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Dose-related stimulation of platelet cyclic adenosine monophosphate by prostacyclin in thrombotic stroke
THROMBOSIS
RESEARCH 45; 669-674, 1987 $3.00 t .OO Printed in the USA. (c) 1987 Pergamon Journals Ltd. All rights reserved.
0049-3848/87
Copyright
BRIEF COMMUNICATION DOSE-RELATED STIMULATION OF PLATELET CYCLIC ADENOSINE MONOPHOSPHATE BY PROSTACYCLIN IN THROMBOTIC STROKE
Creed Pettigrew, Audrey Papp, and Kenneth K. Wu Stroke and Thrombosis Research Center, Department of Internal Medicine, Division of Hematology and Department of Neurology, University of Texas Health Science Center at Houston Houston, TX 77225, U.S.A.
(Received 12.6.1986; Accepted in revised form 4.12.1986 by Editor J. Hawiger)
INTRODUCTION Platelet hyperaggregability is commonly observed in patients with thrombotic stroke (l-5). Evidence of platelet activation and hyperaggregability related to transient ischemic attacks and cerebral infarction includes increased platelet factor 4, B-thromboglobulin levels, shortened platelet survival time, adhesion of radiolabeled platelets to atherosclerotic carotid arteries, increased synthesis of malondialdehyde and thromboxane and accumulation of platelet aggregates. These data suggest a relationship between platelet activation and thrombotic stroke but have not convincingly established that a cause and effect relationship exists. The difficulty lies in part in a lack of understanding of the mechanisms by which platelet hyperaggregability occurs. In the present study, we proposed to test the hypothesis that a defective platelet responsiveness to prostacyclin may represent an important platelet inherent abnormality which attributes to increased platelet activity and thrombotic tendency.
PATIENTS AND METHODS Patients: Thirteen normals and 10 stroke patients were included in the study. The normal group included 8 women and 5 men of mean age 36.2 years
Key words:
prostacyclin, platelets, cyclic AMP, stroke, thrombosis 669
670
PGli2RESPONSE ON STROKE
(range 21-57). Three women and 7 men of mean age 58.3 years (range 38-74) comprised the patients. All were sampled between January of 1985 and February of 1986. Each underwent computerized tomography (CT) of the brain before entering the study. The suprasellar mass discovered on CT of one patient was judged not to have an effect on platelet function. Infarcts occurred in the carotid distribution in 9 patients (3 right and 6 left) and in the brainstem and left cerebellar hemisphere in one. Noninvasive carotid studies and echocardiography showed no potential sources of emboli. All blood samples were obtained from patients within two weeks of their strokes. One patient was taking 325mg of aspirin daily at the time of sampling. The normal donors had not taken any drugs known to inhibit platelet function for at least 10 days prior to the study. Platelet Response to Prostacyclin: We had chosen to study platelet responsiveness to PGI by measuring platelet cyclic AMP elevation in response to several concentrat?ons of PGI because thif4technique is more reliable and sensitive than the platelet agggegation or C-serotonin release procedures (6). Venous blood was withdrawn into a plastic tube containing l/10 vol. of 3.8% sodium citrate. The sample was centrifuged at 2QOxg for 10 minutes at 23°C. Platelet rich plasma 4PRP> was collected and its platelet concentration was standardized to 3 x 10 /ml with zqtologous PPP. PRP was treated with isobutylmethylxanthine (IBMX, 2 x 10 M) at 23°C for 30 minutes to block phosphodiesterase. 0.5ml PRP was placed in a dual-channel Chrono-Log aggregometer cuvette at 37'C for 1 minute followed by addition of authentic sodium prostacyclin (0,10,50, and 100&l) at 37°C for 90 seconds. The samples were under constant stirring throughout the period of study. The reaction was terminated by placing the samples into liquid nitrogen. The samples were stored at -7O'C until assay. To assay for cyclic AMP levels, the samples first underwent several steps of processing. They were thawed, sonicated with a cell disruptor (Heat Systems Ultrasonics, Inc., Plainview, N.Y.) and then placed in boiling water for 4 minutes. The samples were then centrifuged at 13,000xg for 5 minutes and the supernatants were collected and assayed for CAMP by radioimmunoassay using a commercial kit (Amersham, Arlington Heights, IL).
