INFLUENCE OF THE - ADRENOCEPTOR
ANTAGONISTS NAFTOPIDIL AND DOXAZOSIN
ON HUMAN PLATELET FUNCTIONS IN VITRO
by
NAJAH AL-ARAYYED
A THESIS PRESENTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
of the
UNIVERSITY OF LONDON
1995
DEPARTMENT OF MEDICINE DIVISION OF TOXICOLOGY AND PHARMACOLOGY UNIVERSITY COLLEGE AND MIDDLESEX SCHOOL OF MEDICINE ProQuest Number: 10017365
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT
The enhanced platelet activity in hypertension has been suggested to contribute to the increased risk of cardiovascular disease in this condition. Various antihypertensive drugs have been examined both in vitro and in vivo for their ability to inhibit platelet activation.
Antihypertensive drugs which possess selective a^-adrenoceptor blocking activity, e.g. prazosin, doxazosin and urapidil, have been found to inhibit platelet aggregation in some studies. Naftopidil is a new a^-adrenoceptor blocker but its effects on platelet function have not yet been studied. The main objective of the present study, therefore, was to study the effects of naftopidil in comparison with doxazosin on human platelet aggregation and secretory responses in vitro. The effects of the calcium channel blocker nifedipine were also investigated for comparative purposes.
When platelet aggregation was induced by individual agonists, naftopidil but not doxazosin caused slight inhibitions of adrenaline- and serotonin- induced aggregation.
Both drugs, however, failed to inhibit aggregation induced by ADP and collagen.
Moreover, naftopidil markedly and, to a lesser extent, doxazosin inhibited the aggregation induced by sub-threshold concentrations of adrenaline in combination with sub-threshold concentrations of ADP, collagen or serotonin. In a washed platelet system naftopidil and, to a lesser extent, doxazosin inhibited adrenaline-induced aggregation.
In the same system, naftopidil inhibited partially collagen-induced platelet aggregation.
Naftopidil and doxazosin also inhibited the release of the a-granular component, platelet-derived growth factor, induced by adrenaline but not that induced by collagen.
— In the presence of naftopidil and doxazosin apparent increases in platelet serotonin release were observed possibly indicating that these drugs block the uptake mechanism for serotonin in platelets. Nifedipine was found to inhibit collagen-induced platelet aggregation and release of serotonin but it did not inhibit the release of PDGF.
In order to clarify the modes of action of naftopidil, doxazosin and nifedipine as inhibitors of platelet activation, their effects on platelet signal transduction mechanisms were studied in washed platelets. Naftopidil and doxazosin inhibited the mobilization of intra platelet calcium induced by collagen and adrenaline. Naftopidil but not doxazosin inhibited adrenaline- and coUagen-induced TXB 2 generation. The platelet cyclic AMP, which mediates platelet inhibition, was not affected by either naftopidil or doxazosin.
However, both drugs prevented the decline in platelet cyclic AMP stimulated by adrenaline. In comparison with naftopidil and doxazosin, nifedipine produced a greater inhibition of coUagen-induced calcium mobilization and also inhibited the collagen- induced TXB2 production, comparable in its extent to that produced by naftopidil.
Overall, it is concluded that naftopidil and, to a lesser extent, doxazosin inhibit platelet activation, possibly through antagonistic actions on platelet « 2-adrenoceptors and suppressant effects on stimulus-induced calcium mobilization. Experiments with specific (%2-adrenoceptor antagonists will be necessary to establish this mode of action.
Moreover, the inhibitory action of naftopidil on TXA 2 generation may contribute to its marked inhibition of adrenaline-induced platelet activation which is believed to be dependent on the formation of cyclooxygenase metabolites.
- 3 - Table of Contents
Abstract...... 2
Table of Contents ...... 4
Acknowledgments ...... 15
Abbreviations ...... 16
List of Figures ...... 17
List of Tables ...... 21
Chapter 1 General Introduction ...... 25 1.1. General features of platelet anatomy and the related activities .... 26 1.1.1. The peripheral zone ...... 26 1.1.1.1. Plasma membrane...... 26 1.1.1.2. Glycocalyx ...... 29 1.1.1.3. Submembrane area (membrane skeleton) ...... 29 1.1.1.4. The surface-connected open canalicular system ...... 29
1.1.2. Cytoplasmic matrix ...... 30 1.1.2.1. Microfilaments (actin-containing filaments) ...... 30 1.1.2.2. Microtubules (tubulin system) ...... 31 1.1.3. Cytoplasmic organelles ...... 31 1.1.3.1. Mitochondria ...... 32 1.1.3.2. «-granules (protein storage granules) ...... 32 1.1.3.3. Dense granules (amines storage granules) ...... 33 1.1.3.4. Lysosomes and peroxisomes ...... 33 1.1.4. Dense tubular system (membrane system) ...... 34
— Table of Contents
1.2. Physiological platelet stimulatory agonists and their receptors .... 35 1.2.1. . Catecholamines ...... 35 1.2.2. Collagen ...... 36 1.2.3. Adenosine 5 - Diphosphate (A D P ) ...... 36 1.2.4. Vasopressin . . . .' ...... 37 1.2.5. Serotonin (5-hydroxytryptamine, 5-H T ) ...... 37 1.2.6. Thrombin ...... 37
1.2.7. Thromboxane A 2 (TxAj) and prostaglandin H 2 (PGH2) ...... 38 1.2.8. Platelet-activating factor (PAF) ...... 38 1.3.^ Signal transduction mechanisms involved in the regulation of platelet function ...... 39 1.3.1. Phospholipasè C and the phosphoinositide pathway ...... 39 1.3.1.1. Inositol 1, 4, 5-trisphosphate (IP 3) and Calcium Mobilization .... 41 1.3.1.2. Sn-1, 2-DAG and PKC activation ...... 42 1.3.2. Phospholipasè A 2 and the arachidonic acid pathway ...... 42 1.3.3. Regulation of cAMP formation ...... 45
1.4. Role of platelet aggregatory and procoagulant activities in haemostasis and thrombosis ...... 46
1.5. In vitro methods used to assess potential antiplatelet effects of drugs ...... 48 1.5.1. Isolation of human platelets from whole blood for in vitro studies of platelet function ...... 48 1.5.2. Platelet aggregation in platelet-rich plasma ...... 49 1.5.3. Platelet aggregation in whole blood ...... 50 1.5.4. Platelet adhesion ...... 50 1.5.5. Platelet release reaction ...... 51 1.5.6. Platelet biochemical indices ...... 51
- 5 - Table o f Contents
1.6. Involvement of platelets in primary hypertension and coronary heart disease ...... 52 1.6.1. Definition of primary hypertension ...... 52 1.6.2. Mechanism of primary hypertension ...... 53 1.6.3. Disturbances of platelet ion transport as observed in primary hypertension ...... 54 1.6.3 .1. Platelet sodium content and transport in hypertension 54 1.6.3.2. Platelet calcium content and transport in hypertension ...... 55 1.6.4. Platelet serotonin and hypertension ...... 56 1.6.5. Platelets in coronary heart disease (CHD) ...... 57 1.6.5.1. Pathogenesis of CH D ...... 57 1.6.5.2. Platelet hyperactivity in C H D ...... 58
1.7. Antihypertensive treatment and its effects on platelet aggregation. 59 1.7.1. Antihypertensive drugs acting on the sympathetic nervous system . 60 1.7.1.1. a2”^^renoceptor agonists ...... 61 Antihypertensive effects ...... 61 Anti-platelet effects ...... 61 1.7.1.2. tti-adrenoceptorantagonists ...... 62 Anti-hypertensive effects ...... 62 Anti-platelet effects ...... 63 1.7.1.3. p-adrenoceptor antagonists ...... 64 Anti-hypertensive effects ...... 64 Anti-platelet effects ...... 65 1.7.2. Calcium antagonists ...... 65 Anti-hypertensive effects ...... 66 Anti-platelet effects ...... 66 1.7.3. Angiotensin converting Enzyme (ACE) inhibitors ...... 67 Anti-hypertensive effects ...... 67 Anti-platelet effects ...... 68
- 6 - Table of Contents
1.8. The purpose of the study ...... 69
Chapter 2 Materials and Methods ...... 70
2.1. Materials ...... 71 2.1.1. Platelet activators ...... 71 2.1.2. Vasoactive peptides ...... 71 2.1.3. D rugs ...... 71
2.2. Methods ...... 72 2.2.1. Preparation of platelet-rich plasma from b lo o d ...... 72 2.2.2. Preparation of washed and aequorin-loaded platelets ...... 72 2.2.3. Preparation of the fibrinogen solution ...... 74 2.2.4. Platelet aggregation in PRP ...... 74 2.2.5. Calcium mobilization and platelet aggregation in aequorin -loaded platelets ...... 74 2.2.6. Serotonin measurement ...... 75 2.2.7. cAMP Extraction and measurement ...... 75 2 .2 .8 . Measurement of thromboxane B 2 (TXB2) ...... 76 2.2.9. Measurement of platelet derived growth factor (PD GF) ...... 77
2.3. Statistical Analysis ...... 79
Chapter 3 A Study of Some Factors Influencing the Stability of in vitro Platelet Responses...... 80
3.1. Introduction ...... 81
3.2. Aim of the Study ...... 81
- 7 - Table of Contents
3.3. Study Design ...... 82 3.3.1 Storage of PRP samples in the absence or presence of a ir ...... 82 3.3.2. Storage ofPRP samples at 4°C, 13°C,22°C and 37°C ...... 82 3.3.3. PRP dilution ...... 82 3.3.4. Serotonin (5-HT) determination ...... 83
3.4. Results...... 84 3 .4.1. The effect of the absence or presence of air during PRP storage on pH and platelet aggregation ...... 84 3.4.2. The effect of storage temperature on platelet aggregation ...... 86 3.4.3. The effect of PRP dilution on platelet aggregation ...... 89 3.4.3.1. Platelet sensitivity and platelet count ...... 90 3.4.3.2. Storage time and platelet aggregation in diluted and undiluted PRP ...... 93 3.4.4. 5-HT measurement...... 94
3.5. Discussion ...... 99
3.6. Conclusions ...... 102
Chapter 4 A study of the effects of naftopidil and doxazosin on In vitro platelet aggregation induced by single agonists ...... 103
4.1. Introduction ...... 104
4.2. Aim of the study ...... 105
4.3. Study design ...... 106
4.4. Results...... 108 4.4.1. The effects of naftopidil and doxazosin on adrenaline-induced
- 8 - Table of Contents
platelet aggregation ...... 108 4.4.2. The effects of naftopidil and doxazosin on ADP- and collagen-induced platelet aggregation ...... 108 4.4.3 The effects of naftopidil and doxazosin on 5-HT-induced platelet aggregation ...... 108
4.5. Discussion ...... 114 Naftopidil and platelet aggregation ...... 114 Doxazosin and platelet aggregation ...... 115 The dose-response curve approach to studying anti-platelet actions of drugs ...... 115 The relevance of the platelet agonists tested to hypertension-related cardiovascular disease ...... 116
4.6. Conclusions ...... 117
Chapter 5 A Study of the effects of naftopidil and doxazosin on in vitro platelet aggregation induced by synergistic interactions between adrenaline and other agonists ...... 118
5.1. Introduction ...... 119 5.1.1. Endothelin ...... 120 5.1.2. Neuropeptide Y (NPY) ...... 122 5.1.3. Atrial natriuretic peptide (A N P ) ...... 123
5.2. Aim of the Study ...... 125
5.3. Study Design ...... 125
- 9 - Table of Contents
5.4. Results...... 127 5.4.1. The effects of ADP, collagen and 5-HT on adrenaline-induced platelet aggregation ...... 127 5.4.2. The effects of naftopidil and doxazosin on the potentiation by ADP, collagen and 5-HT of adrenaline-induced platelet aggregation...... 127 5.4.3. The effects of the vasoactive peptides endothelin, NPY and ANP on adrenaline-induced platelet aggregation ...... 133
5.5. Discussion ...... 135 5.5.1. The potentiation by 5-HT, collagen and ADP of adrenaline-induced aggregation ...... 135 5.5.2. Utilization of combinations of platelet agonists in drug studies . . 136 5.5.3. The inhibition by naftopidil and doxazosin of the potentiated responses to adrenaline ...... 136 5.5.4. The vasoactive peptides and platelet aggregation ...... 137 Endothelin ...... 137 NPY ...... 138 ANP ...... 138
5.6. Conclusions ...... 139
CHAPTER 6 A study of the effects of naftopidil and doxazosin in comparison with nifedipine on platelet signal transduction mechanisms ...... 140
6.1. Effects of naftopidil, doxazosin and nifedipine on stimulus-induced calcium mobilization ...... 141
- 1 0 - Table or Contents
6.1.1. Introduction ...... 141 Calcium indicators ...... 141 Calcium mobilization ...... 142 Ca^+ influx ...... 142 6.1.2. Aim of the Study ...... 144 6.1.3. Study design ...... 144 6.1.4. Results...... 146 6.1.4.1. Collagen- and adrenaline-induced increase in [Ca“^]j and platelet aggregation ...... 146 6.1.4.2. The effect of naftopidil on the collagen- and adrenaline-induced increase in [Ca^^]j and platelet aggregation ...... 146 6.1.4.3. The effect of doxazosin on the collagen- and adrenaline-induced increase in [Ca^^Jj and platelet aggregation ...... 151 6.1.4.4. The effect of nifedipine on the collagen-induced increase in [Ca^"^]i and platelet aggregation ...... 153 6.1.5. Discussion ...... 154 Adrenaline-induced responses in washed platelets . . ! ...... 154 Inhibition of adrenaline-induced responses by naftopidil and doxazosin ...... 155 Inhibition of collagen-induced responses by naftopidil and doxazosin ...... 156 Inhibition of collagen-induced responses by nifedipine ...... 156
6.2. Effects of naftopidil and doxazosin on stimulus-induced thromboxane Aj generation ...... 158 6.2.1. Introduction ...... 158 6.2.2. Aim of the Study ...... 159 6.2.3. Study Design ...... 159 6.2.4. Results...... 160 6.2.4.1. TxB2 generation and platelet aggregation induced by collagen and adrenaline ...... 160
__ Table o f Contents
6.2.4.2. The effect of naftopidil on the adrenaline- and collagen-induced generation of TxB 2 ...... 160 6.2.4.3. The effect of doxazosin on adrenaline- and collagen-induced generation of TxB 2 ...... 161 6.2.4.4. The effect of nifedipine on the collagen-induced generation of TxB 2 ...... 161 6.2.5. Discussion ...... 163 Effects of naftopidil and doxazosin on the adrenaline-induced
generation of TxA . 2 ...... 164 Effects of naftopidil, doxazosin and nifedipine on the
collagen-induced generation of TxA 2 ...... 164
6.3. Effect of naftopidil and doxazosin on platelet cAMP ...... 165 6.3.1. Introduction ...... 165 6.3.2. Aim of Study ...... 165 6.3.3. Study Design ...... 165 6.3.4. Results...... 166 6.3.5. Discussion ...... 168
6.4. Conclusions ...... 169
CHAPTER 7 A study of the effects of naftopidil, doxazosin and nifedipine on stimulus-induced platelet granular release ...... 170
7.1. Introduction ...... 171 Uptake and release of 5-HT by platelets ...... 173 Specificity of the platelet 5-HT transporter ...... 174 Platelet storage compartments of 5-HT ...... 175 PDGF- origin and release by platelets ...... 175 Inhibition of the platelet release reaction ...... 176
- 12 - Table o f Contents
7.2. Aim of the study ...... 177
7.3. Study design ...... 177 7.4. Results...... 178 7.4.1. 5-HT release induced by collagen and adrenaline ...... 178 7.4.2. The effects of naftopidil on collagen- and adrenaline-induced 5-HT release...... 178 7.4.3. Effect of doxazosin on collagen- and adrenaline-induced 5-HT release...... 180 7.4.4. Effect of nifedipine on collagen-induced 5-HT release ...... 181 7.4.5. PDGF release induced by collagen and adrenaline ...... 183 7.4.6. Effect of naftopidil on collagen and adrenaline-induced release of PD G F...... 183 7.4.7. Effect of doxazosin on collagen- and adrenaline-induced release of PD G F ...... 184 7.4.8. Effect of nifedipine on collagen-induced release of PDGF 184
7.5. Discussion ...... 186 Drug effects on collagen-induced 5-HT release ...... 186 Influence of naftopidil and doxazosin on adrenaline-induced 5-HT release...... 187 Hypothetical mechanism for adrenaline-induced 5-HT release and platelet aggregation ...... 188 Inhibition of PDGF release ...... 189
7.6. Conclusions ...... 190
- 1 3 - Table of Contents
Chapter 8 General discussion and concluding remarks ...... 192
8.1. Stability of in vitro platelet responses ...... 193
8.2. The effect of the vasoactive peptides on adrenaline-induced platelet aggregation ...... 194 8.3. Comparative studies on the effects of naftopidil, doxazosin and nifedipine on platelet aggregation ...... 194
8.4. Plasma protein binding and the masking of drug effects ...... 195
8.5. Influence of drug vehicles on platelet aggregation ...... 196
8 .6. Effects of naftopidil and doxazosin in comparison with nifedipine on 5- HT release ...... 196 8.7. Comparative studies of the effects of naftopidil, doxazosin and nifedipine on platelet signal transduction mechanisms ...... 197
8 .8 . Effect of naftopidil and doxazosin on a-granular release ...... 198
8.9. Summary ...... 198
Publications Pertaining to the thesis ...... 201
Appendix; Tables ...... 202
References...... 234
- 1 4 - Acknowledgments
I am exceedingly grateful to Professor Brian Prichard and Dr. Christopher Smith for their continued advice, encouragement and guidance in carrying out this research, and for their most valuable and constructive comments on the thesis. I also thank Dr.
