectively. The
Repre5entative
tracings
of aggregation suspended in
and aequorin-indicated HEPES-buffered saline
Collagen (5.0 pg) was addedat the time indicated
by the arrows. The platelet suspension (1.0 ml) was pre-incubated for 1.0 min with 1.0~1 of (A) 95% ethanol or (B) 36 p compound I in 95% ethanol. Luminescence was recorded at a gain of 0.2.
890
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Figure 2. Aggregation and [Ca2+]. mobilization in the presence of (A) 1.0 mMEGTA, (B) 2.0 mM EGTA, (C) '2.0 mM EGTA added 30 seconds after pre-incubation of the platelet preparation with 9.3 p compound I, (D) 0.1 mMEGTA. Collagen (20 pg) was added at time indicated by the arrows. Luminescencewas recorded at a gain of 0.5.
aggregation response, as can be expected, was weak even after increasing the 2+ concentration of agonist to 20 ug. A rise in [Ca Ii was first seen at the time of shape change, reached peak levels during the shape change and declined to baseline as aggregation occurred. A 98% inhibition of [Ca2+] i was noticed when compoundI (9.3 uM) was pre-incubated with the platelet preparation containing 2 mMEGTA (Figure 2C). Unlike higher concentrations of EGTAwhich elicited a smaller single peak, a lower concentration (0.1 mM) of this chelator in the external medium resulted in a biphasic [Ca2+li response (Figure 2D). The first Ca2+ peak coincided with shape change and occurred between 1 - 2 min. A very large second peak originated during shape change, became prominent by 2.5 min post-agonist and persisted for many minutes without fully returning to baseline fluorescence. This protracted luminescence peak appears to be qualitatively different from the peak (Figure LA) obtained in the presence of 1 mMCa2+ which returned to baseline as aggregation approached maximum CompoundI inhibited both peaks by approxiabout 5 minutes post-agonist. mately the sameextent (Figure 3). Thus, a 14.59 uM concentration inhibited the first and the second peaks by 46% and 50%, respectively (Figure 3B). The (Figure corresponding values at 23.87 uM were 97% and lOO%, respectively 3C). DISCUSSION
The agonist-induced rise in [Ca2+l i is caused by its discharge from intracellular stores and/or by influx across the platelet plasma membrane Our results demonstrate that when human platelets are suspended in a (2,6). medium in the presence of 0.1 mM EGTA, collagen nominally Ca2+-free 891
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Representative tracings of aggregation and [Ca 2+]. mobilization tl.0 ml) was suspension ~&.ence of 0.1 mM EGTA. The platelet pre-incubated with 1.0 ,ul of (A) 95% ethanol, (B) a solution of compound I (14.59,uM) in 95% ethanol, or (C) a solution of compound I (23.87 ,uM) in 95% ethanol. Collagen (20 pg) was added at time indicated by the arrows. Luminescence was recorded at a gain of 0.5.
evokes a biphasic [Ca 2+ Ii response, as indicated 2+ Ca peak coincides with shape change and the Compound I, a platelet aggregation-inhibitor,
stimulation
by
aequorin.
The first second peak with inhibits both aggregation. phases of the [Ca2+]. response in a concentration dependent manner. At high [Ca2+;o (1.0 mM), luminescence approached baseline at 4-5 min 2+ lo (0.1 mM EGTA), a very large second peak post-agonist, whereas at low [ca developed by that time, reaching a peak sometime after 5 minutes (compare 2+ Figure lA and 2D). Since luminescence is Ca -dependent, the stronger and more protracted fact that the EGTA in the
signal second
suspending
this peak is caused did not significantly lated
with
release
shape of
initial
at lower [Ca’+]o is difficult peak is suppressed by increasing medium
0.1 mM to 1.0 or
2.0
by Ca2+-influx. At the same time, change the magnitude of the first
change;
this
Ca2+
peak,
from
to
from internal arising from the
suggests
that
stores. mobilization
the
first
rationalize. the concentration mM suggests
The of that
the increase in EGTA peak which is correpeak
At a higher [Ca2+lo of intracellular
is
caused
by
(1.0 mM), stores,
the is
overwhelmed extracellular
by influx and results in a single peak. By optimizing the 2+ Ca concentration, it appears possible to separate the 2+ aequorin-indicated [Ca Ii rise into two phases during platelet stimulation. Earlier
reports
(21,22)
stimulated stimulation
platelets involved of aequorin-loaded
containing
medium was reported
of
biphasic
different
[Ca2+li
experimental
rat platelets by Nakano et al.,
release conditions.