RESULTS AND DISCUSSION Gorman et al have previously shown a dose-response stimulation of platelet cyclic AMP by PGI in normal subjects (7). To evaluate individual variations in normal subjec?z s, we assayed platelet CAMP stimulated by several concentrations of PG12_ Our data revealed that the dose-response stimulation in 13 healthy subjects could be divided into 2 distinct subgroups. Ten had a normal dose-response stimulation while 3 were hyporesponsive (Table 1). The distinct difference in the dose-response curve is further illustrated in Fig. 1. Based on the results from 10 normal responders, the mean CAMP values stimulated by 10, 50 and 1OOnM of PGI were 257.3 ? S.D. 40.8 pmol (range 165.7-348.9 pmol), 707.1 c 129.0 pmol trange 449.1-965.1 pmol) and 1071.3 ? 259.8 pmol (range 551.7-1590.9 pmol) per 10' platelets respecti.vely. The dose-response stimulation of 7 stroke patients was within the normal range while 2 were hyporesponders and 1 had an increased sensitivity (Table 1). The mean value of the 7 normal responders was similar to the 10 normoresponsive healthy subjects (Fig. 1). The dose-response curves of the two hyporesponders were similar to the 3 healthy subjects with low responsiveness. The PG12 responsiveness in the patients and normals did not bear any relationship to basal CAMP values, sex or age of the subject. Using PGI2 dose-response stimulation of platelet cyclic AMP, we have
Vol. 45, No. 5
identified
a subset
671
PG12 RESPONSE ON STROKE
of patients
with
thrombotic
stroke who have a subnormal
TABLE 1 Dose-response Stimulation of Cyclic AMP by PGI, in Healthy Donors and Patients ,with Thrombotic Stroke Subjects Healthy Donors
Stroke Patients
No A--
Age
CAMP (pmol/lO' pl)
Sex
Normoresponsive 1 26 F 2 32 F 3 25 M 4 36 F 5 30 M 6 21 F 7 39 M 8 24 F 9 42 F 10 26 M Hyporesponsive 11 23 F 12 57 M 13 56 F Normoresponsive 1 67 M 2 60 M 3 62 M 4 73 M 5 74 F 6 63 F 7 38 M Hyper-responsive 8 44 F Hyporesponsive 9 64 M IO 49 M
§ Basal platelet CAMP level. 1OOnM of PG12 respectively.
09 37.3 76.0 64.7 44.7 46.7 42.8 61.3 36.0 31.3 29.3
lo* 210.0 224.7 299.3 292.0 273.3 317.1 260.0 276.7 206.7 213.3
50* 533.3 497.3 681.3 724.7 767.3 901.3 612.7 825.3 719.3 808.0
100* 771.8 636.7 953.3 909.3 1231.3 1330.3 1396.7 1089.3 1026.7 1367.3
2.9 12.0 74.0
56.0 158.0 164.7
145.5 242.7 144.5
157,8 136.0 289,5
7.8 77.3 94.7 32.0 12.6 94.2 52.2
239.2 258.7 137.3 217.3 166.6 182.1 144.9
749.0 712.0 447.3 564.0 586.0 712.4 1158.4
836.9 931.3 664.7 792.0 754.0 853.4 1435.3
93.4
534.7
2022.1
1759.7
14.0 42.3
34.0 90.7
72.0 181.3
203.0 303.3
* CAMP levels in response to lOnM, 50nM and
response to PGI , Three asymptomatic normal subjects also exhibit hyporesponsiveness t$ PGI,. Previous studies have shown that PG12 binds to platelet membrane receptors whereby it activates adenylate cyclase and causes cyclic AMP elevation (8-10). PGI receptor appears to be coupled to the guanine nucleotide regulatory bind&g proteins which regulate the adenylate cyclase activity (11). A reduced PG12 dose-response stimulation of platelet cyclic AMP may be due to defective receptors, the regulatory proteins or the enzyme, adenylate cyclase. Since the regulatory proteins and adenylate cyclase are ubiquitous and a defect in either probably will lead to multiorgan malfunction (12), we suspect that the defect in the 2 stroke patients and 3 normal subjects may be confined to the prostacyclin receptor. Further studies using direct stimulators of adenylate cyclase such as forskolin are needed to document that the defect is not at adenylate cyclase level. The high
672
PGIz RESPONSE ON STROKE
Vol.