Christopher Smith for performing the 5-hydroxytryptamine measurements which are reported in two of the studies.
I am most grateful to Dr. Mike Cooper for his skilful advice during these research activities, especially during the conduct of the studies of calcium mobilization in platelets.
I am greatly indebted to Mr. Barrie Graham for his kind friendship and for his help in the preparation of figures, and in the analysis of the data generated by these studies.
I am very grateful to Miss Elaine Samuel who typed the earlier version of the thesis, and to my brother, Afif, who provided continued help in completing the final version.
I am grateful to Asta Pharma (Frankfurt, Germany) and Pfizer Ltd. (Sandwich,
UK) for gifts of supplies of Nafl;opidil and Doxazosin, and also for their support to the
Department.
Finally, I thank the Bahrain Ministry of Health for the financial support.
- 1 5 - Abbreviations
P“TG p-thromboglobulin 5-HT 5-hydroxytryptamine, serotonin ADP Adenosine 5- diphosphate ANP Atrial natriuretic peptide ATP Adenosine triphosphate cAMP Cyclic adenosine mono phosphate CHD Coronary heart disease DAG Diacylglycerol DMSO Dimethylsulphoxide
EC50 Elective concentration (interpolated from concentration- response curve) producing 50% response ET Endothelin GP Glycoprotein
IC50 Inhibitory concentration (interpolated from concentration- response curve ) producing 50% inhibition NPY Neuropeptide Y PAF Platelet activating factor PDGF Platelet derived growth factor PG Prostaglandin
PLA2 Phospholipasè A 2 PPP Platelet poor plasma PRP Platelet rich plasma
T1A2 Thromboxane A 2
TxBj Thromboxane B 2 Intracellular calcium ion concentration
1 6 - List of figures
List of Figures
Figure Page 1.1 Diagram summarizing ultrastructural features observed in thin sections of discoid human platelet ...... 28
1.2 Pathways for hydrolysis and resynthesis of phosphatidyl inositol 4, 5-biphosphate in platelets ...... 40
1.3 Arachidonic acid release by phospholipasè A 2 and its metabolism by cyclo-oxygenase and lipo-oxygenase enzymes 44 in platelets ......
3.1 Platelet aggregation induced by ADP threshold concentrations in PRP stored in the presence and absence of air ...... 85
3.2 Platelet aggregation induced by adrenaline threshold concentrations in PRP stored in the presence and absence of air...... 85
3.3 Effect of temperature on platelet aggregation induced in PRP by threshold concentrations of ADP and adrenaline ...... 88
3.4 Dose-response curves for platelet aggregation induced by ADP and adrenaline in diluted and undiluted PRP samples. .. 91
3.5 Correlations between the EC 50 values for ADP and adrenaline in diluted and undiluted PRP samples ...... 92
3.6 Effect of time of PRP storage on platelet aggregation induced by threshold concentrations of ADP and adrenaline in diluted and undiluted PRP...... 95
3.7 Potentiation by adrenaline of platelet aggregation induced by ADP in diluted and undiluted PRP after 24 hours of PRP storage ...... 97
- 1 7 - List o f figures
3.8 Traces showing time-dependent changes in platelet aggregation induced by ADP and adrenaline in diluted PRP . . 98
4.1 Effect of naftopidil and doxazosin on adrenaline-induced platelet aggregation in PRP ...... 110
4.2 Effect of naftopidil and doxazosin on collagen- induced platelet aggregation in PRP ...... I ll
4.3 Effect of naftopidil and doxazosin on ADP-induced platelet aggregation in PRP ...... 112
4.4 Effect of naftopidil and doxazosin on 5-HT-induced platelet aggregation in PRP ...... 113
5.1 Potentiation by ADP, 5-HT and collagen of adrenaline- induced platelet aggregation in PRP ...... 128
5.2 Effect of naftopidil and doxazosin on potentiation by ADP of adrenaline-induced platelet aggregation in PRP ...... 129
5.3 Effect of naftopidil and doxazosin on potentiation by collagen of adrenaline-induced platelet aggregation in P R P ...... 130
5.4 Effect of naftopidil and doxazosin on potentiation by 5-HT of adrenaline-induced platelet aggregation in PRP ...... 131
5.5 Effect of endothelin on adrenaline-induced platelet aggregation in P R P ...... 133
5.6 Effect of NPY on adrenaline-induced platelet aggregation in PR P...... 134
5.7 Effect of ANP on adrenaline-induced platelet aggregation in PR P...... 134
6.1 A tracing showing a collagen-induced aequorin calcium signal in washed platelets ...... 148
- 1 8 - List o f figures
6.2 A tracing showing an adrenaline-induced aequorin calcium signal in washed platelets ...... 148
6.3 Effect of naftopidil on the collagen-induced increase in platelet [Ca^^Ji and platelet aggregation in washed platelets ...... 149
6.4 Effect of naftopidil on the adrenaline-induced increase in platelet [Ca^^Jj and platelet aggregation in washed platelets . . 149
6.5 Inhibitory concentration-response curve of the adrenaline- induced rise in platelet [Ca^^], in washed platelets ...... 150
6.6 Inhibitory concentration-response curve of the adrenaline- induced platelet aggregation in washed platelets ...... 150
6.7 Effect of doxazosin on the collagen-induced increase in platelet and platelet aggregation in washed platelets . . 152
6.8 Effects of doxazosin on the adrenaline-induced increase in platelet [Ca^^Jj and platelet aggregation in washed platelets .. 152
6.9 Effect of nifedipine on the collagen-induced increase in platelet [Ca^^]i and platelet aggregation in washed platelets ...... 153
6.1 CD Effect of naftopidil on the adrenaline-induced generation of plateletTxB2 in washed platelets ...... 162
6.11 Effect of doxazosin on the adrenaline-induced generation of plateletTxBz in washed platelets ...... 162
6.12 Effect of naftopidil and doxazosin on platelet basal cAMP levels ...... 167
6.13 Effect of naftopidil and doxazosin on the adrenaline-induced decline in platelet cAMP ...... 167
7.1 Effect of naftopidil on the collagen-induced release of platelet 5-HT ...... 179
- 19 List o f figures
7.2 Effect of naftopidil on the adrenaline-induced release of platelet 5-H T ...... 179
7.3 Effect of doxazosin on the collagen-induced release of platelet 5-H T ...... 181
7.4 Effect of doxazosin on the adrenaline-induced release of platelet 5-H T ...... 181
7.5 Effect of nifedipine on the collagen-induced release of platelet 5-H T ...... 182
7.6 Effect of naftopidil on the adrenaline-induced release of platelet P D G F ...... 185
7.7 Effect of doxazosin on the adrenaline-induced release of platelet P D G F ......
- 2 0 - List of Tables
List of Tables
Table Page 3.1 EC50 values and threshold concentrations for ADP and adrenaline in diluted and undiluted P R P ...... 89
3.2 The declines with time in responses to the ADP EC 50 in diluted and undiluted P R P ...... 96
3.3 The declines with time in responses to the adrenaline EC 50 in diluted and undiluted P R P ...... 96
4.1 Effect of naftopidil and doxazosin on EC 50 values for adrenaline, collagen and ADP ...... 109
5.1 % inhibition by naftopidil and doxazosin of the aggregatory responses to adrenaline potentiated by 5-HT, collagen and ADP 132
1 Effect of absence or presence of air in diluted PRP of platelet aggregation induced by the ADP threshold concentration 203 2 Effect of absence or presence of air in diluted PRP of platelet aggregation induced by the adrenaline threshold concentration . 203
3 Effect of storage of PRP at 4°C, 13°C and 22°C on platelet aggregation induced by the ADP threshold concentration .... 204
4 Effect of storage of PRP at 4°C, 13°C and 22°C on platelet aggregation induced by the adrenaline threshold concentration . 204
5 Effect of storage of PRP at 22°C and 37°C on platelet 205 aggregation induced by the adrenaline threshold concentration .
6 Effect of storage of PRP at 22°C and 37°C on platelet aggregation induced by the adrenaline threshold concentration . 205
- 2 1 - List of Tables
7 Platelet aggregation induced by the ADP threshold concentration in diluted and undiluted PRP ...... 206
8 Platelet aggregation induced by the adrenaline threshold concentration in diluted and undiluted PRP ...... 206
9 Changes with time of platelet aggregation induced by the ADP threshold concentration in diluted and undiluted PR P ...... 207
10 Changes with time of platelet aggregation induced by the adrenaline threshold concentration in diluted and undiluted PRP 207
11 Potentiation by adrenaline of ADP-induced platelet aggregation after 24 hours storage of PRP ...... 208
12 Effects of naftopidil and doxazosin on adrenaline-induced platelet aggregation ...... 209
13 Effect of naftopidil and doxazosin on collagen-induced platelet aggregation ...... 211
14 Effect of naftopidil and doxazosin on ADP-induced platelet aggregation ...... 212
15 Effect of naftopidil on serotonin-induced platelet aggregation. . 213
16 Effect of doxazosin on serotonin-induced platelet aggregation. 213
17 Potentiation by serotonin, ADP or collagen of adrenaline- induced platelet aggregation ...... 213
18 Effect of naftopidil and doxazosin on the potentiation by ADP of adrenaline-induced platelet aggregation in P R P ...... 214
19 Effect of naftopidil and doxazosin on the potentiation by collagen of adrenaline-induced platelet aggregation in PRP ... 215
- 2 2 - List of Tables
20 Effect of naftopidil and doxazosin on the potentiation by serotonin of adrenaline-induced platelet aggregation in PRP ... 216
21 Effect of endothelin on adrenaline-induced platelet aggregation 217
22 Effect of neuropeptide Y on adrenaline-induced platelet aggregation ...... 217
23 Effect of atrial natriuretic peptide on adrenaline-induced platelet aggregation ...... 218
24 Effect of naftopidil on collagen-induced [Ca^^]j increase and platelet aggregation in washed platelets ...... 219
25 Effect of naftopidil on adrenaline-induced [Ca^^]j increase and platelet aggregation ...... 220
26 % Inhibition by naftopidil of adrenaline- induced [Ca^^Ji increase and platelet aggregation in washed platelets ...... 221
27 Effect of doxazosin on collagen-induced [Ca^^]^ increase and platelet aggregation in washed platelets ...... 222
28 Effect of doxazosin on adrenaline-induced [Ca^^]j increase and platelet aggregation ...... 223
29 Effect of nifedipine on collagen-induced [Ca^^]j increase and platelet aggregation ...... 224
30 Effect of naftopidil and doxazosin on adrenaline-induced TXA 2 release and platelet aggregation ...... 225
31 Effect of naftopidil on collagen-induced TXA 2 release ...... 225
32 Effect of doxazosin on collagen-induced TXA 2 release 226
33 Effect of nifedipine on collagen-induced TXA 2 release ...... 226
- 2 3 - List o f Tables
34 Effect of naftopidil and doxazosin on basal levels of cAMP in washed platelets ...... 226
35 Effect of naftopidil and doxazosin on cAMP level in presence of adrenaline ...... 227
36 Effect of naftopidil on collagen-induced platelet serotonin release in washed platelets ...... 227
37 Effect of naftopidil on adrenaline-induced platelet serotonin release in washed platelets ...... 228
38 Effect of doxazosin on collagen-induced platelet serotonin release in washed platelets ...... 228
39 Effect of doxazosin on adrenaline-induced platelet serotonin release in washed platelets ...... 229
40 Effect of nifedipine on collagen-induced platelet serotonin release in washed platelet ...... 230
41 Effect of naftopidil on collagen-induced PDGF release in washed platelets ...... 230
42 Effect of naftopidil on adrenaline-induced PDGF release in washed platelets ...... 231
43 Effect of doxazosin on collagen-induced PDGF release in washed platelets ...... 232
44 Effect of doxazosin on adrenaline-induced PDGF release in washed platelets ...... 232
45...... Effect of nifedipine on coUagen-induced PDGF release ...... 233
- 2 4 - Chapter 1
General Introduction
- 2 5 - Chapter 1 : General Introduction
1.1. General features of platelet anatomy and the related activities Platelets are anucleate cells derived from bone marrow megakaryocytes. They circulate in blood together with the molecules of the plasmatic coagulation system in an inactive state but become activated at sites of vessel injury to play their primary physiological role in maintaining haemostasis. Platelets in the inactive state have a characteristic discoid shape. Four anatomical features consisting of peripheral zone, cytoplasmic matrix, organelles and dense tubular system can be distinguished in a thin section of a platelet (White 1992) (figure. 1.1).
1.1.1. The peripheral zone
This zone consists of a typical plasma membrane unit covered by an exterior coat
(glycocalyx) and underlayed by a cytoplasmic layer that contains a relatively regular system of filamentous elements (Submembrane area). The membrane and its covering invaginate at some sites into the cytoplasm to form a network of the surface-connected open canalicular system.
1.1.1.1. Plasma membrane
The platelet plasma membrane is composed of phospholipids, cholesterol, glycolipids, proteins and glycoproteins. Two types of phospholipids have been identified in the membrane: choline phospholipids (phosphatidyl choline and sphingomyelin) and acidic phospholipids (phosphatidylethanolamine, phosphatidyl serine and phosphatidyl inositol) (Schick 1994). These phospholipids are arranged in bilayer with their hydrophilic polar groups oriented towards the external and internal cytoplasmic sides of the bilayer and with their lipophilic acyl groups facing each other. The distribution of
- 26 - Chapter 1 : General Introduction phospholipids in the membrane is asymmetrical as the acidic phospholipids are primarily located in the inner layer and the choline phospholipids in the outer layer of the plasma membrane (Schick 1994).
Phospholipids are important functionally and their fatty acid compositions have a major influence on membrane-mediated biological activities such as platelet coagulant activities and the production of eicosanoids. The sequestration of acidic phospholipids within the inner layer of the plasma membrane minimizes the coagulant activity of unstimulated platelets, whereas the location of phosphatidyl choline in the outer leaflet of the plasma membrane helps in the exchange of fatty acids between platelets and plasma.
Free cholesterol is the predominant neutral lipid in the platelet plasma membrane.
Because platelets are anucleate they cannot synthesize cholesterol and therefore renew their cholesterol content via an exchange mechanism with plasma cholesterol (Schick
1994). The sterol composition of the platelet plasma membrane influences the fluidity and physiological activities of platelets.
Glycolipids participate in membrane-mediated activities and act as co-factors or auxiliary receptors in signal transduction. Acidic glycolipids (gangliosides) can bind serotonin but the extent and relevance of this binding to platelet function are not known
(Schick 1994).
A variety of proteins and glycoproteins are wholly or partially embedded in the phospholipid bilayer and are held in this state by interaction between the lipids and hydrophobic domains of the proteins.
- 2 7 - Chapter 1 : (General Introduction
Figure 1.1 A diagram summanzing ultrastructural features observed in thin sections of discoid human platelets cut in the equatonai plane (left) or in cross section (nght) (Wliite 1994).
Microtubules Surface-connected canalicular system.
/ Dense tubular system
Glycogen
Dense aranules
-granule
Mitochcndrion
Glycocalyx
Submembrane filaments
Plasma membrane
- 2 8 - Chapter 1 : General Introduction
1.1.1.2. Glycocalyx
Many membrane proteins and glycoproteins, with their multi branched
carbohydrate moieties, extend into the plasmatic environment 20-50nm from the outer
surface of the lipid bilayer to form the ‘glycocalyx’ layer that covers the membrane.
These proteins and glycoproteins serve as receptors involved in mediating different
aspects of platelet function.
1.1.1.3. Submembrane area (membrane skeleton)
The submembrane area is a spectrin-rich network containing actin filaments that
are connected with the plasma membrane through a number of membrane-associated proteins (Hartwig and DeSisto 1991). This area forms a barrier between the organelles
inside the matrix and the cell wall, and actin filaments may cooperate with
circumferential microtubules to maintain platelet discoid shape and play a role in
pseudopod formation (White 1992).
1.1.1.4. The surface-connected open canalicular system
The surface-connected open canalicular system is formed by invagination of the
cell membrane which permeates the entire cytoplasm forming a network of interconnecting channels, thereby providing the means for plasma substances to reach the cell interior and reaction products to reach the cell exterior. Moreover, the canalicular membranes may contribute to the increase in platelet surface area occurring
during the process of shape change (White and Escolar 1993).
- 2 9 - Chapter I : General Introduction
1.1.2. Cytoplasmic matrix
The platelet cytoplasmic matrix contains at least two systems of fibres including microfilaments and microtubules. Glycogen is distributed in the matrix as both masses and discrete particles.