in
collagenCollagen mM Ca 2+ -
suspended in a 1.0 (21). Using quin-2 as the fluorescent indicator, Ardlie et al. (22) observed that collagen elicited a 2+ biphasic elevation of [Ca2+] i in the presence of 1.0 mu Ca as well as in the absence of [Ca 2+ lo. A small first peak which occurred at 15-20 seconds 892
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BIOCHEMICAL
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was attributed to initial adhesion of a small number of platelets to collagen. This was followed by a second larger peak at 1-2 min. We could not discern a biphasic response in the presence of 1.0 mM external calcium as did these workers. It should be noted that Nakano et al. (21) studied rat platelets and Ardlie et al. (22) used guin-2 as the fluorescent indicator. It is known that aequorin and quin-2 differ in their regional intraplatelet distribution (5,23). A biphasic (Ca2+li response to collagen in aequorin-loaded human platelets has not been reported to our knowledge. Compound I reduces intracellular calcium mobilization and blocks 2+ transmembrane Ca -influx. Its platelet aggregation-inhibitory properties appear to be mediated through the inhibition of [Ca 2+ Ii.
We wish to express our appreciation to Dr. Marion Dugdale, Division Hematology for her assistance and counsel, and to Ms. Stella A. Bain secretarial help. This research was supported by USPHS grant, HL 22236.
of for
REFERENCES 1. 2. 3. 4. 5. 6. i: 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Rink, T.J. (1988) Experientia 44, 97-100. Seiss, W. (1989) Physiol.Rev. 69, 58-178. Rink, T.J., and Sage, S.O. (1990) Annu.Rev.Physiol. 52, 431-449. Rink, T.J., Smith, S.W., and Tsien, R.Y. (1982) FEBS Lett. 148, 21-26. Johnson, P.C., Ware, J-A., Cliveden, P.B., Smith, M., Dvorak, A.M., and Salzman, E.W. (1985) J.Biol.Chem. 260, 2069-2076. Ware, J.A., Johnson, P.C., Smith, M., and Salzman, E.W. (1986) J.Clin.Invest. 77, 878-886. 220, 345-360. Berridge, M.J. (1984) Bi0chem.J. Quist, E.E., and Barker, R.C. (1983) Arch.Biochem.Biophys. 222, 170-178. Berridge, M.J., and Irvine, R.F. (1984) Nature (London) 312, 315-321. Nishizuka, Y. (1984) Nature (London) 308, 693-698. Daniel, J-L., Dangelmaier, C.A., and Smith, J.B. (1987) Bi0chem.J. 246, 109-114. Lasslo, A., and White, J.G. (1984) Biochim.Biophys.Acta 777, 37-40. Folie, B.J., McIntire, L.V., and Lasslo, A. (1988) Blood 72, 1393-1400. Lawrence, W.H., Gollamudi, R., Dillinqham, E-O., Carter-Burks, G., Tisdelle, P.A., and Lasslo, A. (1988) J.Pharm.Sci. 77, 464-465. Marzec, U.M., Kelly, A.B., Hanson, S.R., Lasslo, A., and Harker, L.A. (1990) Arteriosclerosis 10, 367-371. Lawrence, W.H., Tisdelle, P.A., Turner, J.E., Dillingham I, E.O., Gollamudi, R., Carter-Burks, G., and Lasslo, A. (1988 Eundam.Appl.Toxicol. 10, 499-505. Quintana, R.P., Lasslo, A., Dugdale, M., and Goodin, L. (1981) Thromb.Res. 22, 665-680. Lasslo, A., Quintana, R-P., Johnson, R.W., Naylor, J-L., and Dugdale, M. (1984) Biochim.Biophys.Acta 172, 84-92. Quintana, R.P., Smith, T.D., and Lorenzen, L.F. (1965) J.Pharm.Sci. 54, 185-787. Yamaguchi, A., Suzuki, H., Tanoue, K., and Yamazaki, H. (1986) Thromb.Res. 44, 165-174. Nakano, T., Terawaki, A., and Arita, H. (1986) J.Biochem. 99, 1285-1288. Ardlie, N.G., Garrett, J.J., and Bell, L.K. (1986) Thromb.Res. 42, 115-124. 62, 1094-1099. Lages, B., and Weiss, H.J. (1989) Throrrb.Haemost. 893
177,
June
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
14, 1991
CYTOSOLIC INFLUENCE
IONIZED CALCIUM IN HUMAN PLATELETS: OF COLLAGEN AND A NOVEL ANTIPLATELET
888-893
TEE AGENT
Ranmchander Gollautudi, Elwood 0. Dillingham, Stephen E. Bond and Beverly A. Lyman Department of Medicinal Chemistry, College of Pharmacy, The University of Tennessee, Memphis, 26 South Dunlap, Memphis, Tennessee 38163
Received May 9, 1991 Two phases of calcium mobilization were observed when aequorin-loaded humanplatelets, suspended in a nominally calcium-free medium containing 0.1 mMEGTA, were stimulated with collagen. The first phase coincided with platelet shape change, and the second phase corresponded to aggregation. On the other hand, only one [Ca2+]. peak was found in systems containing 1.0 m.M compoundu,u'-bis Ca, or 1.0 or 2.0 mM EGTA: A novel antiplatelet [3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, inhibited both [C$+]. peaks. It is suggested that inhibition of the mobilization of intrapiatelet calcium stores as well as the blocking of transmembrane calcium flux may be responsible for the platelet aggregation-inhibitory action of this compound. 0 1991Academic Press,Inc.