45, No. 5
affinity binding sites for PGI on human platelet has a Kd of 12nM (8,9). Lack of a good dose-response to GI at concentrations ranging from lOnM-bOOnM would support the possibility that % hese individuals have a defect in the high affinity binding sites. Work is in progress to further characterize the defects by using more direct approaches such as ligand binding. Prostacyclin is considered to be an important autocoid which is produced and accumulated locally at vascular damage sites (13). It inhibits and reverses platelet aggregation via its interaction with its specific receptors and the consequential elevation of CAMP. Defective platelet response to PGI may lead to enhanced platelet aggregation and risk for thromboembolic d&orders such as thrombotic stroke. We recognize that the number of subjects included in this study is too small to draw any conclusion concerning the pathophysiologic significance of low PGI2 responsiveness. We plan to confirm the preliminary observation by studying a larger number of normal subjects and patients. We believe that
PQi2 (nM)
FIG. 1 Dose-response stimulation of platelet cyclic AMP by PGI2 in healthy donors and patients with thrombotic stroke. The closed circles denote the mean ?:S.D. of Closed triangles 10 healthy subjects who had a normal response to PGI2* denote the mean + S.D. of 7 stroke patients whose response to PGI were comparable to the normals. Open circles denote the healthy donors an?i open triangles the stroke patients with reduced responsiveness.
Vol. 45, No. 5
PG12 RESPONSE ON STROKE
673
assay of dose-response stimulation of CAMP wi:Llbe valuable for further understanding of the mechanism of platelet hyperaggregability and its role in thrombotic stroke.
ACKNOWLEDGEMENT The authors wish to thank Ms. Toni Rudersdorf for her excellent We are thankful to Ms. Nancy Fernandez for her technical assistance. secretarial work. The work is supported in part by a program project grant from National Institute of Health (NS-23327) and the Cullen Trust for Health Correspondence to K. K. Wu, M.D., Division of Care of Houston, Texas. Hematology, University of Texas Medical School, 6431 Fannin, Houston, Texas 77030.
REFERENCES 1.
WU, K.K., and HOAK, J.C. Increased platelet aggregates in patients with transient ischemic attacks. Stroke 6, 521-524, 1975.
2.
SHAH, A.B., BEAMER, N., COULL, B.M. Enhanced in vivo platelet activation in subtypes of ischemic stroke. ~Stroke, -16, 643-647, 1985.
3.
FRANCESCO, V., CESARE, A., LUIGI, I., ET AL. Malondialdehyde-like material and beta-thromboglobulin plasma levels in patients suffering from transient ischemic attacks. Stroke, 16, 14-16, 1985.
4.
STEWART, M.E., DOUGLAS, J.T., LOWE, G.D.O., ET AL. Prognostic value of beta-thromboglobulin in patients with transient cerebral ischemia. Lancet, 1, 479-482, 1983.
5.
DAVIS, H.H., II, SIEGEL, B.A., SHERMAN, L.A., ET AL. Scintigraphic detection of carotid atherosclerosis with Indium-111-labeled autologous platelets. Circulation, 61, 982-988, 1980.
6.
PAN, J., HALL, E.R., and WU, K.K. Alteration of platelet responses to metabolites of arachidonic acid by oral contraceptives. Brit. J. Haem., & 317-323, 1984.
7.
GORMAN, R.R., BUNTING, S., and MILLER, O.V. Modulation of human platelet adenylate cyclase by prostacyclin. Prostagl., 13, 377-388, 1977.
a.
SIEGL, A.M., SMITH, J.B., SI5VER, M.J. NICOLAOU, K.D., and AHERN, D. Selective binding site for [ H] prostacyclin on platelets. J. Clin. Invest., 63, 215-220, 1979.
9.
SCHAFER, A.I., COOPER, B., O'HARA, D., and HANDIN, R.I. Identification of platelet receptors for prostaglandins I2 and D2. J. Biol. Chem., 254, 2914-2917, 1979.
10.
TATESON, J.E., MONCADA, S., and VIZNE,J.R, Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostagl., 13, 389-397, 1977.
Guanine nucleotide-binding regulatory proteins and dual 11. GILMAN, A.G. control of adenylate cyclase. J. Clin. Inv&, )73 1-4, 1984.
674
PG12 RESPONSE ON STROKE
Vol. 45, No. 5
12. MOTULSKY, H.J. HUGHES, R.J., BRICKMAN, A.S., FARFEL, Z., BOURNE, H.R., and INSEL, P.A. Platelets of pseudo-hypoparathyroid patients: evidence that distinct receptor-cyclase coupling proteins mediate stimulation and inhibition of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A., 79, 4193-4197, 1982. 13.
PAPP, A.C., CROWE, L., PETTIGREW, L.C. and WU, K.K. Production of eicosanoids by deendothelialized rabbit aorta: Interaction between platelets and vascular wall in the synthesis of prostacyclin. Thromb. Res., 62, 549-556, 1986.