The cytoplasmic matrix is sometimes termed the ‘sol-gel zone’ which describes its gel consistency. The term ‘cytoskeleton’ is frequently used to describe the detergent- resistant elements of the matrix (Tuszynski et al, 1985) and to define its role in supporting the discoid shape of inactivated platelets and in accomplishing the shape change which occurs with platelet activation (Nachmias 1983).
1.1.2.1. Microfilaments (actin-containing filaments)
The microfilament system is an extensive network, composed of the contractile protein actin, which extends fi*om the submembrane area into the interior of the cell and entraps the cytoplasmic granules (Tuszynski et al 1985). The detergent-insoluble polymerized actin filaments (F-actin) constitute about 40% of the total actin in unstimulated platelets and the remainder exists in a monomeric form (G-actin) which is detergent-soluble (Fox and Phillip 1983). Although the concentration of G-actin exceeds the critical concentration of about 30 pM (Gordon et al 1977) required for F-actin
assembly, its effective concentration may be lowered by proteins that bind to it, e.g.
profilin which forms a 1:1 complex with G-actin (profilactin) (Markey et al 1978). On
platelet activation a rapid polymerization of cytoplasmic G-actin to F-actin occurs
(Tuszynski et al 1985) with the organization of the newly assembled filaments into shells
around centralized granules (White 1992) and parallel bundles that fill the pseudopods
formed (Nachmias 1983, White 1968a). The fall in G-actin concentration may occur in __ Chapter 1 ; General Introduction a reversible or biphasic maimer reflecting, respectively, the reversible or biphasic mode of platelet aggregation (Heptinstall et al 1992).
Myosin is the other major contractile protein present in platelets although the amounts present are considerably less than those of actin (100:1, Actin: myosin) (Hartwig and De Sisto 1991). On platelet activation myosin can interact with F-actin to form a contractile gel (Nakata and Hirokawu 1987).
Actin and actin-associated proteins comprise 40-50% of the total platelet protein and have attracted much attention as potential targets for drugs interfering with platelet responses.
1.1.2.2. Microtubules (tubulin system)
Microtubules are formed from a single coiled-up filamentous tubule that lies around the circumference of the cell wall (Nachmias 1983) and are separated from the cell surface by the submembrane filaments (White 1992). Following platelet activation the proteins of the microtubule ring undergo reorganisation and, under the influence of actin molecular polymerization, the bundle of microtubules becomes constricted into a tight ring around clumped organelles in the central region of the cell (White 1968a).
After the release of the organelle contents the microtubule ring fractures or disperses.
However, if no release takes place the aggregated cells resume their discoid shape with randomly dispersed cytoplasmic organelles and circumferential microtubules.
1.1.3. Cytoplasmic organelles
Several types of organelle, including mitochondria and storage granules, are randomly dispersed throughout the cytoplasm of platelets. Based upon differences in their contents, ultrastructural appearance and electron opacity, storage granules are
- 3 1 - Chapter 1 : General Introduction divided into «-granules (protein storage granules), dense granules (amine storage granules, electron-dense bodies), lysosomes and peroxisomes. As platelets retain only a limited capacity for protein synthesis, most proteins and enzymes contained in the various storage granules are products of synthetic processes occurring at the megakaryocyte level. Some of the proteins found in storage granules may be synthesized elsewhere and enter the megakaryocyte or platelet by receptor-mediated endocytosis
(Niewiarowski 1994).
1.1.3.1. Mitochondria
Platelet mitochondria contain the enzymes of the tricarboxyhc acid cycle and of fatty acid oxidation together with the systems required for coupled oxidative phosphorylation. In resting platelets oxidation of long chain fatty acids appears to make a major contribution to ATP generation in contrast with stimulated platelets in which glycolysis is the major source of ATP (Crawford and Scrutton 1994).
1.1.3.2. «-granules (protein storage granules)
«-granules contain a wide range of proteins with different origins. Some of these proteins occur exclusively or predominantly in platelets, e.g. platelet factor 4 and p- thromboglobulin. Others are synthesized in other cells as well as in megakaryocytes but occur at higher concentrations in platelets than in plasma, e.g. thromobospondin. Von
Willbrand factor, factor V, platelet-derived growth factor and amyloid p-protein precursor, and some are identical or similar to plasma proteins, e.g. fibrinogen, albumin and fibronectin (Niewiarowski 1994). After secretion, «-granular proteins express their activity as procoagulant factors (fibrinogen. Von Willbrand factor), antiheparin factors
(platelet factor 4, p-thromboglobulin) or as growth promoters and mitogens (platelet- derived growth factor, low affinity factor 4) (Niewiarowski 1994).
- 3 2 - Chapter 1 : General Introduction
1.1.3.3. Dense granules (amine storage granules)
In electron micrographs dense granules show a markedly greater electron density than «-granules and lysosomes and have a 'bulls-eye' appearance (White 1992). The principal constituents of dense granules are serotonin (5-hydroxytryptamine, 5-HT),
ATP, ADP, GTP, pyrophosphate and divalent cations, e.g. Ca^^ and Mg^^. The relative amounts of these constituents are species specific. For example, in human platelets ADP is the predominant nucleotide and Ca^^ the predominant divalent cation, whereas in rabbit and pig platelets ATP and Mg^^ predominate (Crawford and Scrutton 1994).
Approximately, 60% of the total cellular adenine nucleotides and Ca^^ are present in dense granules (storage pools) and do not readily exchange with adenine nucleotides and Ca^^ in the cytosol. In contrast, 5-HT is believed to be taken up by dense granules fi’om the cytosol through a selective transport system (5-HT/H^ symport) in the granular membrane (Fishkes and Rudnick 1982). Most of the dense granular constituents are present in high molecular weight complexes. Thus, 5-HT is stored in a physiologically inactive state and fi-ee 5-HT is only liberated on exocytosis of the granular contents when
5-HT /nucleotide complexes dissociate due to dilution (Crawford and Scrutton 1994).
1.1.3.4. Lysosomes and peroxisomes
Lysosomes are organelles containing a variety of acid hydrolases (enzymes with an optimal activity in the pH range of 3.5-5.5) that are involved in the degradation of extracellular and intracellular materials (de Duve 1983). In platelets, lysosomes are morphologically distinct fi*om «-granules and dense granules (Bentfeld-Barker and
Bainton 1982). Moreover, while maximal secretion of dense and «-granule contents on platelet activation is rapid and almost complete (80-100%), and is observed with all __ Chapter 1 : Ciencral Introduction
platelet agonists, lysosomal secretion is slow, incomplete and requires high
concentrations of strong agonists like thrombin and collagen (Holmsen and Day 1970):
The secreted lysosomal enzymes may be involved in the clearing of platelet thrombi
(Oosta et al 1982). Peroxisomes in human platelets are relatively few in number and
contain catalase which degrades hydrogen peroxide.
1.1.4. Dense tubular system (membrane system)
The dense tubular system originates from the rough endoplasmic reticulum in the parent megakaryocyte (White 1992). It consists of narrow tubules dispersed randomly
in the cytoplasm, and contains an amorphous material which is separated from the
surrounding cytoplasm by a membrane unit (intracellular membrane) (White 1992). The lipid and enzymatic compositions of the intracellular membrane are different from those
of the surface membrane. In particular, the contractile proteins, actin and myosin, the
actin-binding protein filamin and most of the glycoproteins present in the surface membrane are absent in the intracellular membrane (Crawford and Scrutton 1994). The dense tubular system is the calcium-sequestering site in platelets (Menashi et al 1984).
Calcium accumulation is associated with Ca^^/Mg^^ ATPase activity which is localized in the dense tubules (Cutler et al 1978) and operates a Ca^^ pump (Hack et al 1986), therefore maintaining the cytoplasmic level of Ca^^ in the range of 50-100nM in the resting platelet. A specific peroxidase activity has been identified in the channels of the dense tubular system (Breton-Gorius and Guichard 1972) together with the enzymes involved in prostaglandin synthesis. Therefore, the dense tubular system is the site of platelet prostaglandin synthesis (Gerrard et al 1976, 1978) as well as the storage site for calcium (Manashi et al 1984).
- 3 4 - Chapter 1 : General Introduction
1.2. Physiological platelet stimulatory agonists and their receptors 1.2.1. Catecholamines
The catecholamines adrenaline and noradrenaline which are released from the adrenal medulla and sympathetic neurons to the systemic circulation during stress
(î^emdahl et al 1991) and to the coronary circulation during myocardial ischaemia may stimulate platelet aggregation (Kjeldsen et al 1989).
In vitro, adrenaline is about eight times more potent than noradrenaline in stimulating platelets (Siess 1989) but adrenaline concentrations required for platelet activation in vitro (> 0.1 pM) are much higher than the concentrations measured in circulating blood (O.lnM) (Ardlie et al 1985). However, combinations of relatively low concentrations of adrenaline and other platelet agonists can induce platelet aggregation, suggesting that circulatory adrenaline may have a role in sensitizing platelets (Ardlie et al 1985).
Independent of its induction of platelet aggregation adrenaline inhibits adenylate cyclase, both effects being mediated through the activation of (% 2-adrenoceptors of which there are 150-450 sites per platelet (Siess 1989). Human platelets also carry P 2- adrenoceptors (60 per platelet) which mediate platelet inhibition and are coupled to adenylate cyclase stimulation (Siess 1989). In a small proportion of the population, some
«i-adrenoceptors may exist as well, although their physiological role is unclear (Grant and Scrutton 1979).
3 5 - Chapter 1 : General Introduction
1.2.2. Collagen
The connective tissue protein collagen becomes exposed to circulating platelets at vascular sites when endothelial damage occurs. Platelets adhere to the exposed collagen fibrils (mainly types I and HI) and become activated with subsequent arachidonic acid metabolism, degranulation and aggregation.
Various platelet membrane proteins have been proposed as collagen receptors but no conclusive evidence has been obtained so far in favour of any of these. Proposed receptors for collagen include GP IV and the GPIg-Hg complex ( Hourani and Cusack
1991).
1.2.3. Adenosine 5 - Diphosphate (ADP)
ADP is an exclusive intracellular substance and appears in the circulation initially through lysis of tissue or blood cells at sites of vascular injury and then by dense granule secretion firom platelets (Bom and Kratzer 1984).
In vitro, ADP causes shape change, aggregation and thromboxane A 2 (TxA%) formation with subsequent release of granule contents. ADP also inhibits adenylate cyclase but controversy exists as to whether aggregation and the inhibition of adenylate cyclase are mediated by one or two types of ADP receptors (Siess 1989). The nature of the ADP receptor, its coupling mechanism and its physiological importance are not clearly understood (Hourani and Cusack 1991). Importantly, adrenaline increases the affinity of ADP for the ADP receptor by 10-fold (Siess 1989). This may be a mechanism by which adrenaline induces sensitization of platelets to low concentrations of ADP.
- 36 - Chapter 1 : General Introduction
1.2.4. Vasopressin
Vasopressin is released by the posterior pituitary gland and acts as an antidiuretic
hormone. Vasopressin, at a threshold concentration of l.OnM activates platelets through
the YI receptor which is coupled to polyphosphoinositide hydrolysis and intracellular
Ca^^ mobilization (Siess 1989). The significance of vasopressin-induced platelet
aggregation, however, remains unclear since the circulating levels of vasopressin are too
low to activate platelets. Nevertheless, vasopressin may influence platelet responsiveness
when present in combination with other agonists such as ADP and adrenaline. (Hourani
and Cusack 1991).
1.2.5. Serotonin (5-hydroxytryptamine, 5-HT)
Serotonin, in micro molar concentrations, induces weak and generally reversible
platelet aggregation (Baumgartner and Bom 1968) mediated through 5-HT2 receptors
(De Clerk et al 1984) which are coupled to phospholipase C and phosphoinositide
hydrolysis (De ChafiFoy de Courcells et al 1988). Serotonin, released fi*om aggregating
platelets, contributes to the amplification mechanism by which aggregation is enhanced
and circulating platelets recruited to a thrombus.
1.2.6. Thrombin
Thrombin (Factor Ha), a glycosylated trypsin-like proteinase, is formed during
the blood coagulation cascade, through the action of factor X^, from the circulating
plasma protein prothrombin. It cleaves fibrinogen (Factor 1) to yield fibrin which forms the fibrillar basis of the clot.
- 3 7 - Chapter 1 : General Introduction
At physiological concentrations, thrombin acts as a very powerful platelet
stimulus causing receptor-mediated aggregation and secretion of dense granules, oc-
granules and lysosomal contents. The thrombin receptor is coupled to phospholipase C via a G-protein. Another specific interaction of thrombin with the platelet surface is
related to its proteolytic cleavage of glycoprotein V.
The significance of the two specific interactions of thrombin with platelets in
haemostatic platelet activation is uncertain, presumably because other mechanisms for
activating platelets exist (Siess 1989, Hourani and Cusack 1991).
1.2.7. Thromboxane (TxA^) and prostaglandin Hj (PGHj)
TxA2 and, less potently, PGIf play an important role in the amplification of
platelet aggregation. TXA 2 causes shape change, aggregation and release of granular
contents via membrane receptors (TXA 2, TXA2/PGH2 or TxA 2/endoperoxide receptor),
and may act as an intracellular ionophore releasing Ca^^ directly, or through different
effector systems on the same receptor. TXA 2 also inhibits platelet adenylate cyclase via
a G-protein ( Q ) coupled presumably to the same receptor as that inducing aggregation.
1.2.8. Platelet-activating factor (PAF)
PAF (PAF-acether) is released from many cells including platelets, and activates
human platelets at concentrations of 1-lOnM. PAF causes shape change, aggregation
and TXA2 formation with subsequent secretion of granule contents. These effects are
mediated by specific PAF receptors (Hourani and Cusack 1991).
- 3 8 - Chapter 1 : General Introduction
1.3. Signal transduction mechanisms involved in the regulation of platelet function The interaction between platelet agonists and their receptors on the cell surface is the first step in the signal transduction pathway in which G proteins, hetero trimeric regulatory proteins bound to guanine nucleotides, mediate activation of enzymes that generate second messengers. These enzymes include phospholipases C and A 2 which are involved in platelet activation, and adenylate cyclase which regulates the formation of cyclic adenosine monophosphate (cAMP) which mediates platelet inhibition. The two intracellular pathways that mediate platelet activation begin with the enzymatic hydrolysis of specific phospholipids in the plasma membrane.
1.3.1. Phospholipase C and the phosphoinositide pathway
Phospholipase C (PLC) in platelets is present in both the membranes and the cytosol, and hydrolyses specifically inositol-containing phospholipids with a preferential hydrolysis of polyphosphoinositides (Siess 1989 ). Activation of PLC initiates the phosphoinositide pathway by hydrolysing phosphatidyl inositol 4, 5-bisphosphate (PIP 2) to form inositol 1,4, 5-trisphosphate (IP 3) and 1, 2 diacylglycerol (DAG) ( Figure. 1.2), both of which serve as second messengers mediating the activation and aggregation of platelets. The IP 3 released into the cytoplasm induces calcium mobilization whereas
DAG activates protein kinase C (PKC) (Nishizuka 1988).
- 3 9 - Chapter 1 : General Introduction
Figure 1.2. Pathways for hydrolysis and resynthesis of phosphatidyl inositol 4,5- bisphosphate ( PI 4,5-P2 ) in platelets. Abbreviations: I, Inositol; PI, Phosphatidyl inositol; DAG, Diacylglycerol; PA,Phosphatidic acid; PLC, Phospholipase C. (Modifiedfrom Siess 1989).
ATP ATP
Plasma membrane
PI Pi 4-P Pi 4,5-P;
PLC
Cytosol
i 1-P i 1.4-P; i 1,4,5-P; Cytldlne (IP2) (IPs)
Plasma membrane OTP ATP
CMP-PA PA DAG
- 4 0 - Chapter 1 : General Introduction
1.3.1.1. Inositol 1, 4, 5-trisphosphate (IP 3) and Calcium Mobilization
IP3 functions as an intracellular second messenger when it binds to its receptor to mobilize stored calcium and promote the influx of extracellular calcium (Berridge
1993).
IP3 receptors represent intracellular calcium channels and are found on the membranes of intracellular calcium stores which are normally located on modified portions of the endoplasmic reticulum (Berridge 1993). Specific calcium channels operated by IP 3, perhaps acting together with IP 4 (inositol 1, 3, 4, 5-tetrakisphosphate), are also present within the plasma membranes (Siess 1989).
IP3 may also facilitate Ca~^ influx indirectly by depleting intracellular Ca^^ stores and thereby allowing a "capacitative entry” of extracellular Ca^^ (Putney 1986).
However, there is little evidence to support the idea of this indirect mechanism of calcium entry (Berridge 1993). The formation of IP 3 in human platelets is rapid and transient (Nakashima et al 1991). Thus within 10-20 sec IP 3 is converted either to inositol 1, 4-bisphosphate (IP 2) by a specific cytosolic phosphatase or to its isomer inositol 1, 3, 4-P3 through the formation, by a specific 3-kinase, of IP 4 (Siess 1989). The role of IP 3 isomer and IP 4 are unknown but they may be responsible for sustaining the elevation of intracellular Ca^^ concentration promoted by IP 3.