Stimulus-response coupling in platelets is regulated by a complex signal transduction mechanism, in which calcium plays a key role (1-3). A 2+ steep concentration gradient exists between the free Ca levels in plasma (1 mu) and the platelet cytosol. The cytosolic ionized calcium ([Ca 2+]i) in resting humanplatelets varies with the calcium indicator used, being 100 nh when measured with quin-2 (4) and 2-4 uh when measured with aequorin (5). Agonist binding to platelet receptors is accompanied by a sharp rise in [Ca2+li which originates from one or more dischargeable intracellular stores as well as from the extracellular fluid (2,6). Receptor binding by a number of platelet agonists results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C to form the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) (7,8). The primary role of IP3 is to release Ca2+ from nonmitochondrial intracellular stores (9) whereas DG activates protein kinase C and causes phosphorylation of a 40-47 kDa protein (10). On the other hand, the mechanism controlling Ca2+-influx is more complex and appears to be mediated by at least three pathways (3). Other phosphoinositides, inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate have also been implied as possible Ca2+-mobilizing agents (11). Any interference with this chain of events is anticipated to inhibit the [CaZcli levels.
Vol.
177,
No.
2, 1991
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AND
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One of the most promising inhibitors of human blood platelet aggregation developed in this laboratory isop'-bis[3-(N,N-diethylcarbamoyl)piperidino]-pxylene dihydrobromide (I). This compound was effective in inhibiting human blood platelet aggregation in vitro (12), platelet clustering induced by polymer surfaces in whole human blood in vitro (13), platelet aggregation in dogs in vivo (14), and platelet deposition in baboons in vivo (15), while showing relatively low toxicity in mice and rats In experiments with monolayer films, compound I showed substantial (16). interaction with anionic phospholipids (phosphatidylinositol and phosphatidylserine) but not with phosphatidylethanolamine or phosphatidylcholine A concentration of I (10 uM) affecting substantial inhibition of (17). ADP-induced aggregation in vitro, failed to retain that effect when 5.0 mM Ca2+ was added to the test medium. Considerably higher concentrations of I 2+ were required to inhibit aggregation in the presence of 5.0 mM Ca or when the Ca2+-ionophore A23187 was used as the agonist (18). Based on these observations, it was postulated that this compound may restrain [Ca2+li mobilization (18). We report for the first time, a biphasic aequorin-indicated [Ca 2+1 i response to stimulation of human platelets with collagen and blockade of this by compound I.