RESEARCH 45; 669-674, 1987 $3.00 t .OO Printed in the USA. (c) 1987 Pergamon Journals Ltd. All rights reserved.
0049-3848/87
Copyright
BRIEF COMMUNICATION DOSE-RELATED STIMULATION OF PLATELET CYCLIC ADENOSINE MONOPHOSPHATE BY PROSTACYCLIN IN THROMBOTIC STROKE
Creed Pettigrew, Audrey Papp, and Kenneth K. Wu Stroke and Thrombosis Research Center, Department of Internal Medicine, Division of Hematology and Department of Neurology, University of Texas Health Science Center at Houston Houston, TX 77225, U.S.A.
(Received 12.6.1986; Accepted in revised form 4.12.1986 by Editor J. Hawiger)
INTRODUCTION Platelet hyperaggregability is commonly observed in patients with thrombotic stroke (l-5). Evidence of platelet activation and hyperaggregability related to transient ischemic attacks and cerebral infarction includes increased platelet factor 4, B-thromboglobulin levels, shortened platelet survival time, adhesion of radiolabeled platelets to atherosclerotic carotid arteries, increased synthesis of malondialdehyde and thromboxane and accumulation of platelet aggregates. These data suggest a relationship between platelet activation and thrombotic stroke but have not convincingly established that a cause and effect relationship exists. The difficulty lies in part in a lack of understanding of the mechanisms by which platelet hyperaggregability occurs. In the present study, we proposed to test the hypothesis that a defective platelet responsiveness to prostacyclin may represent an important platelet inherent abnormality which attributes to increased platelet activity and thrombotic tendency.
PATIENTS AND METHODS Patients: Thirteen normals and 10 stroke patients were included in the study. The normal group included 8 women and 5 men of mean age 36.2 years
Key words:
prostacyclin, platelets, cyclic AMP, stroke, thrombosis 669
670
PGli2RESPONSE ON STROKE
(range 21-57). Three women and 7 men of mean age 58.3 years (range 38-74) comprised the patients. All were sampled between January of 1985 and February of 1986. Each underwent computerized tomography (CT) of the brain before entering the study. The suprasellar mass discovered on CT of one patient was judged not to have an effect on platelet function. Infarcts occurred in the carotid distribution in 9 patients (3 right and 6 left) and in the brainstem and left cerebellar hemisphere in one. Noninvasive carotid studies and echocardiography showed no potential sources of emboli. All blood samples were obtained from patients within two weeks of their strokes. One patient was taking 325mg of aspirin daily at the time of sampling. The normal donors had not taken any drugs known to inhibit platelet function for at least 10 days prior to the study. Platelet Response to Prostacyclin: We had chosen to study platelet responsiveness to PGI by measuring platelet cyclic AMP elevation in response to several concentrat?ons of PGI because thif4technique is more reliable and sensitive than the platelet agggegation or C-serotonin release procedures (6). Venous blood was withdrawn into a plastic tube containing l/10 vol. of 3.8% sodium citrate. The sample was centrifuged at 2QOxg for 10 minutes at 23°C. Platelet rich plasma 4PRP> was collected and its platelet concentration was standardized to 3 x 10 /ml with zqtologous PPP. PRP was treated with isobutylmethylxanthine (IBMX, 2 x 10 M) at 23°C for 30 minutes to block phosphodiesterase. 0.5ml PRP was placed in a dual-channel Chrono-Log aggregometer cuvette at 37'C for 1 minute followed by addition of authentic sodium prostacyclin (0,10,50, and 100&l) at 37°C for 90 seconds. The samples were under constant stirring throughout the period of study. The reaction was terminated by placing the samples into liquid nitrogen. The samples were stored at -7O'C until assay. To assay for cyclic AMP levels, the samples first underwent several steps of processing. They were thawed, sonicated with a cell disruptor (Heat Systems Ultrasonics, Inc., Plainview, N.Y.) and then placed in boiling water for 4 minutes. The samples were then centrifuged at 13,000xg for 5 minutes and the supernatants were collected and assayed for CAMP by radioimmunoassay using a commercial kit (Amersham, Arlington Heights, IL).