The primary role of the elevated cytosolic Ca^^ is to regulate the activity of phospholipase A 2 which causes arachidonic acid (AA) liberation and thromboxane
(TxAj) formation, so eliciting typical responses such as aggregation and secretion (Siess
1989).
-41 - Chapter 1 : General Introduction
1.3.1.2. Sn-1, 2-DAG and PKC activation
DAG functions as a lipid second messenger when it binds to and activates PKC, a calcium-dependent cytosolic serine/threonine kinase. The major substrate of PKC in platelets is a cytosolic 40,000-47,000 molecular weight protein, also known as P47
( Siess 1989).
The production of DAG in the plasma membrane is normally transient and
corresponds to the formation of IP 3 , but a more sustained formation of DAG frequently follows its initial production and may result from a hydrolysis of phosphatidyl choline
(Nishizuka 1992).
DAG is largely converted by the action of diacylglycerol kinase to phosphatidic acid which remains within the membrane and influences Ca^"^ translocation across the membrane (Siess 1989). Déacylation of phosphatidic acid by a specific phospholipase
A2 produces lysophosphatidic acid which can be released to the extracellular medium and induce platelet activation through binding to a G protein-coupled receptor (Siess 1989).
1.3.2. Phospholipase A 2 and the arachidonic acid pathway
In platelets, a Ca^ - dependent phospholipase A 2 (PLA2) with a preference for arachidonic acid (AA) is present in the cytosol (CPLA 2) (Takayama 1991, Dennis 1994).
In response to Ca^^ signals CPLA 2 is translocated to the membrane where it may interact with its substrates (Lisovith 1994). Phosphatidyl choline is the preferred substrate for
PLA2 but phosphatidylethanolamine, phosphatidyl serine and, to a lesser extent, phosphatidyl inositol are also hydrolysed by PLA 2 to release AA (Kroll and Schafer
1989). AA can also be produced from DAG by the action of DAG-lipase.
-4 2 Chapter 1 : General Introduction
The liberated AA may fiinction as a lipid second messenger by enhancing the
D AG-dependent activation of PKC (Nishizuka 1992) and inducing Ca^^ mobilization
(Tohmatsu et al 1989). In the cytosol, AA is subsequently metabolized by cyclo- oxygenase and 12-lipoxygenase to various bioactive lipid mediators ( Figure. 1.3).
Thromboxane A 2 (TxAj is the principal cyclo-oxygenase product in platelets and is formed from its transient intermediate product endoperoxide PGH 2. TXA2 is released by platelets and binds to its G-protein linked receptor which mediates its stimulatory action through the PLC/phosphoinositide pathway (Baldassare et al 1993).
In addition to TXA 2, PGD2, PGp 2a and PGE2 are also formed as lesser products of AA and may act as inhibitors of platelet responses. Significant quantities of 12- monohydroperoxy eicosatetraenoic acid (12-HPETE) and 12-monohydroxy eicosatetraenoic acid (12-HETE) are also formed through oxygenation of AA by 12- lipoxygenase but their physiological relevance is not yet known (Kroll and Schafer
1989).
- 4 3 - Chapter 1 : General Introduction
Figure 1.3. A rachidonic acid release by phospholipase A? and its m etabolism by cyclo-oxygenase and lipoxygenase enzym es in platelets (M odified fi-om M arcus 1994).
0-CH2-R
<ÎXC I— ^-Choline
Phosphaadylcholine
Phospholipase A2
12-lipo-oxyge«ia» Aracfaidoaic Acid
Cyclo-oxygeaase
ÔOH 1 2 - H P E T E
12-HETE
Thrombowne symhase
OH PGFza
- 44 - Chapter 1 : General Introduction
1.3.3. Regulation of cAMP formation
cAMP is formed from ATP by the catalytic action of adenylate cyclase and
hydrolysed by various phosphodiesterases, so that the intracellular concentration
represents a steady-state level. The cAMP concentration in resting platelets is low
(approximately Ipmol per 10* cells) (Manning and Brass 1991).
The intracellular concentration of cAMP is an important determinant of platelet
reactivity thus, an elevated cAMP level makes platelets less responsive to agonists. The
decline in the level of cAMP is not, however, the major mechanism for coupling the
agonist to platelet activation (Seiss 1989).
The rise in platelet cAMP levels dampens platelet responsiveness presumably by
activating cAMP-dependent protein kinases (Brass et al 1993) and subsequently
stimulating the dense tubular uptake of Ca^^ (Tao et al 1992).
The formation of platelet cAMP is stimulated by various prostaglandins such as
PGI2, PGEj, PGD2 and by adenosine (Jakobs et al 1986). This process is mediated by the stimulatory component of the guanine nucleotide-binding regulatory protein, Gg
(Jakobs et al 1986, Ashby 1990). On the other hand, most platelet agonists suppress
cAMP formation by an action on platelet adenylate cyclase which is mediated by the
inhibitory component of the guanine nucleotide-binding regulatory protein (Gi).
- 4 5 - Chapter 1 : General Introduction
1.4. Role of platelet aggregatory and procoagulant activities in haemostasis and thrombosis The main function of circulating platelets is to participate in primary haemostasis by interaction, at sites of vascular injury, with the exposed sub-endothelial layers of the vessel wall. Specifically, platelets adhere to collagen in the sub-endothelium resulting in their aggregation and the release of their bioactive granule constituents, e.g. ADP and serotonin that together with the formation of TXA 2 (Oates et al 1988) may promote further aggregation to form a primary haemostatic plug and cause vasoconstriction. This process is associated with a change in platelet shape fi'om a discoid configuration to a spiny sphere resulting in re-arrangement of membrane components and the formation of a catalytic procoagulant surface. Thus, procoagulant membrane surfaces are formed by the exposure of anionic phosphatidyl serine at the platelet outer surface, resulting in the acceleration of reactions of the coagulation cascade (Steen and Holmsen 1987, Zwaal
1992). These reactions lead to the production of thrombin, the key enzyme in haemostasis, which catalyses the conversion of fibrinogen to fibrin and activates platelets. Haemostasis is therefore achieved by the organized assembly of platelets and fibrin in a thrombus.
Thrombosis, on the other hand, can be considered as abnormal haemostasis and is generally characterized by excessive production of thrombin (Zwaal et al 1992). An imbalance of the proteolytic enzyme cascade of the coagulation system and the counteracting anticoagulant and fibrinolytic pathways is considered to be the mechanism underlying thrombosis. The thrombus occurs almost always at sites of pathological vascular damage, for example, at deeply fissured or ulcerated atherosclerotic plaques in
4 6 - Chapter 1 : General Introduction the vessel wall(Fuster et al 1992). Recurrent fissuring of these plaques, as well as slow release of incorporated thrombin from lipid-rich plaques (Davies 1990, Szczeklik et al
1992) underlies the agonistic capacity of such vascular lesions to promote further platelet adhesion, activation and recruitment which is not seen with injured healthy vessels (Marcus and Safier 1993).
The pathological thrombi can enlarge to embolize or to totally occlude vessels, leading to ischaemia or death (Marcus and Safier 1993).
- 4 7 - Chapter 1 : General Introduction
1.5. In vitro methods used to assess potential antiplatelet effects of drugs Recognition of the role of platelets in primary haemostasis has led to the development of techniques for assessing platelet function in vitro.
Apart from their haemostatic role platelets are also involved in thrombosis which has been recognized as an important mechanism contributing to the development of myocardial infarction and stroke. This has led to increasing investigation and use of antithrombotic agents in the prophylaxis and treatment of these disorders. New drugs are being developed and are undergoing laboratory assessment. Thus, methods for the measurement of various aspects of platelet function such as adhesion, shape change, aggregation and the release reaction are utilized in studies of the antiplatelet effects of drugs, as well as in screening for abnormalities of platelet function.
1.5.1. Isolation of human platelets from whole blood for in
vitro studies of platelet function
Platelets are isolated by centrifugation or gel filtration (Hutton et al 1974) from whole blood commonly anticoagulated with citrate; heparin is not appropriate as it causes some platelet aggregation (Eika 1972, Zucker 1975) and EDTA produces platelet damage (White 1968).
The low concentrations of ionized calcium present in citrated platelet-rich plasma may lead to results not truly reflective of the physiological situation. Thus, activation of platelets in this medium by ADP, for example, leads to thromboxane formation and the release of granular contents whereas, in a medium with approximately physiological concentrations of ionized calcium activation is associated with platelet aggregation but
__ Cliapter 1 : Ci^neral Introduction not thromboxane formation and granular release (Mustard et al 1989).
Platelets may be washed and suspended in artificial media in which physiological concentrations of ionized calcium are present. In these media glucose is added as a source of metabolic energy, a protective protein, commonly albumin, is used to prevent platelet adherence to the sides of storage containers,.and lessen the chance of activation during isolation, and the pH is controlled at the pH of plasma i.e. 7.35 (Mustard et al
1989).
1.5.2. Platelet aggregation in platelet-rich plasma
The phenomenon of platelet aggregation, i.e. metabolically-dependent platelet- platelet adhesion, has been applied in the assessment of platelet function most commonly by using the turbidimetric method first described by Bom (Bom 1962) and O'Brien
(O'Brien 1962), and reviewed by TiflTany (Tiffany 1983) and Zucker (Zucker 1989). In this method platelet aggregation, induced by a variety of agonists, is measured by recording changes in light transmission through a sample of platelet-rich plasma (PRP).
Aggregation of platelets suspended in artificial media can also be measured but fibrinogen and calcium ions are required in the suspending medium.
The limitation of such a method in the assessment of the antithrombotic action of drugs relates to the use of anticoagulated blood which may ironically lead to platelet activation, loss of platelet sub-populations and the separation of platelet aggregation fi'om the overall process of coagulation.
- 4 9 - Chapter 1 : General Introduction
1.5.3. Platelet aggregation in whole blood
In addition to platelet aggregation as measured in PRP, pharmacological studies have also utilized methods in which platelet activation in whole blood is detected by the alteration in electrical impedance at an electrode (Cardinal and Flower 1980) or by counting residual individual platelets after stirring with an agonist (Saniabadi et al 1983).
Hence, the inhibitory effect of the phosphodiesterase inhibitor, dipyridamole, on platelet aggregation was clearly shown in whole blood, whereas in PRP it had little or no effect on aggregation. (Cresele et al 1983, Heptinstall et al 1986).
1.5.4. Platelet adhesion
Adhesion is an important aspect of platelet function and is defined as the adherence of platelets to a non-platelet surface such as occurs in vivo at areas of damage in a blood vessel. The measurement of platelet adhesion is technically diflBcult. Adhesion is studied in vitro using artificial surfaces e.g. glass or collagen (George 1972, Cazenave et al 1976). Glass beads packed in columns have been used in drug assessment (David et al 1979) although this method in reality measures a combination of aggregation and adhesion processes. More physiological methods have been devised which use perfusion chambers containing de-endothelialised segments of mammalian blood vessels mounted on probes (Baumgartner and Haudenschild 1972, Escolar et al 1985). Another approach involves culturing vascular endothelial cells to generate a sub-endothelial matrix which resembles that of the vascular lamina, thus providing a good model for the study of platelet/sub-endothelium interactions (Müller et al 1990).
- 5 0 - Chapter 1 : General Introduction
1.5.5. Platelet release reaction
Various platelet granular constituents can be released following the application of appropriate platelet agonists. Release can be induced in vitro by thrombin, collagen, arachidonic acid, endoperoxides, thromboxane A 2, ADP, adrenaline, vasopressin, ristocetin, ionophore A23187, fluoride, aggregated immunoglobulin, Newcastle disease virus and latex particles (Holmsen 1994).
Many drugs inhibit the platelet release reaction (Holmsen 1994) and the assessment of any potential antiplatelet agent should include an assessment of drug’s effect on this release process.
1.5.6. Platelet biochemical indices
The mechanism (s) whereby a new drug produces an antiplatelet action can be investigated by studying its effects on biochemical indices of platelet function. These
include second messengers such as cAMP and IP 3 , mobilization of free intracellular Ca^^ and TXA2 production (Kroll and Schafer 1989). Other biochemical variables that are frequently examined include various agonist receptors on the platelet membrane and uptake mechanisms for serotonin and catecholamines (Coller 1992).
- 5 1 - Chapter 1 : General Introduction
1.6. Involvement of platelets in primary hypertension and coronary heart disease 1.6.1. Definition of primary hypertension
Abnormally raised resting blood pressure as defined by the World Health Organization is 140-159/90-94 mmHg for borderline hypertension and 160/95 or above for sustained hypertension.
For clinical purposes primary hypertension, which accounts for about 95% of the hypertension occurring in the hypertensive population, can be defined as "blood pressure elevation which is sufBcient to pose a clinically significant cardiovascular risk, and which is not attributable to a single discrete cause" (Swales 1994). Thus, this definition takes account of the fact that hypertension is associated with cardiovascular morbidity (Millar and Sever 1990), and the higher the blood pressure the greater the cardiovascular risk
(Pickering 1992).
The association between hypertension and cardiovascular disease has been confirmed in clinical trials of antihypertensive treatment which have proven conclusively that treatment reduces cardiovascular morbidity in the general population although not necessarily in the individual patient. Regarding this aspect, however, it should be noted that hypertension is but one of many risk factors for cardiovascular disease and the co existence of other risk factors with hypertension, for example glucose intolerance, smoking and/or hyper cholesterolaemia, tend to have multiplicative interactive effects
(Pickering 1992).
Another criterion considered in the definition is that primary hypertension is not attributable to a single discrete cause but is a product of a large number of factors both
__ Chapter 1 : General Introduction environmental and genetic. Environmental factors include psychosocial stress, excess calorie intake (manifested by obesity), physical inactivity, heavy alcohol consumption, high sodium intake and low potassium intake (Swales 1994). On the other hand, genetic factors that predispose to the development of hypertension remain virtually unknown as few genetic studies in hypertensive subjects have been reported (Swales 1994).
1.6.2. Mechanism of primary hypertension
It is difficult to determine the primary mechanism (s) responsible for primary hypertension in susceptible individuals because of the variety of systems involved in the regulation of arterial pressure, and the complexity of the relationships between these systems. One of the mechanisms implicated in the pathogenesis of primary hypertension is alterations in cellular cation transport (Heagerty et al 1986). Thus, generalized abnormalities of ion transport across the cell membrane in various tissues may result in the development of ‘cell resetting’ with simultaneous changes in hormone-target interactions which are, for example, expressed in augmented corticosteroid secretion, increased activity of the sympathetic nervous system and hyperinsulinaemia (Postnov
1993).
Abnormalities of ion transport across the vascular smooth muscle cell membrane can contribute to enhanced vascular reactivity (Folkow 1982), the development of vascular hypertrophy and the proliferation of vascular smooth muscle cells (Lever 1986).
Structural changes in resistance vessels affect the real barostat and the functioning of cardiovascular baroreceptors and volume receptors, and result in increased peripheral resistance, in the face of normal cardiac output, and sustained elevated blood pressure.
- 5 3 - Chapter 1 : General Introduction
1.6.3. Disturbances of platelet ion transport as observed in
primary hypertension
Vascular smooth muscle is clearly the tissue principally involved in hypertension.
However, as samples of blood vessels from human subjects cannot be easily obtained, in vitro studies have tended to utilize more easily accessible tissues i.e. blood cells particularly erythrocytes and leukocytes. The assumption has been made that abnormalities regarding ion transport in these cells reflect a more generalized phenomenon, although they may not have a direct involvement in the mechanisms of hypertension. More recently platelets have been shown to share properties with vascular smooth muscle cells such as the possession of (X 2“adrenoceptors, an adenyl cyclase system (Erne et al 1983) and a calcium-dependent contraction-coupling mechanism.
1.6.3.1. Platelet sodium content and transport in hypertension
An increase in cellular sodium, which may be related to decreased Na^-K^ pump
(ATPase) activity, was proposed as a leading event in the process of blood pressure elevation (Blaustein 1977, 1984). Most studies on human and experimental hypertension have reported increased leukocyte contents of sodium (Beron et al 1985), while findings for erythrocytes have been less consistent (Hilton 1986).
Platelet sodium was reported to be significantly higher in hypertensive than normotensive subjects (Touyz 1992). In platelets as well as leukocytes, Na^-K^ ATPase activity was found to be decreased in hypertensive patients (Touyz 1992, Hilton 1986).
The reduction in the activity of the Na^-K^ pump was proposed to be due to increased fluid volume which stimulates the secretion of a digitalis-like hypothalamic natriuretic hormone that inhibits the Na^-K^ ATPase pump (De Wardener and Clarkson
__ Chapter 1 : General Introduction
1985) resulting in an increase in renal sodium excretion with the restoration of fluid volume but at the same time leading to hypertension as a result of increases in intracellular sodium content. Otherwise the transport of sodium may be affected by genetically conditioned alterations in the structure of the cell membrane (Williams et al
1989).
I.6.3.2. Platelet calcium content and transport in hypertension
Ca^^ is the key ion which determines peripheral vascular tone, and alterations in cellular Ca^^ handling leading to Ca^^ overload has been implicated in the pathogenesis of hypertension (Robinson 1984).