Materials. The test compound, a,o'-bis[3-(N,N-diethylcarbamoyl)piperidino]mdihydrobromide (I), was synthesized in this laboratory as described Aequorin was obtained from Friday Harbor Photoproteins, Friday (19). Harbor, WA. Ethylene glycol tetraacetate (EGTA), prostaglandin E (PGE 1, albumin (bovine, fraction V, powder), and dextrose were obtained $ rom Si.b Chemical Co., St. Louis, MO. Collagen was obtained from Hormon Chemie Mtichen, GmbH, Miinchen, Germany. Fibrinogen (grade L lyophilized powder) was purchased from Kabi Vitrum, Franklin, OH. Dimethyl sulfoxide (DMSO) was obtained from Pierce Chemical Co., Rockford, IL. The Platelet Ionized Calcium Aggregometer (PICA) Chrono-Log Corp., Ltd., Havertown, PA was used in all assays. Platelet Preparation. The basic procedure of Yamaguchi et al. (20) was Blood was collected from six blood donors, aged 24-38 years, who followed. had fasted overnight and affirmed abstinence from alcohol, caffeine and medications of any kind for a period of at least one week prior to donation. Platelet rich plasma (PRP) was obtained by centrifugation (12Og, 15 min) of titrated venous blood (3.8% trisodium citrate, final concentration) and 1.0 0.009 of the volume of PRP, was added. Platelets were M citric acid, collected (8OOg, 15 min), resuspended in and washed (8OOg, 15 min) with HEPES-buffered saline (140 mM NaCl, 2.7 mM KCl, 0.1% bovine albumin, 0.5% The glucose, 3.8 mM HEPES, pH 7.6) with added EGTA (5 mM) and PGE (1 uM). platelet pellet was resuspended in 90 ul of the same buffer and added to 20 Six ul of 3 mg/ml aequorin solution in a 1.5 ml Eppendorf centrifuge tube. 1 ul aliquots of DMSO were added at 90 second intervals with brief, gentle 2 min mixing on a Vortex mixer and the platelet suspension was incubated gently One ml of HEPES-buffered saline was added, after the last addition. were pelleted (lOOOg, mixed, and again incubated for 2 min. The platelets and pelleted again. The final resuspended in the same buffer, 30 set), pellet was resuspended in HEPES-buffered saline to which was added either 1 (calcium mM Mg Cl2 (calcium-free buffer) or 1 mM Mg Cl2 and 1 mM Ca Cl The platelet count was idjusted to depending on the experiment. buffer) 3-4 x lb5 platelets/ul. 889
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The PICA was used to obtain simultaneous Experimental Procedure. aggregation and luminescence data under the following experimental (1) platelet suspension, 1 ml, was equilibrated at 37 C with conditions: stirring at 1100 rpm; (2) 100 ul (1.2 ug) of fibrinogen was added; (3) the vehicle (95% ethanol) or the test compound in vehicle was added; (4) when 10 ul of the desired concentration of EGTA was added 30 seconds needed, prior to addition of the agonist; then (5) the agonist (collagen, 5 ug or 20 ug) was added and the time noted. At one minute after addition of agonist in Channel 1, a platelet sample containing fibrinogen in Channel 2 was lysed with 10 ul of a 1:l solution of Triton X-100 (final concentration 0.005%) and 10 mM CaCl (final concentration 0.5 mM). Treated and control fluorescence dat 2 were corrected for time dependent decay of fluorescence using data from the untreated lysed controls. RESULTS
Representative tracings of the collagen-induced [Cazili response in aequorin-loaded human platelets are shown in Figure 1. Platelets (3-4 x 105/u1) were resuspended in HEPES-buffered saline containing 1 mM Ca2+. Luminescence tracings were recorded at the gain settings indicated. The addition of collagen to the suspension medium containing 1 mM Ca 2+ ([Ca2+lo) resulted in the appearance of a single peak within 1 min (Figure IA). The appearance of this peak coincided with shape change and returned to baseline as the aggregation response approached maximum. Treatment of platelet suspensions with compound I (36 uM) inhibited the [Ca2+] i peak by 59% (Figure 18). The influence of 1 and 2 mM EGTA added to the nominally Ca '+-free suspending medium is shown in Figures 2A and B, respectively. The
Repre5entative
tracings
of aggregation suspended in
and aequorin-indicated HEPES-buffered saline
Collagen (5.0 pg) was addedat the time indicated
by the arrows. The platelet suspension (1.0 ml) was pre-incubated for 1.0 min with 1.0~1 of (A) 95% ethanol or (B) 36 p compound I in 95% ethanol. Luminescence was recorded at a gain of 0.2.
890
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Figure 2. Aggregation and [Ca2+]. mobilization in the presence of (A) 1.0 mMEGTA, (B) 2.0 mM EGTA, (C) '2.0 mM EGTA added 30 seconds after pre-incubation of the platelet preparation with 9.3 p compound I, (D) 0.1 mMEGTA. Collagen (20 pg) was added at time indicated by the arrows. Luminescencewas recorded at a gain of 0.5.