RESULTS AND DISCUSSION Gorman et al have previously shown a dose-response stimulation of platelet cyclic AMP by PGI in normal subjects (7). To evaluate individual variations in normal subjec?z s, we assayed platelet CAMP stimulated by several concentrations of PG12_ Our data revealed that the dose-response stimulation in 13 healthy subjects could be divided into 2 distinct subgroups. Ten had a normal dose-response stimulation while 3 were hyporesponsive (Table 1). The distinct difference in the dose-response curve is further illustrated in Fig. 1. Based on the results from 10 normal responders, the mean CAMP values stimulated by 10, 50 and 1OOnM of PGI were 257.3 ? S.D. 40.8 pmol (range 165.7-348.9 pmol), 707.1 c 129.0 pmol trange 449.1-965.1 pmol) and 1071.3 ? 259.8 pmol (range 551.7-1590.9 pmol) per 10' platelets respecti.vely. The dose-response stimulation of 7 stroke patients was within the normal range while 2 were hyporesponders and 1 had an increased sensitivity (Table 1). The mean value of the 7 normal responders was similar to the 10 normoresponsive healthy subjects (Fig. 1). The dose-response curves of the two hyporesponders were similar to the 3 healthy subjects with low responsiveness. The PG12 responsiveness in the patients and normals did not bear any relationship to basal CAMP values, sex or age of the subject. Using PGI2 dose-response stimulation of platelet cyclic AMP, we have
Vol. 45, No. 5
identified
a subset
671
PG12 RESPONSE ON STROKE
of patients
with
thrombotic
stroke who have a subnormal
TABLE 1 Dose-response Stimulation of Cyclic AMP by PGI, in Healthy Donors and Patients ,with Thrombotic Stroke Subjects Healthy Donors
Stroke Patients
No A--
Age
CAMP (pmol/lO' pl)
Sex
Normoresponsive 1 26 F 2 32 F 3 25 M 4 36 F 5 30 M 6 21 F 7 39 M 8 24 F 9 42 F 10 26 M Hyporesponsive 11 23 F 12 57 M 13 56 F Normoresponsive 1 67 M 2 60 M 3 62 M 4 73 M 5 74 F 6 63 F 7 38 M Hyper-responsive 8 44 F Hyporesponsive 9 64 M IO 49 M
§ Basal platelet CAMP level. 1OOnM of PG12 respectively.
09 37.3 76.0 64.7 44.7 46.7 42.8 61.3 36.0 31.3 29.3
lo* 210.0 224.7 299.3 292.0 273.3 317.1 260.0 276.7 206.7 213.3
50* 533.3 497.3 681.3 724.7 767.3 901.3 612.7 825.3 719.3 808.0
100* 771.8 636.7 953.3 909.3 1231.3 1330.3 1396.7 1089.3 1026.7 1367.3
2.9 12.0 74.0
56.0 158.0 164.7
145.5 242.7 144.5
157,8 136.0 289,5
7.8 77.3 94.7 32.0 12.6 94.2 52.2
239.2 258.7 137.3 217.3 166.6 182.1 144.9
749.0 712.0 447.3 564.0 586.0 712.4 1158.4
836.9 931.3 664.7 792.0 754.0 853.4 1435.3
93.4
534.7
2022.1
1759.7
14.0 42.3
34.0 90.7
72.0 181.3
203.0 303.3
* CAMP levels in response to lOnM, 50nM and
response to PGI , Three asymptomatic normal subjects also exhibit hyporesponsiveness t$ PGI,. Previous studies have shown that PG12 binds to platelet membrane receptors whereby it activates adenylate cyclase and causes cyclic AMP elevation (8-10). PGI receptor appears to be coupled to the guanine nucleotide regulatory bind&g proteins which regulate the adenylate cyclase activity (11). A reduced PG12 dose-response stimulation of platelet cyclic AMP may be due to defective receptors, the regulatory proteins or the enzyme, adenylate cyclase. Since the regulatory proteins and adenylate cyclase are ubiquitous and a defect in either probably will lead to multiorgan malfunction (12), we suspect that the defect in the 2 stroke patients and 3 normal subjects may be confined to the prostacyclin receptor. Further studies using direct stimulators of adenylate cyclase such as forskolin are needed to document that the defect is not at adenylate cyclase level. The high
672
PGIz RESPONSE ON STROKE
Vol.