Ca^^-ATPase in the plasma membrane is involved in maintaining low [Car^Jj in intact cells, by pumping Ca^^ from the cytosolic compartment to the extracellular medium. Therefore, a decrease in Ca^^’-ATPase activity will result in intracellular Ca^"^ accumulation. Moreover, reductions in Na^-K^ ATPase activity will result in increased
Ca^^ entry through membrane calcium channels or via the Na'"-Ca^^ exchanger (Blaustein
1988). Intracellular Ca^^ overload, altered membrane binding and defective efflux mechanisms have been reported in human hypertension (Buhler and Resink 1988,
Bing et al 1987).
In platelets, reduced membrane Ca^^-ATPase activity and raised cytosolic Ca^^ levels have been reported in hypertension with a strong positive correlation between platelet Ca^^ and blood pressure (Touyz et al 1992, Erne et al 1984). In addition, platelets from hypertensive patients show increased responsiveness to various agonists as measured by agonist-induced responses i.e. shape change, adhesion, aggregation, thromboxane formation and the release reaction. (De Clerk 1986, Nyrop and Zweifler
1988). These platelet processes are controlled by the elevation of [Ca^'"]i.
- 5 5 - Chapter 1 : General Introduction
1.6.4. Platelet serotonin and hypertension
Serotonin (5-HT) is derived from the amino acid tryptophan and is mainly found
in the gut, the brain and platelets. The enterochromaffin cells of the gut represent the
major site of 5-HT synthesis, whereas platelets represent the storage site as they actively
take up 5-HT from plasma against a high concentration gradient, and package it in their
dense granules. Accordingly, except in cases of 5-HT-secreting tumours (carcinoid
syndrome), plasma levels of 5-HT are extremely low (<50 ng/ml) (Ahlman 1985). The
stored platelet 5-HT constitutes about 90% of the total blood 5-HT (Tranzer et al 1966)
and represents the major endogenous source of 5-HT in the circulation.
5-HT has been suggested to play a role in the development and maintenance of primary hypertension. However, the haemodynjunic effects of endogenous and exogenous
5-HT are complex as, depending upon the vascular bed, it may cause vasodilation,
mediated by 5-HTi receptors (Kalkman et al 1984), as well as vasoconstriction,
mediated by 5-HT2 receptors (Van Zwieten et al 1990). Effects of endogenous 5-HT on
the vascular system can be expected to occur as a result of platelet aggregation which
causes an increased release of 5-HT from platelet dense granules. However, since 5-HT
is rapidly deactivated by enzymatic degradation and also avidly taken up by platelets, it
seems unlikely that platelet aggregation causes a persistent elevation of 5-HT in the
general circulation (Van Zwieten et al 1990).
It was hypothesized that the availabihty of free 5-HT in plasma may be increased
in hypertension (Vanhoutte 1982) and the results of studies have tended to support this
hypothesis (Biondi et al 1984, Valtier 1986). The uptake of 5-HT by platelets has been
reported to be reduced in hypertensive subjects (Bhargrava et al 1979, Kamal et al 1984,
__ Chapter 1 : General Introduction
Fetkovska et al 1990, Nityanand et al 1990, Jafri et al 1992). However, the role of elevated levels of peripheral plasma 5-HT in the development of hypertension is unclear, especially when considering the rare association of hypertension with carcinoid syndrome (Ahlam 1985). Furthermore, 5-HT cannot be considered as a general endogenous pressor agent as 5-HT2 receptors present at post synaptic sites in resistance vessels are not effectively stimulated by circulating 5-HT (Van Zwieten et al 1990).
Therefore elevated peripheral 5-HT levels do not seem to cause hypertension but it probably plays an important role in the development of the thromboembolic complications associated with hypertension.
1.6.5. Platelets in coronary heart disease (CHD)
The atherosclerotic process in the coronary artery is the principal contributor to the pathogenesis of CHD that manifests itself in the form of acute ischaemic coronary syndromes, stable or unstable angina, acute myocardial infarction and sudden cardiac death.
The development of atherosclerosis in response to endothelial injury involves the incorporation of lipid into the arterial wall and the deposition of fibrin and platelets with the subsequent growth of fibroblasts and smooth muscle cells (Ross 1993, Badimon et al 1993).
1.6.5.1. Pathogenesis of CHD
The association between blood pressure and the incidence of CHD is strong suggesting a direct causal relationship (MacMahon 1990). However, the results of randomized trials on the drug treatment of hypertension indicate only a small reduction in the risk of CHD (about 16%), whereas the risk of cerebral stroke is reduced by about
__ Chapter 1 : General Introduction
38% (Collins et al 1990). This might indicate that the association between blood pressure and CHD is not a causal one, but rather a consequence of common atherogenic biochemical abnormalities in hypertensive patients (Kjeldsen et al 1992).
The elevated sympathetic activity in hypertension may also play an important pathogenetic role in predisposition to CHD. The direct constrictor action on coronary vessels and the cellular proliferative properties of noradrenaline, which are mediated by
«j-adrenoceptors, (Anderson et al 1972, Thyberg et al 1990, Schwartz et al 1990) might affect the coronary pathology by inducing cardiac and vascular hypertrophy (Julius and
Gudbrandsson 1992).
1.6.5.2. Platelet hyperactivity in CHD
Sympathetic overactivity may contribute to increased platelet activity which, together with a higher blood viscosity, increases the chance of coronary thrombosis, particularly in patients with additional risk factors such as lipidAnsulin-related coronary endothelial injury (Kjeldsen et al 1992). In fact, positive correlations between plasma p- thromboglobulin (P-TG), an indicator of the platelet release reaction, and both total serum cholesterol levels and raised arterial plasma adrenaline have been found in patients with primary hypertension (Kjeldsen et al 1983).
Platelet dysfunction in hypertensive subjects (De Clerk 1986, Nyrop and Zweifler
1988, Kjeldsen et al 1989) has been suggested as a potential cause of increased cardiovascular morbidity. Platelets may accumulate on subendothelial collagen, exposed at sites of arterial damage, causing partial vascular obstruction as evidenced by the presence of platelet thrombi on the acute lesions of atherosclerotic plaques in the main epicardial arteries in acute myocardial infarction (Stehbens 1985). The platelet derived growth factor (PDGF), released from activated platelets, may also contribute to the __ Chapter 1 : General Introduction smooth muscle hyperplasia of atherosclerotic lesions (Cercek et al 1991). Moreover, aggregating platelets have been reported to cause the contraction of human isolated coronary arteries being mediated by released 5-HT and TXA 2, which are now believed to be the major vasoconstrictors involved in platelet-induced vasoconstriction
(Forstermann et al 1988, Yang et al 1991, Bax et al 1994). In fact, elevated levels of 5-
HT (Van den Berg et al 1989) and TXB 2 (Tada et al 1981, De Boer et al 1982) have been reported in patients with coronary artery lesions and angina. Furthermore, a TXA 2 receptor antagonist, a 5-HT2 receptor antagonist or low dose of aspirin, taken orally, were found to attenuate platelet-induced contraction of human isolated coronary arteries
(Bax et al 1994).
The importance of platelet thrombus formation in acute ischaemic coronary syndromes has been emphasized by clinical trials showing that antiplatelet agents are useful in the prevention of acute coronary events (Dunbabin et al 1994).
1.7. Antihypertensive treatment and its effects on platelet aggregation. Pharmacological treatment of hypertension has been shown to greatly reduce the incidence of stroke and congestive heart failure, but not to have much impact on the morbidity and mortality related to coronary heart disease. This may be because the currently used antihypertensive drugs do not prevent, or may even exacerbate some of the pathological events contributing to atherosclerosis.
Because, apparently, platelet activation plays an important role in the initiation and maintenance of atherosclerosis (Packham and Mustard 1986), the various types of antihypertensive drugs and their effects on platelet function in humans are reviewed in this section.
-59- Chapter 1 : General Introduction
1.7.1. Antihypertensive drugs acting on the sympathetic
nervous system
The central and peripheral actions of adrenaline and noradrenaline are mediated by adrenoceptors («i, cLj and Pi, P2) which are found in nearly all peripheral tissues and on many neuronal populations within the central nervous system (CNS). The a- adrenoceptors mediate mainly excitatory functions and p-adrenoceptors principally inhibitory functions, with the exception of the cardiac excitatory P-adrenoceptors
(Cruickshank and Prichard 1994).
Apparently, the central and peripheral sympathetic pathways which interact with
«1- and 02-adrenoceptors play a major role in the control of blood pressure. In addition,
Oj- and 02-adrenoceptors may mediate coronary artery constriction and, hence, play a role in the regulation of coronary artery blood flow.
The excitatory P^-receptors in the heart may contribute to the elevation of blood pressure through increased conduction velocity, excitability and force of contraction.
Stimulation of presynaptic P 2-receptors results in increased NA release from sympathetic nerve endings, however, the significance of this effect in the control of blood pressure is not yet certain (Dahlof 1993).
In both the CNS and peripheral tissues, activation of «i-adrenoceptors causes increased membrane phospholipid metabolism and that of « 2-adrenoceptors inhibition of adenylate cyclase. However, increased production of cAMP, via a complex mechanism, occurs in response to a^-adrenoceptor activation in the CNS but not in peripheral tissues, except the liver. Activation of p-adrenoceptors stimulates the G protein-adenylate cyclase system resulting in an elevation in intracellular cAMP (Port et al 1992).
-60- Chapter 1 : General Introduction
1.7.1.1. ttj-adrenoceptor agonists
Antihypertensive effects
02-adrenoceptor agonists, such as o-methyldopa, guanfacine and guanbenz, have been utilized clinically in the treatment of hypertension because of their stimulatory action on central postsynaptic 02-adrenoceptors which results in enhancement of parasympathetic and inhibition of sympathetic outflow to the periphery (Emsberger et al 1994). The involvement of central non-adrenergic imidazoline receptors in the effects of imidazoline antihypertensive drugs that also possess 02-adrenoceptor agonist actions
(e.g. clonidine) has also been proposed (Emsberger et al 1990).
The use of o£2-adrenoceptor agonists in the treatment of hypertension has been limited because of their sedative side effect which results from the stimulation of a different sub-population of central a 2“^drenoceptors ( Oliver and Christen 1994.
Anti-platelet effects
Although (%2-adrenoceptors on platelets have been shown to mediate adrenaline- induced aggregation, many ^ 2-adrenoceptor agonists, e.g. clonidine, do not usually induce human platelet aggregation or only produce a small aggregatory response (Hsu et al 1979, Barnes et al 1982). This is probably because of their low efficacy combined with the low density of « 2-adrenoceptors on platelets. More potent and selective q - adrenoceptor agonists however can induce levels of platelet aggregation comparable to that induced by adrenaline (Clare et al 1984).
-61- Chapter 1 : General Introduction
I.7.I.2. a,-adrenoceptor antagonists
Anti-hypertensive effects
The rationale for the use of «i-adrenoceptor antagonists to lower blood pressure is that increased sympathetic activity contributes to the development and maintenance of hypertension. Thus, in hypertension higher circulating concentrations of catecholamines are seen resulting in stimulation of vascular a^- and «2-adrenoceptors causing increased arteriolar and venous tone and hence raised blood pressure. The «j- antagonists may have some advantages over ‘conventional’ first-line treatments, e.g. based on thiazide diuretics and p-blockers, because they reduce the total peripheral vascular resistance which is the fimdamental homodynamic abnormality in hypertension.
An additional advantage of «^-adrenoceptor antagonists in the treatment of hypertension is their favourable effect on the plasma lipoprotein profile. Thus, these drugs have been associated with a decrease in low-density lipoprotein (LDL) cholesterol levels and/or an increase in high-density lipoprotein (HDL) cholesterol (Pool et al 1990), which suggests that «^-adrenoceptor antagonists may have long-term beneficial effects on the development of atherosclerosis.
The selective «^-adrenoceptor antagonists are preferable to non-selective antagonists because they do not interfere significantly with the modulation of neuronal
NA release which is mediated by pre-synaptic «2 receptors, and cause only a modest reflex tachycardia indicating that their hypotensive action is largely unopposed by baroreceptor reflexes.
Prazosin was the first selective «^-adrenoceptor antagonist shown to be effective in the management of hypertension but many other selective «^-adrenoceptor antagonists
62- Chapter 1 : General Introduction have been introduced and others are under clinical investigation. These include prazosin analogues, such as terazosin (Titmarsh and Monk 1989), doxazosin, alfuzosin (Sega et al 1991) and bunazosin (Baba et al 1990), as well as structurally different antagonists, such as indoramin (Holmes and Sorkin 1986), urapidil and naftopidil.
Anti-platelet effects
Prazosin therapy in hypertensive patients was reported to be associated with normalization of elevated plasma P-TG levels and decreased platelet aggregation in response to ADP (Ikeda et al 1985). A possible mechanism by which prazosin inhibits platelet function is through the reduction of the total peripheral resistance and blood pressure, which may be important in relation to platelet hyperaggregability in hypertension. In in vitro studies, prazosin had no effect on platelet aggregation (Hsu et al 1979, Smith et al 1990), although it should be noted that the maximal prazosin concentrations used (IpM and 2|iM) in these studies may be low when binding of the drug to plasma protein, which is approximately 95%, (Baughman et al 1980), is considered.
Doxazosin, which is structurally related to prazosin and terazosin, was also reported to normalize platelet hyperaggregability in hypertensive patients (Hernandez
et al 1991a) and inhibit platelet aggregation in vitro (Hernandez et al 1991b). Moreover, urapidil, a phenyl piperazine-substituted derivative of uracil, has been reported to exert inhibitory actions on in vitro platelet responses to adrenaline and other aggregating agents (Emanuelli et al 1988, Smith et al 1990).
-63- Chapter 1 : General Introduction
1.7.1.3, P-adrenoceptor antagonists
Since the first observation by Prichard and Gillam (1964) that propranolol, a p- adrenoceptor antagonist, possessed antihypertensive properties, enormous clinical attention has been given to the efficacy of p-adrenoceptor antagonists in the treatment of hypertension in addition to ischaemic heart disease and certain arrhythmias. The p- adrenoceptor antagonists can be grouped according to their relative affinity for p^ and
P2 receptors. The non-selective antagonists include drugs such as propranolol, timolol and pindolol whilst selective Pj-antagonists include atenolol, metoprolol and bisoprolol.
Additional properties exhibited by p-adrenoceptor antagonists include sympathomimetic activity (partial agonist activity), blockade of a-adrenoceptors (vasodilating activity) and lipophilicity. Some P-adrenoceptor antagonists such as propranolol, labetalol and oxprenolol possess membrane-stabilizing activity which bears no relationship to their p- adrenoceptor blocking activity and may reflect the ability of these drugs to exert either quinidine effects or local anaesthetic activity (Cruickshank and Prichard 1994).
Anti-hypertensive effects
The modes of anti-hypertensive action of p-adrenoceptor antagonists depend on their affinity for Pj and P 2 receptors and on the other properties that distinguish the drugs (Cruickshank and Prichard 1994). Thus the blockade of cardiac P-adrenoceptors by classical non-selective p-blockers and p^-selective blockers results in lowering of the blood pressure by reducing cardiac output. Other possible modes of antihypertensive action for p-blockers include centrally-mediated actions, inhibition of peripheral sympathetic activity, renin-blocking activity, baroreceptor resetting, stimulation of vasodilator prostaglandins and increases in the plasma levels of atrial natriuretic factor
__ Chapter 1 ; General Introduction
(Cruickshank and Prichard 1994).
Anti-platelet effects
Pharmacologically, blockade of p 2 adrenoceptors would be expected to enhance platelet sensitivity to adrenaline due to unopposed « 2-stimulation, and would not interfere with the responses induced by other agonists. In fact, P 2-blockade by propranolol reveals « 2-mediated pro-aggregatory effects of adrenaline at low concentrations (Larsson et al 1992). However, propranolol does not influence in vivo platelet responses to high physiological concentrations of adrenaline, which seem to be dominated by « 2-adrenoceptors (Larsson et al 1992).
The in vitro effects of p-adrenoceptor antagonists on platelet function have been studied in healthy volunteers and the results obtained have been conflicting as inhibition of platelet function has been reported in some studies, whilst in others no effects have been seen (Hjemdahl et al 1991, Larsson et al 1992).
Ex vivo studies in platelets from hypertensive subjects showed that propranolol and timolol (non-selective blockers) lowered platelet cAMP and increased platelet sensitivity to adrenaline and ADP (Hansen et al 1982, Winther et al 1986). On the other hand, cAMP content was increased with the p^-selective blocker, metoprolol, (Winther and Trap-jensen 1988).
In patients with ischaemic heart disease, reduced platelet aggregability during propranolol (P-non selective) and metoprolol (p^-selective) treatment has been reported although not in all studies (Hjemdahl et al 1991).
-65 Crhapter 1 : (General liitroductiun
The anti-aggregatory effects of P-blockers are not related to P-blockade but, as hypothesized, is mediated by non-receptor interactions of p-blockers with platelet membrane phospholipids. Thus, P-blockers penetrate platelet membranes according to their lipid solubility and, as cations, interact with anionic phospholipids making them less susceptible to enzymatic hydrolysis.