aggregation response, as can be expected, was weak even after increasing the 2+ concentration of agonist to 20 ug. A rise in [Ca Ii was first seen at the time of shape change, reached peak levels during the shape change and declined to baseline as aggregation occurred. A 98% inhibition of [Ca2+] i was noticed when compoundI (9.3 uM) was pre-incubated with the platelet preparation containing 2 mMEGTA (Figure 2C). Unlike higher concentrations of EGTAwhich elicited a smaller single peak, a lower concentration (0.1 mM) of this chelator in the external medium resulted in a biphasic [Ca2+li response (Figure 2D). The first Ca2+ peak coincided with shape change and occurred between 1 - 2 min. A very large second peak originated during shape change, became prominent by 2.5 min post-agonist and persisted for many minutes without fully returning to baseline fluorescence. This protracted luminescence peak appears to be qualitatively different from the peak (Figure LA) obtained in the presence of 1 mMCa2+ which returned to baseline as aggregation approached maximum CompoundI inhibited both peaks by approxiabout 5 minutes post-agonist. mately the sameextent (Figure 3). Thus, a 14.59 uM concentration inhibited the first and the second peaks by 46% and 50%, respectively (Figure 3B). The (Figure corresponding values at 23.87 uM were 97% and lOO%, respectively 3C). DISCUSSION
The agonist-induced rise in [Ca2+l i is caused by its discharge from intracellular stores and/or by influx across the platelet plasma membrane Our results demonstrate that when human platelets are suspended in a (2,6). medium in the presence of 0.1 mM EGTA, collagen nominally Ca2+-free 891
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No.
BIOCHEMICAL
2, 1991
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Representative tracings of aggregation and [Ca 2+]. mobilization tl.0 ml) was suspension ~&.ence of 0.1 mM EGTA. The platelet pre-incubated with 1.0 ,ul of (A) 95% ethanol, (B) a solution of compound I (14.59,uM) in 95% ethanol, or (C) a solution of compound I (23.87 ,uM) in 95% ethanol. Collagen (20 pg) was added at time indicated by the arrows. Luminescence was recorded at a gain of 0.5.
evokes a biphasic [Ca 2+ Ii response, as indicated 2+ Ca peak coincides with shape change and the Compound I, a platelet aggregation-inhibitor,
stimulation
by
aequorin.
The first second peak with inhibits both aggregation. phases of the [Ca2+]. response in a concentration dependent manner. At high [Ca2+;o (1.0 mM), luminescence approached baseline at 4-5 min 2+ lo (0.1 mM EGTA), a very large second peak post-agonist, whereas at low [ca developed by that time, reaching a peak sometime after 5 minutes (compare 2+ Figure lA and 2D). Since luminescence is Ca -dependent, the stronger and more protracted fact that the EGTA in the
signal second
suspending
this peak is caused did not significantly lated
with
release
shape of
initial
at lower [Ca’+]o is difficult peak is suppressed by increasing medium
0.1 mM to 1.0 or
2.0
by Ca2+-influx. At the same time, change the magnitude of the first
change;
this
Ca2+
peak,
from
to
from internal arising from the
suggests
that
stores. mobilization
the
first
rationalize. the concentration mM suggests
The of that
the increase in EGTA peak which is correpeak
At a higher [Ca2+lo of intracellular
is
caused
by
(1.0 mM), stores,
the is
overwhelmed extracellular
by influx and results in a single peak. By optimizing the 2+ Ca concentration, it appears possible to separate the 2+ aequorin-indicated [Ca Ii rise into two phases during platelet stimulation. Earlier
reports
(21,22)
stimulated stimulation
platelets involved of aequorin-loaded
containing
medium was reported
of
biphasic
different
[Ca2+li
experimental
rat platelets by Nakano et al.,
release conditions.
in
collagenCollagen mM Ca 2+ -
suspended in a 1.0 (21). Using quin-2 as the fluorescent indicator, Ardlie et al. (22) observed that collagen elicited a 2+ biphasic elevation of [Ca2+] i in the presence of 1.0 mu Ca as well as in the absence of [Ca 2+ lo. A small first peak which occurred at 15-20 seconds 892
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was attributed to initial adhesion of a small number of platelets to collagen. This was followed by a second larger peak at 1-2 min. We could not discern a biphasic response in the presence of 1.0 mM external calcium as did these workers. It should be noted that Nakano et al. (21) studied rat platelets and Ardlie et al. (22) used guin-2 as the fluorescent indicator. It is known that aequorin and quin-2 differ in their regional intraplatelet distribution (5,23). A biphasic (Ca2+li response to collagen in aequorin-loaded human platelets has not been reported to our knowledge. Compound I reduces intracellular calcium mobilization and blocks 2+ transmembrane Ca -influx. Its platelet aggregation-inhibitory properties appear to be mediated through the inhibition of [Ca 2+ Ii.
We wish to express our appreciation to Dr. Marion Dugdale, Division Hematology for her assistance and counsel, and to Ms. Stella A. Bain secretarial help. This research was supported by USPHS grant, HL 22236.
of for
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