45, No. 5
affinity binding sites for PGI on human platelet has a Kd of 12nM (8,9). Lack of a good dose-response to GI at concentrations ranging from lOnM-bOOnM would support the possibility that % hese individuals have a defect in the high affinity binding sites. Work is in progress to further characterize the defects by using more direct approaches such as ligand binding. Prostacyclin is considered to be an important autocoid which is produced and accumulated locally at vascular damage sites (13). It inhibits and reverses platelet aggregation via its interaction with its specific receptors and the consequential elevation of CAMP. Defective platelet response to PGI may lead to enhanced platelet aggregation and risk for thromboembolic d&orders such as thrombotic stroke. We recognize that the number of subjects included in this study is too small to draw any conclusion concerning the pathophysiologic significance of low PGI2 responsiveness. We plan to confirm the preliminary observation by studying a larger number of normal subjects and patients. We believe that
PQi2 (nM)
FIG. 1 Dose-response stimulation of platelet cyclic AMP by PGI2 in healthy donors and patients with thrombotic stroke. The closed circles denote the mean ?:S.D. of Closed triangles 10 healthy subjects who had a normal response to PGI2* denote the mean + S.D. of 7 stroke patients whose response to PGI were comparable to the normals. Open circles denote the healthy donors an?i open triangles the stroke patients with reduced responsiveness.
Vol. 45, No. 5
PG12 RESPONSE ON STROKE
673
assay of dose-response stimulation of CAMP wi:Llbe valuable for further understanding of the mechanism of platelet hyperaggregability and its role in thrombotic stroke.
ACKNOWLEDGEMENT The authors wish to thank Ms. Toni Rudersdorf for her excellent We are thankful to Ms. Nancy Fernandez for her technical assistance. secretarial work. The work is supported in part by a program project grant from National Institute of Health (NS-23327) and the Cullen Trust for Health Correspondence to K. K. Wu, M.D., Division of Care of Houston, Texas. Hematology, University of Texas Medical School, 6431 Fannin, Houston, Texas 77030.
REFERENCES 1.
WU, K.K., and HOAK, J.C. Increased platelet aggregates in patients with transient ischemic attacks. Stroke 6, 521-524, 1975.
2.
SHAH, A.B., BEAMER, N., COULL, B.M. Enhanced in vivo platelet activation in subtypes of ischemic stroke. ~Stroke, -16, 643-647, 1985.
3.
FRANCESCO, V., CESARE, A., LUIGI, I., ET AL. Malondialdehyde-like material and beta-thromboglobulin plasma levels in patients suffering from transient ischemic attacks. Stroke, 16, 14-16, 1985.
4.
STEWART, M.E., DOUGLAS, J.T., LOWE, G.D.O., ET AL. Prognostic value of beta-thromboglobulin in patients with transient cerebral ischemia. Lancet, 1, 479-482, 1983.
5.
DAVIS, H.H., II, SIEGEL, B.A., SHERMAN, L.A., ET AL. Scintigraphic detection of carotid atherosclerosis with Indium-111-labeled autologous platelets. Circulation, 61, 982-988, 1980.
6.
PAN, J., HALL, E.R., and WU, K.K. Alteration of platelet responses to metabolites of arachidonic acid by oral contraceptives. Brit. J. Haem., & 317-323, 1984.
7.
GORMAN, R.R., BUNTING, S., and MILLER, O.V. Modulation of human platelet adenylate cyclase by prostacyclin. Prostagl., 13, 377-388, 1977.
a.
SIEGL, A.M., SMITH, J.B., SI5VER, M.J. NICOLAOU, K.D., and AHERN, D. Selective binding site for [ H] prostacyclin on platelets. J. Clin. Invest., 63, 215-220, 1979.
9.
SCHAFER, A.I., COOPER, B., O'HARA, D., and HANDIN, R.I. Identification of platelet receptors for prostaglandins I2 and D2. J. Biol. Chem., 254, 2914-2917, 1979.
10.
TATESON, J.E., MONCADA, S., and VIZNE,J.R, Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostagl., 13, 389-397, 1977.
Guanine nucleotide-binding regulatory proteins and dual 11. GILMAN, A.G. control of adenylate cyclase. J. Clin. Inv&, )73 1-4, 1984.
674
PG12 RESPONSE ON STROKE
Vol. 45, No. 5
12. MOTULSKY, H.J. HUGHES, R.J., BRICKMAN, A.S., FARFEL, Z., BOURNE, H.R., and INSEL, P.A. Platelets of pseudo-hypoparathyroid patients: evidence that distinct receptor-cyclase coupling proteins mediate stimulation and inhibition of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A., 79, 4193-4197, 1982. 13.
PAPP, A.C., CROWE, L., PETTIGREW, L.C. and WU, K.K. Production of eicosanoids by deendothelialized rabbit aorta: Interaction between platelets and vascular wall in the synthesis of prostacyclin. Thromb. Res., 62, 549-556, 1986.