1.7.2. Calcium antagonists
The calcium antagonists can be divided chemically into three main groups, the dihydropyridines e.g. nifedipine, amlodipine, felodipine, isradipine, nicardipine and nislodipine, the benzothiazepines e.g. diltiazem, and the phenylalkylamines e.g. verapamil. The three groups differ not only in their chemical structures but also in their pharmacodynamic profiles and therapeutic uses (van Zweiten and Pfaffendorf 1993).
Anti-hypertensive effects
Calcium antagonists were introduced into vascular pharmacology during the
1960s (Fleckenstein 1983), and are now amongst the most commonly used drugs in the therapy of hypertension and of cardiac and cerebral ischaemia. Calcium antagonists are also used in the treatment of certain neurological disorders such as migraine, vertigo and epilepsy.
The beneficial effects of calcium antagonists in cardiovascular disorders are primarily through their stimulation of vascular relaxation which results from the binding of the drugs to a specific receptor domain situated on the sub-unit of the high threshold (L), voltage-gated channels in the membrane of VSM and myocardial tissues.
This consequently results in inhibition of cellular calcium entry and modulation of the sensitivity of the contractile proteins to calcium.
- 66 - Chapter 1 : (General Introduction
Anti-platelet effects
Calcium antagonists have been shown to inhibit platelet activation in some systems (Nyrop and Zweifler 1988). The effects of calcium antagonists on platelet function in vitro appear to have been studied only in platelets from healthy volunteers and inhibition of platelet aggregation to a variety of agonists has been reported
(Takahara et al 1985, Valone 1987, Glusa et al 1989) although this has not been consistently reproduced by other investigators (Fernandes et al 1993). Ex vivo inhibition of platelet aggregation (Kribben et al 1987, Tschope et al 1988) and/or inhibition of platelet activation as measured by decreased plasma p-TG levels have also been reported
(Sinzinger et al 1992, Sengelov and Wmther 1989), however, in some studies significant increases (Takahara et al 1985, Mundal 1993) or no change (Islim et al 1992) in plasma
P-TG levels were seen after treatment with nifedipine.
1.7.3. Angiotensin converting Enzyme (ACE) inhibitors
ACE is an integral part of the renin-angiotensin system, a dual tissue and hormonal system for cardiovascular control, and catalyses the conversion of angiotensin
I to angiotensin II. ACE is most prominently localized at the luminal surface of the vascular endothelium but is also present in epithelial cells of the kidney, the gastrointestinal and reproductive tracts, and in epithelial and neuronal elements in the brain (Gohlke and Unger 1994).
ACE inhibitors are peptide analogues which bind to the zinc ion at the active site of ACE. Chemically ACE inhibitors can be classified as sulphydryl-containing inhibitors e.g. captopril; carboxyl-containing inhibitors e.g. enalapril and phosphorus-containing inhibitors e.g. fosinopril (Gohlke and Unger 1994).
- 67 - Chapter 1 : General Introduction
Anti-hypertensive effects The acute hypotensive effect of ACE inhibitors is produced via a reduction in circulating angiotensin II thereby preventing angiotensin-induced vasoconstriction, reducing aldosterone release, suppressing sympathetic activity and, in the long term, inhibiting the trophic actions of angiotensin II on vessels and myocardium (Schelling et al 1991). The chronic antihypertensive effects of ACE inhibitors, however, cannot be exclusively explained on the basis of reductions in circulating angiotensin II levels because ACE is not a particularly specific enzyme and its inhibition also leads to changes in the kallikrein-kinin system and local accumulation of bradykinin with, possibly, potentiation of the endothelium derived growth factor (EDRF)- or prostaglandin- mediated hypotensive effects of endogenous kinins (Gohlke and Unger 1994). The tissue renin-angiotensin system may make an important contribution to blood pressure control and to cardio-vascular function and structure (Johnston 1992).
Anti-platelet effects Platelets contain and release angiotensin II (Deleuw et al 1984, Ferri et al 1988), and also possess specific angiotensin II receptors (Moore and Williams 1981). Angiotensin II increases [Ca^^], in platelets through a transmembrane flux (Haller et al 1987) but does not produce aggregatory effects itself (Someya et al 1984), although it potentiates adrenaline-induced platelet aggregation in vitro (Ding et al 1985).
Treatment of hypertensive patients with ACE inhibitors (enalapril, captopril, lisinopiil) has been shown to be associated with inhibition of platelet activity as indicated by lower plasma p-TG levels. Also, in hypertensive patients captopril inhibits ex vivo platelet aggregation (Someya et al 1984, James et al 1988) and concomitant TxAz formation (James et al 1988). The influence of ACE inhibitors on platelet activity may be related to their reduction of calcium uptake in platelets since captopril and lisinopril were demonstrated to inhibit the ex vivo influx of calcium into human platelets (Gill et al 1988, 1992).
-68- Chapter 1 : General Introduction
1.8. The purpose of the study The purpose of the in vitro work presented in this thesis was to examine the effects of the «i-adrenoceptor antagonists naftopidil, a new antihypertensive drug currently under clinical investigation, and doxazosin, an established antihypertensive drug, on human platelet aggregation and platelet release reactions. In addition, the effects of naftopidil and doxazosin on some of the processes involved in platelet signal transduction, i.e. calcium mobilization, thromboxane Aj generation and cAMP depression, were investigated in an effort to gain an insight into the mechanisms of action of the two drugs. The effects of the calcium channel blocker nifedipine on these processes were examined for comparison.
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Materials and Methods
70- Chapter 2 : Materiab and Methods
2.1. Materials 2.1.1. Platelet activators
The platelet activators used in this study were adenosine 5-diphosphate (ADP; sodium salt), 5-hydroxytryptamine (serotonin) HCL and (-) adrenaline, all of which were obtained from the Sigma Chemical Co. Ltd. ( Poole, Dorset, UK), and fibrillar collagen which was obtained from Nycomed Arzneimittel (Munich, Germany). Solutions of these activators were prepared in normal saline.
2.1.2. Vasoactive peptides
Neuropeptide Y(human; synthetic; purity 99%), endothelin 1 (human, porcine; synthetic; purity 98%) and atrial natriuretic peptide (human; purity 97%) were obtained from Sigma Chemical Co. Ltd (UK). Solutions of these peptides were prepared in distilled water, although in the case of atrial natriuretic peptide a few drops of dilute acetic acid were added to aid in solubilization. 2.1.3. Drugs
Naftopidil was supplied by Asta Pharma (Frankfurt, Germany). Solutions were freshly prepared by dissolving naftopidil free base in dimethylformamide acidified with acetic acid (3.2:1 v/v), followed by dilution with distilled water to the required concentrations so that the final concentrations of dimethylformamide and acetic acid in the aggregometer cuvette did not exceed 0.01% v/v and 0.003% v/v respectively, so avoiding any solvent effects on platelet function.
Doxazosin mesylate was obtained from Pfizer Ltd, (Sandwich, UK) and its solutions were freshly prepared in distilled water.
Nifedipine was obtained from Sigma Chemical Co. Ltd (Poole, Dorset UK).
Solutions were freshly prepared in dimethyl sulphoxide, and kept in the dark to avoid drug decomposition.
-71- Chapter 2 : Materials and Methods
2.2. Methods 2.2.1. Preparation of platelet-rich plasma from blood
Antecubital venous blood samples were collected via a 19 gauge butterfly needle
* into tubes containing 3.13% trisodium citrate buffered with citric acid to pH 7.4, the anticoagulant: blood ratio being 1:9. Platelet-rich plasma (PRP) was prepared by centrifugation of the blood at 300x g for 10 minutes. Platelet-poor plasma (PPP) was obtained by further centrifugation of the blood at 2000x g for 20 minutes. Platelet counts and mean platelet volume (MPV) were determined for PRP samples using a model
STKS Coulter counter (Coulter Electronics Inc, Hialeah, FI., USA) and, when required, the count was adjusted to 200x10^/L with autologous PPP. PRP samples were allowed to equilibrate for 1 hr at room temperature (22°C), 75 min elapsing between venesection and commencement of the experiment. This procedure was followed to avoid the variability in platelet responses reported to occur over the first hour after venesection
(Rossi and Louis 1975, Terres et al 1986).
2.2.2. Preparation of washed and aequorin-loaded platelets
Antecubital venous blood samples were collected via a 19 gauge butterfly needle into tubes containing 1 volume acid citrate-dextrose anticoagulant (citric acid 0.8% w/v,
* trisodium citrate 2.8% w/v, glucose 2.4% w/v) per 9 volumes of blood. PRP was obtained by centrifugation of the blood at 300x g for 10 minutes at 22 “C, and acidified to pH 6.0 with M. citric acid solution in order to inhibit binding of fibrinogen to its receptor (Peerschke 1985). The platelets were pelleted by centrifugation of the acidified
PRP at 800x g for 20 minutes, and washed once with HEPES (N-[2-hydroxyethyl] piperazine-]Sr-[2-ethane-sulphonic acid]) -buffered saline (140mMNaCl, 2.7mM KCl,
dihydrate - 72 - Chapter 2 : Materials and Methods
lOmM HEPES, 0.1% w/v bovine serum albumin, 5mM glucose, pH 7.3) containing
5mM EGTA. After washing, the platelets were sedimented as before and resuspended in 5Dpi of the same buffer to give a thick suspension. Platelets were loaded with aequorin (Friday Harbor Photoprotein, Friday Harbor, WA, USA ) by a method based on a dimethyl sulphoxide (DMSO) permeabilization procedure (Yamaguchi et al 1986).
The original protocol was modified by the omission of prostacyclin (PGF) treatment of the platelets (Cooper et al 1994). The loading procedure was carried out at room temperature (22°C) as follows; The volume of the platelet suspension was measured
(typically this was approximately lOOpl) and aequorin (90pg in 30pl of 5mM EGTA solution) added. DMSO was added in three equal aliquots to a final concentration of 6%
(v/v), the additions being spaced at intervals of 2 min and the cells being mixed on a haematological roller during the loading process. After the final portion of DMSO had been added, the cells were incubated for a further 4 min and then transferred to a 1,5ml microcentiifuge tube and diluted with 1 ml of HEPES -buffered saline containing 5mM
EGTA. Platelets were pelleted by centrifugation at 12,000x g for 15 seconds. The platelet pellet was resuspended in the HEPES-buffered saline and pelleted twice. Finally, the platelets were resuspended in the HEPES-bufifered saline to give a platelet count of
300x10^ platelets/L. CaClg and MgClz were added to the platelet suspension to a final concentration of ImM. The platelets were allowed to stabilize for 20 minutes at room temperature (22 °C) before commencement of the experiment.
-73- Chapter 2 : Materials and Methods
2.2.3. Preparation of the fibrinogen solution
Fibrinogen (lOOmg) (Sigma Chemical Co. Ltd., UK) was suspended in deionized water (4ml) and dialysed overnight in HEPES-buffered saline (140mM NaCl, 2.7mM
KCl, lOmM HEPES, 5mM glucose, pH 7.3) with continuous stirring at 4°C. The protein concentrations in the solution were measured by the method of Bradford (Bradford
1976) and then adjusted with HEPES-buffered saline to 3mg/ml.
2.2.4. Platelet aggregation in PRP
Platelet aggregation induced by various agonists was measured as % light transmission according to the method of Bom and Cross (Bom and Cross 1963) in
270|il aliquots of PRP, stirred at 1000 rpm at 37°C in a Payton-dual channel aggregometer. 0% and 100% light transmission were calibrated using PRP and PPP respectively. The change in light transmission during aggregation was recorded on a
Rikadenki chart recorder.
2.2.5. Calcium mobilization and platelet aggregation in
aequorin-loaded platelets
Platelet aggregation and aequorin luminescence were recorded simultaneously in 1ml aliquots of aequorin-loaded platelets containing fibrinogen (0.06% final concentration), using a platelet ionized calcium aggregometer (PICA, Chrono Log Corp.
Ltd, Penn, USA). Aggregation was measured as the percentage of light transmission with 0% and 100% light transmission being that recorded for the platelet-fi-ee buffer (PFB) and the unstimulated platelet suspension, respectively. The difference between light transmission through PFB and platelet suspension was set automatically at the start of each test.
-74- Chapter 2 : Materiab and Methods
Intracellular calcium ion concentration [Ca^^Jj was calculated from the fractional luminescence (L/Lmax) obtained from the luminescence (L) of the aequorin-loaded platelet suspension, and the maximal luminescence (L max) recorded from 1ml HEPES- buffered saline containing 100pi aliquots of aequorin-loaded platelets lysed by the addition of Triton X-100 (20pl, final concentration 0.1%). Log^o L/L max was then converted to [Ca^^]i by reference to a calibration curve relating fractional aequorin luminescence to [Ca^^] in the presence of ImM Mg^"^.
2.2.6. Serotonin measurement
Samples of PR? or washed platelet suspension were centrifuged at 12,000g for
4 minutes at 4°C to yield PPP or buffer respectively, which were frozen at -20°C for later measurement of serotonin using high performance liquid chromatography with electrochemical detection. Plasma (platelet-poor) or supernatant samples (125pi) were mixed with normal saline (875 pi) and treated with N-acetyl-serotonin internal standard
(lOul, lOOpmol) and perchloric acid (0.4M, final concentration) and centrifuged.
Supernatants were then injected on to the chromatographic system which incorporated a Spherisorb S 3 ODS2 analytical column (Phase Separations Ltd, UK), separation being achieved using an isocratic solvent system consisting of an acetate-citrate buffer containing EDTA (3 mmol/L) and 17.5% methanol (Mefford 1981).
2.2.7. cAMP Extraction and measurement
Intracellular cAMP was extracted from washed platelet suspensions (0.5ml) containing fibrinogen (0.1ml, 0.06% final concentration). Ice-cold ethanol (0.5ml) was added to the suspension to give a final concentration of 65% ethanol and the suspension was allowed to settle. The supernatant was drawn off into Eppendorf tubes and the
-75- Chapter 2 : Materials and Methods remaining precipitates were washed with 65% ice-cold ethanol and the washings were added to the appropriate tubes. The extracts were centrifuged at 12,000 g for 5 minutes at 4°C and the supernatant transferred to glass tubes and evaporated to dryness at 90°C in a Techne Dri-Block (DB-3) and then stored at -20°C for later assay. The dried extracts were redissolved in 1ml 0.05M acetate buffer with sodium azide for assay of cAMP.
A cAMP [^^I] scintillation proximity assay (SPA) system (dual range), purchased from Amersham (Amersham International PLC, UK), was used for the measurement of cAMP concentrations. The assay is based on the competition between unlabelled cAMP and a fixed quantity of labelled cAMP for a limited number of binding sites on a cAMP specific antibody. With fixed amounts of antibody and radioactive ligand, the amount of radioactive ligand bound by the antibody will be inversely proportional to the concentration of added non-radioactive ligand. The antibody-bound cAMP reacts with the SPA reagent which contains anti-rabbit second antibody bound to fluomicrospheres.
Therefore, any [^^I] cAMP that is bound to the primary rabbit antibody will be immobilised on the fiuomicrosphere which will produce light.
Concentrations of cAMP were determined using the non-acetylation assay protocol which measures cAMP in the range of 0.2-12.8 pmol/tube. The amount of cAMP bound to the fluomicrospheres was determined by counting the vials in a P- scintillation counter for 2 minutes. The concentrations of unlabelled cAMP in the sample were then determined by interpolation from a standard curve.
- 76 - Chapter 2 : Materials and Methods
2.2.8. Measurement of thromboxane (TxB^)
Samples of washed platelet suspension (0.5ml) were centrifuged at 12,000g for
4 minutes in an Eppendorf centrifuge at 4°C and the supernatants frozen at -20°C for later measurement of TXB2, the stable metabolite of TXA 2.
A TXB2 [^H] scintillation proximity assay (SPA) system, purchased from
Amersham (Amersham International PLC, UK), was used for the measurement of TXB 2 concentrations in the range of 5-300pg/tube. The principle of the TXB 2 assay is similar to that for cAMP with the competition between unlabelled TXB 2 and a fixed quantity of
[^H] TXB2 for a limited number of binding sites on a TfcB specific antibody. The antibody-bound TXB 2 then reacts with the SP A-anti-rabbit reagent, which contains anti rabbit IgG bound to fluomicrospheres. Any [^T x B 2 that is bound to the primary rabbit antibody is immobilized on the anti-rabbit fiuomicrosphere which will produce light.
Concentrations of TxBj were determined using an overnight assay protocol. The amounts of TXB 2 bound to the fluomicrospheres were determined by counting the tubes in a P-scintillation counter for 4 minutes. The concentrations of unlabelled TXB 2 in the samples were then determined by interpolation from a standard curve.
2.2.9. Measurement of platelet derived growth factor
(PDGF)
Samples of washed platelet suspensions (0.5ml) were centrifuged at 12,000g for
4 minutes and the supernatants were frozen at -20°C for later measurement of PDGF, a cationic dimeric protein wich can be either homodimeric (PDGF-AA, PDGF-BB) or heterodimeric (PDGF-AB) (Ostman et al 1992)
-77- Chapter 2 : Materials and Methods
The A Biotrak PDGF-AB, human, ELISA system, purchased from Amersham
(Amersham International PLC, England), was used for the measurement of PDGF concentrations in the range of 31.3-2000pg/ml. The assay system is based on a solid phase ELISA which utilizes a specific monoclonal antibody for PDGF-AA bound to the wells of a micro titre plate, together with a polycolonal antibody to PDGF-BB conjugated to horseradish peroxidase. Standards and samples are pipetted into the wells so that any PDGF-AB present is bound by the immobilized antibody. After washing away any bound proteins, an enzyme-linked polyclonal antibody, specific for PDGF-BB, is added to the wells and allowed to bind to any PDGF-AB that was bound during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and colour develops in proportion to the amount of PDGF-AB bound in the initial step. The optical densities of the standards and the samples were determined in a plate reader at 450nm within 30 minutes of stopping the reaction.
The concentrations of PDGF-AB in the samples were determined by comparing the optical densities of the samples with those for standards, standard curve having been prepared by plotting the optical density versus the concentration of PDGF-AB in standard wells.
- 78 - Chapter 2 : Materials and Methods
2.3. Statistical Analysis The results in all studies presented in this thesis are expressed as means ± standard errors of the means (SEM).
The statistical significance of the differences measured between controls and treated samples was assessed by the student's t-test for paired data or by analysis of variance (ANOVA) and Dunnett's test for multiple comparisons. A value of p<0.05 was considered statistically significant. The 95% confidence intervals (Cl) for the means of the differences were also calculated. The linearity of the performed regression and correlation analyses was confirmed with runs test.
ANOVA was performed using the statistical package SAS (SAS Institute Inc.,
Box 8000, cary. North Carolina 27511, USA) and regression and correlation analysis were done using the package INSTAT ( Graphipad Software v2.04a, 10855 Sorrento
Vally Road #203, San Diego, CA 92121, USA).
-79- Chapter 3
A Study of Some Factors Influencing the Stability of in vitro Platelet Responses.
-80- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.1. Introduction In vitro studies of platelet aggregation, involving Bom aggregometry, are usually completed within 2-3 hours after PRP preparation (Zucker 1989). This is because platelet sensitivity to some agonists declines with time of storage. Also, factors such as temperature and PRP pH have been reported to influence in vitro platelet behaviour
(Kunicki et al 1975, de Korte et al 1990). Moreover, PRP platelet count adjustment using autologous PPP has been reported to cause indeterminate changes in platelet responsiveness (Thaulow et al 1991), although no evidence to support this claim was presented. Previous studies have invariably focussed on individual factors that may influence platelet behaviour, the effects of combinations of factors not having been considered.
Clearly, if those conditions of storage which yield the most reproducible results can be established, it will be possible to maintain platelets in a relatively normal state for longer periods, so facilitating more detailed and comparative studies of platelet function.
3.2. Aim of the Study The aim of the study presented in this chapter was to clarify the conditions of
PRP storage that influence platelet sensitivity and yield optimal stability of platelet responses in vitro. The effects of PRP dilution, temperature of storage, the presence or absence of air and the duration of storage on platelet responses to ADP and adrenaline were therefore tested, either singly or in combination.
- 81 - Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.3. Study Design In all the experiments presented below platelet aggregation was measured as described in Chapter 2 (2.2.4).
3.3.1. Storage of PRP samples in the absence or presence of air
PRP samples with platelet counts adjusted to 200x10^/L were stored at room temperature (22°C) in two 20ml plastic syringes. The air in one of the syringes was retained, the volume ratio of air to PRP being set at 1:1, whereas in the other syringe the air was completely expelled.
Aggregation was induced by threshold concentrations of ADP and adrenaline and measured for both types of sample at 0, 2, 4 and 6 hours following 1 hr equilibration at room temperature. The pH of PRP samples subjected to this treatment was determined after 24 hours storage.
3.3.2. Storage of PRP samples at 4°C, 13°C, 22°C and 37°C
PRP samples, with platelet counts adjusted to 200x10^/L, in syringes fi’om which the air had been expelled were stored at 4°C, 13°C, 22°C or 37°C. Aggregation was measured in response to threshold concentrations of ADP and adrenaline at hourly intervals up to 6 hours, and after 24 hours. Platelet counts were measured in PRP samples stored for 24 hours to determine whether any changes had occurred over this period.
3.3.3. PRP dilution
PRP samples were either adjusted to a platelet count of 200x10^/L or left undiluted (mean platelet count 469x10^/L; range 296 - 659x10^/L), and stored at room temperature in the absence of air. Following equilibration, aggregation in both diluted
- 82 - Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
and undiluted PRP samples was induced by a logarithmic range of ADP and adrenaline
concentrations (0.25-16pmol/L, final concentrations). The threshold concentration
(defined as the minimal concentration of agonist sufficient to induce secondary
irreversible aggregation) and the EC# ( defined as the concentration of agonist required to produce 50% maximal aggregation) were determined. The responses to threshold and
EC# concentrations of each agonist were then tested at hourly intervals up to 6 hours
and then after 24 hours.
3.3.4. Serotonin (5-HT) determination
Diluted (Platelet count adjusted to 200x10^/L) and undiluted ( mean platelet
count: 386xlO^/L) PRP samples, were stored at room temperature in the absence of air
and tested for their responsiveness to threshold concentrations of ADP and adrenaline
at 0, 3, 6 and 24 hours post-equilibration. At each time point samples of PRP were
centrifiiged to yield PPP samples, which were frozen at -20 °C for later measurement of
5-HT as described in Chapter 2 (2.2.6).
-83- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.4. Results 3.4.1. The effect of the absence or presence of air during
PRP storage on pH and platelet aggregation.
When the air from the storage syringes was excluded, the pH of PRP stored for
24 hr was not statistically different from the initial pH measured directly after equilibration (0 hr), values being 7.79±0.03 ( n= 6) and 7.78±0.03 (n= 6), respectively.
In air-exposed samples, however, the pH increased to 8.44±0.07 (n= 6) over the same period (p<0.02 compared to the pH in the air-free sample at Ohr).
Aggregatory responses to threshold concentrations of adrenaline and ADP progressively declined in both air-free and air-exposed PRP samples. For ADP the declines were, at 4 and 6 hr post equilibration respectively, less marked in the air-free samples (20.9±7.9%, p=not significant, and 36±12.0%, P<0.05) than in samples exposed to air (41.4±7.5%, p < 0.01, and 52.5±3.7%, P<0.001 ) ( Figure 3.1. Appendix, Table
1 ), and a statistically significant difference between responses in the air-exposed and air-free samples being measured at 4 hr post equilibration (15.3±2.3% light transmission,
P<0.01). For adrenaline, however, the responses declined equally in air-free and air- exposed PRP samples ( Figure 3.2. Appendix, Table 2), the decreases at 2, 4 and 6hr postequilibration relative to the responses at Ohr being, respectively, 36.6±14.0%
(P<0.05), 46.9±10.4% (P<0.01) and 63.7±8.7% (P<0.001) for the air-free samples, and
41.7±15.4% (P<0.05), 44.9±12.6% (P<0.02) and 64.1±10.1% (P<0.01) for the air- exposed samples.
Because of these findings PRP samples were stored in the absence of air in all subsequent experiments.
- 84 - Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
Figure 3.1 Platelet aggregation (% light transm ission) induced by the A D P threshold concentration (3.00± 0.44|iM ) in PR P stored in the presence and absence o f air. Platelet counts w ere adjusted to 200x10^/L. D ata are also presented in the appendix. T able 1. (**: P<0.001, *; P<0.01 com pared to Ohr, n=6) 100 -
A ir-free PRP | PRP with air 8 0 I
6 0 % 4 0 CD 20 I 2.0 4.0 6.0 Hours (Post-equilibration)
Figure 3.2 Platelet aggregation (% light transm ission) induced by the adrenaline threshold concentration (1.50±0.32pM ) in PR P stored in the presence and absence o f air. Platelet counts w ere adjusted to 200x10^/L. D ata are also in the appendix. T able 2.
100 r : P<0.05 com pared to Ohr, n=6
8 0 A ir-free PRP PRP with air
60
40 jc CD
20
Hours (Post-equilibration)
- 85 - Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.4.2. The effect of storage temperature on platelet
aggregation.
The storage of PRP samples at4°Corl3°C was initially thought to improve
platelet preservation and viability compared with storage at room temperature.
Aggregatory responses to both ADP and adrenaline far exceeded those obtained for
samples stored at room temperature, with little or no changes in response being evident with time of storage ( Figure 3.3A and 3.3C, Appendix, Tables 3 and 4). However, on
closer examination of the aggregatory traces obtained, it was found that the
characteristic oscillations of the trace baseline seen with normal unstimulated (i.e.
discoid) platelets (O'Brien and Heywood 1966) were absent, indicating that chilling had
caused platelet shape change (Kattlove et al 1972). Aggregatory traces produced by threshold concentrations of ADP and adrenaline changed from the normal biphasic mode
to a monophasic mode indicating that platelet sensitivity to these agonists had increased.
After 24 hours storage at low temperature, spontaneous platelet aggregation occurred,
as indicated by a considerable decline in platelet count; at 4°C platelet counts had
declined by 56.3±5.0% (p<0.02) and at 13°C by 44.6±3.0% (p<0.02), whereas the
decline at 22°C was smaller (8.2±1.8%) and not statistically significant.
The pH of PRP samples stored at 4°C for 24 hours was 7.76±0.06 and was not
statistically different from that of samples maintained at room temperature.
Samples stored at 37°C were considerably less responsive than samples
maintained at 22°C ( Figure 3.3B and 3.3D, Appendix, Tables 5 and 6), the decline in
platelet responsiveness being more evident for aggregation induced by adrenaline
( Figure 3.3D) than by ADP ( Figure 3.3C). Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
After 24 hours at 37°C the pH of PRP samples had dropped to 7.29±0.11, compared to the initial pH of 7.77±0.05 recorded 1 hour after PRP preparation
(p<0.05). This would suggest that platelets had changed from aerobic to anaerobic metabolism with a resultant increase in the production of lactic acid (Pisciotto et al
1991).
-87- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
F igure 3.3 Effect of the storage of PR P (platelet count = 200x10^/L) at 4°C , 13°C, 22°C and 37°C on platelet aggregation (% light transm ission) induced by the threshold concentration o f A D P (2.67±0.67pM ) (A , B ) and adrenaline (1.67±0.33pM ) (C , D ). PR P w as stored in the absence o f air. D ata are also presented in the appendix, T ables 3,4,5 and 6. (*:P<0.05 com pared to Ohr, n= 3)
O 22'C 100 • rc O 22°C V 13“C # 57°C ; 80 V
60 '0 h- 40 _c 20
0 2 3 4 5 6 0 2 3 4 5 6
Hours (Post —equilibration) Hours (Post—equilibration)
o 22“C # 4°C 100 V 13°c c o 00 80 00 E c00 60 D 40
20
0 2 3 4 5 6 0 2 3 4 5 6
Hours (Post-equilibration) Hours (Post—equilibration)
- 88 - Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.4.3. The effect of PRP dilution on platelet aggregation
Immediately following equilibration, platelet responses to both ADP and adrenaline were greater for undiluted PRP samples than for samples diluted to a count of200x1(y/L ( Figure 3.4, Appendix, Tables 7 and 8 ), and differences were statistically significant when the ECjq values for ADP and adrenaline, and threshold concentrations for adrenaline were compared (Table 3.1).
Table 3.1 EC50 values and threshold concentrations (pM) calculated fi*om the dose- response curves obtained for ADP and adrenaline in diluted and undiluted PRP.
Adrenaline ADP Adrenaline ADP
EC50 EC50 Threshold Threshold Diluted PRP 1.09 ±0.23 1.56±0.17 1.50 ±0.32 3.00 ±0.44
Undiluted PRP 0.43 ±0.12** 1.20 ±0.13* 0.65 ±0.17** 2.03 ± 0.43
Values (means ± SEM) were determined within 1 hour following the equilibration of PRP at room temperature (22 °G ) in the absence of air. ( Significance: p<0.05 and **p<0.01 when comparing undiluted and diluted PRP samples, n= 6).
-89- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
3.4.3.1 Platelet sensitivity and platelet count
There was no correlation between platelet count in undiluted PRP and platelet sensitivity (as indicated by EC 50 values) to adrenaline (r=0.28, p; not significant, n=9) or ADP (r=0.13, p: not significant, n=12). Platelet count and sensitivity as indicated by threshold concentration values also did not correlate (adrenaline: r= 0.22, p: not significant, n=ll; ADP: r=0.00008, P: not significant, n=12). There was, however, a strong positive linear correlation between the EC 50 values for diluted and undiluted PRP samples with both ADP (r=0.83, p<0.001, n=12) ( Figure 3.5A) and adrenaline (r=0.82, p<0.01, n=9) ( Figure 3.5B) indicating that platelet count adjustment did not affect the ranking order of platelet sensitivity within the subject group. Positive correlations were also seen with the threshold concentrations for adrenaline (r=0.93, p<0.0001, n=l 1) and
ADP (r=0.69, p<0.02, n=12).
-90- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
Figure 3.4 Dose-response curves for platelet aggregation induced by ADP (A) and adrenaline (B) in diluted (platelet count: 200x10^/L) and undiluted ( mean platelet count 469xlO^/L, range 296-659x10^/L) PRP samples. Experiments were completed within 90 minutes following the equilibration of PRP samples for 1 hour at room temperature in the absence ^ f air. Data are also presented in the appendix, Tables 7 and 8 . 100 *: P<0.05 diluted vs undiluted, n =6
O Diluted PRP 80 0 Undiluted PRP c o (/) — E 60 in c o L. I— 4 0 _c Li 20
0.25 0.5 1.0 2.0 4.0 8.0 16.0 ADP Concentration (/xM)
100 O Diluted PRP # U ndiluted PRP 80 c o win E 60 in c o I— sz 40 O)
20
0.25 0.5 1.0 2.0 4.0 8.0 16.0 Adrenaline Concentration (/.iM)
-91- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses
Figure 3.5 Correlations between the EC 50 values for ADP (A) and adrenaline (B) in diluted and undiluted PRP samples. A 2,5 r
CL CL 0_ Tj 2.0 c 1 .5 O in Linear regression U ^ 1.0 Q_ Q 95% Confidence Interva < □ 0 .5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ADP EC^q (/zM) in undiluted PRP B 2 .5 0_ CL CL □ ■u (D 2.0 3 ’"D C 1 .5 o 1.0 LÜ 0) c o 0 .5 Linear regression c(U "O 95 % Confidence Interval < 0.0 ______I______I 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 -4 Adrenaline EC^q (^cM) in undiluted PRP -92- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses 3.4.3.2 Storage time and platelet aggregation in diluted and undiluted PRP With both diluted and undiluted PRP samples there was a progressive decline in the aggregatory responses to threshold and EC 50 concentrations of ADP and adrenaline. The declines in responses to the ADP threshold concentrations were greater for undiluted samples (Figure 3. 6A, Appendix, Table 9). The percentage decline in responses to the ADP EC% in diluted and undiluted PRP samples are presented in Table 3.2. The declines in aggregatory responses to threshold concentrations of adrenaline were not significantly different between diluted and undiluted PRP samples, due to the high inter-individual variability in the responses to this agonist ( Figure 3.6B, Appendix, Table 10). The percentage decline in responses to the adrenaline EC 50 in diluted and undiluted PRP samples are presented in Table 3.3. After 24 hr storage platelet responses to threshold concentrations of ADP were extremely weak for both diluted and undiluted PRP samples and no response to adrenaline occurred, even at maximal concentrations. However, on challenging the platelets with adrenaline (2pM) followed immediately by an ADP threshold concentration, significant responses were restored, particularly with diluted PRP samples ( Figure 3.7, Appendix, Table 11). Examination of the aggregatory traces revealed that it was the secondary, irreversible phase of aggregation that progressively deteriorated, reflecting a gradual decline in platelet granular secretory processes ( Figure 3.8). Platelet counts did not change significantly over 24 hr although the mean platelet volume (MPV) which is a determinant of platelet function, (Saigo et al 1992) was found to have been decreased fi"om 7.9±0.4 fl to 5.9±0.4 fl (p <0.02), agreeing with previous -93- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses reports (Fijnheer et al 1989). This decrease in MPV may be related to the type of anticoagulant used (buffered citrate) (Thompson et al 1983). 3.4.4. 5-HT measurement 5-HT concentrations in PPP samples obtained after the centrifugation of diluted and undiluted PRP samples revealed that no changes in its concentration had occurred over 24 hr of storage. Values measured at 0, 3, 6 and 24 hr after PRP equilibration were respectively 63.0±20.2, 54.9±17.3, 57.4±12.5 and 58.9±13.4 pmol/ml for diluted PRP (n=3) and 56.1±17.3, 66.2±7.4, 54.0±18.5 and 57.0±22.2 pmol/ml for undiluted PRP (n=3). This would indicate that excessive dense granular release had not occurred. -94- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses Figure 3.6 Platelet aggregation (% light transmission) in diluted ( platelet counts: 200x 1 (ffL) and undiluted ( mean platelet count 469x10^; range 296-659xlO^/L ) PRP samples stimulated by the ADP threshold concentrations (3.00±0.44 pM and 2.03±0.4pM for diluted and undiluted PRP, respectively) (A) and adrenaline threshold concentration (1.50±0.32pM and 0.65±0.17pM for diluted and undiluted PRP, respectively) (B). PRP was stored at room temperature in the absence of air. Data are also presented in the appendix. Tables 9 and 10. (*: P<0.05 compared to Ohr, n= 6) 100 80 50 40 O) !_i 20 0 1 2 , 3 4 5 , 6 Hours (Post equilibration) 100 c o 80 V) E C/O 60 c o 1— 40 _c cn !_j 20 0 0 i?ours (^ost equilibration) -95- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses Table 3.2. The percentage decline in responses to the ADP EC 50 ( 1.56±0.17uM and 1.20±0.13uM) in diluted (platelet counts: 200x10^/L) and undiluted ( mean platelet count 469x10^; range 296-659x10^/L ) PRP samples, respectively. Hours ( Post equilibration ) Diluted PRP Undiluted PRP 1 18.5 ±4.4 15.9 ±20.2 2 23.0 ±3.2 30.0 ± 18.3 3 30.3 ±3.2 49.5 ±5.4 4 32.7 ±5.7 56.6 ±6.0 5 35.8 ±5.4 64.5 ±5.3** 6 38.5 ±5.9 63.8 ±4.4* 24 73.7 ±5.1 84.9 ±9.0 *: p < 0.05 and **: p<0.02 when comparing undiluted and diluted PRP samples. (n= 6) Table 3.3. The percentage decline in responses to the adrenaline EC 50 (1.09 ± 0.23uM and 0.43 ± 0.12uM) in diluted (platelet counts: 200x10^/L) and undiluted ( mean platelet count 469x10^; range 296-659x10^/L ) PRP samples, respectively. Hours ( Post equilibration ) Diluted PRP Undiluted PRP 1 18.9 ±25.0 53.4 ±28.4 2 26.5 ± 18.2 28.1 ±33.9 3 57.2 ± 12.3 88.5 ± 1.6* 4 77.1 ±3.2 88.3 ±0.9* 5 77.3 ±2.9 91.6± 1.9** 6 81.7±5.0 95.4 ±2.2** 24 100 ± 0.0 100 ± 0.0 p<0.05 and **: p<0.02 when comparing undiluted and diluted PRP samples. (n= 6) -96- CTuipter 3 : Factors Influencing Stability of In Vitro Platelet Responses Figure 3.7. Potentiation b> adrenaline (2fiM ) of platelet aggregation induced by the threshold concentration o f A D P in diluted (3 ,00±0.44pM ) and undiluted (2.03±0.43p.M ) PR P sam ples stored for 24hour. A ggregation at da\ 0 and day 1 w ere m easured, respectively, at 1 and 24 hour post- equilibration. A drenaline on its ow n did not have any aggregatoiy effect at 24 hour. PR P sam ples w ere stored at room tem perature (22°C ) in the absence o f air. D ata are also presented in the appendix. T able 11. (*P<0.01 com pared to day 1, n=6) 1 0 0 m i Day 1 KxJM Day 1+Adrenaline 8 0 6 0 4 0 " _ i 20 Diluted PRP Undiluted PRP - 9 7- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses Figure 3.8 Examples of aggregatory traces showing time-dependent changes in platelet responsiveness to threshold concentrations of ADP (2pM) (A) and adrenaline (IpM) (B). Addition of ADP or adrenaline is indicated bv the arrow. Hours ( Post Equilibration ) 0 12 3 4 QO B 0 —, JZ. QO -98- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses 3.5. Discussion The present study was undertaken in order to establish definitively those conditions of PRP storage that maintain platelets in a relatively normal state for longer periods so that more detailed studies of platelet function are made possible. It has been reported that storing PRP under an atmosphere of 5% C 02-95 % O2 stabilizes both pH and platelet activity (Rogers 1972, Coller et al 1976). The results presented in this chapter, however, confirm that storing PRP in the absence of air is a much simpler means whereby stabilisation of PRP pH can be achieved (Watts et al 1985) and platelet sensitivity to ADP, but not to adrenaline (Rossi and Louis 1975), maintained. The temperature at which PRP was stored was found to strongly influence platelet sensitivity. As previously reported, storage at 4°C and 13 °C maintained platelet activity but caused shape change (Murphy and Gardner 1969), whereas storage at 37°C caused a reduction in the sensitivity of platelets to adrenaline (O'Brien 1964) and, to a lesser extent, ADP (Praga and Pogliani 1973, Holme and Holmsen 1975, Watts et al 1986). Indeed, it has been demonstrated that at 4°C platelets irreversibly lose their discoid configuration and assume a spherical one (Kattlove et al 1972), whereas at 37°C platelets maintain the elliptical conformation characteristic of the non-activated state (Watts et al 1986). It has also been reported that at 37°C the platelet receptor binding sites for fibrinogen and other adhesive proteins involved in platelet aggregation, are converted firom an "open" to a "closed" configuration (Willigen and Akkerman 1991). Therefore, enhanced platelet activity at low temperatures needs to be balanced against reduced platelet activity but greater stability at physiological temperature. On the basis -99- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses of the results obtained in this study it appears that storage at 22°C provides an acceptable compromise. Dilution of PRP to a standard count of 200x10^/L caused a reduction in platelet sensitivity to ADP and adrenaline. This adjustment, however, did not affect the ranking order of platelet sensitivity within the subject group as strong positive correlations between both the EC^ values and threshold concentrations for diluted and undiluted PRP were found. However, the between subject variation in platelet sensitivity was less for diluted PRP, especially with adrenaline. The observed reduction in platelet sensitivity caused by PRP dilution supports the claim that platelet count adjustment influences platelet responsiveness (Thaulow et al 1991). It is also reported that PRP dilution masks the effect of exercise on platelet aggregation (Rendra et al 1988). î^emdahl et al (1991) have also questioned the practice of standardising the platelet count in PRP samples for Bom aggregometry. The progressive decline in platelet sensitivity to ADP or adrenaline with storage seen in this study has been reported previously, although a satisfactory explanation for this phenomenon has yet to be proposed. Many factors are probably involved including, perhaps, a gradual deterioration in receptor-coupling mechanisms. Significant responses were, however, obtained with sub-optimal concentrations of adrenaline and ADP in combination, even after 24 hours of storage, agreeing with previous reports (Di Minno et al 1982, 1983). Some investigators have suggested that stored platelets develop defects in both their dense and a-granules and in their ability to maintain ATP homeostasis (Rao et al 1981, de Korte et al 1990), perhaps resulting in the appearance of platelet factors in the - 100- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses plasma and changes in platelet behaviour. One such factor is the dense granule component 5-HT which is a platelet stimulator. Measurement of 5-HT levels in PPP derived from PRP stored up to 24 hour, however, revealed no variations, indicating that it has no part in influencing the observed variations of the aggregatory responses. Fijnheer et al (1992) tested the hypothesis that stored PRP may contain an inhibitor of platelet function and, indeed, showed that platelets release nucleotides that inhibit platelet function. The finding in this study that the aggregatory responses for undiluted PRP declined to a greater extent than those for diluted PRP may therefore reflect differences in the concentrations of inhibitory nucleotides. -101- Chapter 3 : Factors Influencing Stability of In Vitro Platelet Responses 3.6 Conclusions This study confirms that platelet activity is highly dependent upon the conditions under which PRP is stored. Therefore, before embarking upon a study on platelet function, the experimental conditions to be employed should be carefully considered. Failure to do so may lead to the generation of spurious results. The results presented suggest that in order to ensure optimal platelet stability, PRP should be stored at room temperature (22 °C) in the absence of air and tested within 4 hours of sampling. Also a decision about platelet count adjustment should be made. The present findings would indicate that differences between individuals in platelet aggregation arise fi’om intrinsic differences in platelet sensitivity rather than differences in platelet count. Thus, if the experimental objective is to compare platelet responsiveness to adrenaline or ADP in different individuals no adjustment of platelet count should be made, as indeterminate changes in platelet sensitivity may occur. If, however, the objective is to test the effects of drugs on platelet aggregation, standardisation of the platelet count may be necessary to standardise the ratio of ligand to receptor. 102- Chapter 4 A study of the effects of naftopidil and doxazosin on In vitro platelet aggregation induced by single agonists. - 103- Chapter 4: Drugs' effects on platelet aggregation Induced by single agonists 4.1. Introduction It has been suggested that enhanced platelet activity in hypertension may contribute to the increased cardiovascular risk in this condition (Kjeldsen et al 1989, Nyrop and Zweifler 1988). Accordingly, drugs that reduce not only blood pressure but also platelet aggregation could represent a therapeutic advantage. On the other hand, drugs which increase platelet activity may exacerbate the complications of primary hypertension. Various antihypertensive drugs in current use have been examined for their effects on platelet function and been found to have inhibitory, excitatory or no effects on various processes associated with platelet activation, i.e. adhesion, secretion and aggregation (Hjemdahl et al 1991, Rostagno et al 1991, Islim et al 1992). The modes whereby drugs induce an anti-platelet action include, among others, cyclo-oxygenase inhibition e.g. by aspirin (Taylor et al 1992), cyclic AMP elevation e.g. by iloprost (El-Gamal et al 1992), thromboxane A 2 synthase inhibition e.g. by ridogrel (Weber et al 1992) and thrombaxne Aj receptor antagonism e.g. by vapiprost (Takiguchi et al 1992). Platelet membranes possess «2“^^renoceptors which mediate adrenaline-induced aggregation, and the antagonism of these receptors, by yohimbine for example, has been reported to inhibit platelet aggregation induced by adrenaline and other agonists (Grant and Scrutton 1979, Berlin et al 1991). Interestingly, the aj-selective adrenoceptor blocker prazosin has also been shown to inhibit platelet aggregation in some studies (Ikeda et al 1985), although not in others (Grant and Scrutton 1980, Smith et al 1990). Urapidil, a highly selective «j-adrenoceptor blocker was also reported to interfere with platelet activation (Emanuelli et al 1988, Smith et al 1990). Doxazosin, a quinazoline -104- Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists derivative related to prazosin with selective post-junctional a^-adrenoceptor inhibitory activity (Elliott et al 1986) and which in humans attains a maximal therapeutic serum concentration of about 0.27pM (Young and Brogden 1988), has been reported to inhibit in vitro platelet aggregation indued by adrenaline, collagen and ADP (Hernandez et al 1991b). Naftopidil, ±-4-(2-Methoxyphenyl)-a-( 1 -naphthalenoxy)methyl)-1 -piperazine- ethanol, is a new a^-adrenoceptor blocker currently under clinical investigation (Sponer et al 1992), although its effects on human platelet aggregation had not yet been reported. Pharmacodynamic investigations in different animal models have revealed the following properties of naftopidil; a) The binding affinity of naftopidil to the a^-adrenoceptors is about 100 times lower than that of prazosin but is three times higher than that of urapidil (Sponer et al 1992). b) The afSnity of naftopidil for binding to (Xj- or p-adrenoceptors is about 100 times less than that of «i-adrenoceptors (Sponer et al 1992). Pharmacokinetic studies in humans have shown that the maximal therapeutic serum concentrations of naftopidil ranged between 0.31 pM and 1.91 pM (according to the manufacturer Asta Pharma). 4.2. Aim of the study The aim of the study presented in this chapter was to examine the effects of the antihypertensive drugs naftopidil and doxazosin on platelet aggregation induced by agonists beheved to play important roles in thrombosis and atherosclerosis, i.e. adrenaline, collagen, ADP and 5-HT. -105- Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists 4.3. Study design Eight healthy non-smoking males were studied (median age 30 years, range 20- 39 years) none of whom had consumed any aspirin-like drugs for at least 2 weeks before the study. Four concentrations of naftopidil (0.4, 2, 10, and 40 pM) and doxazosin (0.3, 1.5, 7.5, and 30 pM), equivalent, respectively, to the reported therapeutic plasma free drug concentration and 5 times, 25 times, and 100 times that concentration, were tested. However, when taking into account the extents of the binding of the two drugs to plasma proteins, which is 95-97% for naftopidil (according to the manufacturer, Asta Pharma) and 98-99% for doxazosin (Young and Brogden 1988), the lowest concentrations of naftopidil, i.e. 0.4pM, and doxazosin, i.e. 0.3 pM, tested would probably give rise to extremely low free drug concentrations in plasma (0.02pM and 0.01 pM for naftopidil and doxazosin, respectively), whilst the highest concentrations would be close to the reported plasma free drug concentrations (2pM and 0.6pM for naftopidil and doxazosin, respectively). These free drug concentrations were calculated by subtracting the applied drug concentrations from the estimated bound concentrations, according to the following formula: Free drug concentration = A - (A x Percent of protein binding) where A is equivalent to the applied drug concentration. PRP samples (270 pi), prepared as described in chapter 2 (2.1), with platelet counts adjusted to 200x1 0^/L, were incubated at 37°C for one minute prior to the addition of 15 pi of naftopidil (0.4, 2,10 and 40 pM, final concentrations) or doxazosin (0.3, 1.5, 7.5 and 30 pM, final concentrations), incubation then being continued for __ Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists another minute whereupon platelet aggregation was induced by the addition of 15 pi of adrenaline (0.25, 0.5, 1, 2, 4, 8 and 16 pM, final concentrations), ADP (0.25, 0.5, 1, 2, 4, 8 and 16 pM, final concentrations), collagen (0.25, 0.5, 1, 2, 4 and 8 pg/ml, final concentrations) or 5-HT (10 pM, final concentration). PRP samples, pre-incubated with distilled water (naftopidil and doxazosin vehicle, see section 2.1.3.) served as controls, and platelet aggregation was measured as described in chapter 2 (2.2.4). -107- Chapter 4: Drugs’ effects on platelet aggregation induced by single agonists 4.4. Results 4.4.1. The effects of naftopidil and doxazosin on adrenaline- induced platelet aggregation The dose-response curve obtained with adrenaline-induced platelet aggregation was shifted slightly to the right by naftopidil but only at the highest concentration tested, i.e. 40 pM, ( Figure 4.1 A, Appendix, Table 12).This shift was accompanied by an increase of 47.6±16.3 % (95% Cl from 9.32 to 85.9%, P<0.05, n =8 ) in the EC# ( Table 4.1). Doxazosin, however, had no effect on adrenaline-induced aggregation ( Figure 4. IB, Appendix, Table 12) or on the adrenaline EC# at any of the concentrations tested. 4.4.2. The effects of naftopidil and doxazosin on ADP- and collagen-induced platelet aggregation The dose-response curves obtained for platelet aggregation induced by collagen (Figure 4.2, Appendix, Table 13) and ADP (Figure 4.3, Appendix, Table 14) were unaffected either by naftopidil or doxazosin, with no significant differences in the EC# values being observed (Table 4.1). 4.4.3 The effects of naftopidil and doxazosin on 5-HT- induced platelet aggregation 5-HT (10 pM) induced platelet shape change and weak reversible aggregation. This weak aggregation was significantly inhibited by naftopidil but, as with adrenaline, only at the highest concentration tested, i.e. 40 pM, (Figure 4.4A, Appendix, Table 15), % inhibition being 23.9±5.6 (95% Cl from 10.7 to 37.1%; P<0:01, n=8).0n the other hand, doxazosin did not inhibit 5-HT-induced platelet aggregation at any of the concentrations tested ( Figure 4.4B, Appendix, Table 16). - 108 - Chapter 4: Drugs’ effects on platelet aggregation induced by single agonists Table 4.1 Efifect of naftopidil and doxazosin on EC# values calculated from the dose-response curves plotted for adrenaline, collagen and ADP. (*: P < 0.05 compared with control). EC50 in presence of EC50 in presence of Agonist ECjo control Naftopidil (40pM) Doxazosin (30pM) Adrenaline 1.23 ±0.25 pM 1.73 ±0.36 pM* 1.24 ±0.23 pM Collagen 0.88 ± 0.17 pg/ml 0.97 ± 0.17 pg/ml 0.95 ±0.11 pg/ml ADP 1.46 ±0.21 pM 1.46 ±0.19 pM 1.51 ±0.21 pM - 109- Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists F igure 4.1 Platelet aggregation induced by adrenaline (0.25-16|iM ) in PR P, in the absence (control) and presence o f naftopidil 40 pM (A ) and doxazosin 30pM (B ). D ata are also presented in the appendix, T able 12. (*; P < 0.05, and **: P< 0.01 com pared w ith control, n=8) 100 80 c o CiO — E 60 (/) c o I— 40 20 Adrenaline Concentration (/xM) 100 80 c o — E 60 cCO L.o I— 40 L i 20 Adrenaline Concentration (yCzM) - 110- Chapter 4: Drugs' efTects on platelet aggregation induced by single agonists Figure 4.2 Platelet aggregation induced by collagen (0.25-8|ig/ml) in PRP, in the absence (control) and presence of naftopidil (A) and doxazosin (B). Data are also presented in the appendix. Table 13. (n= 8 ) A 100 r o Control • Naftopidil O.AfxM V N aftopidil II uLM 80 c T Naftopidil 10/LtM o □ Naftopidil 40/J.M E 60 - (/) C D L. 40 - x: CD 20 - 0 0.25 0.5 1.0 2.0 4.0 Collagen Concentration (/xg/ml) B 100 O Control □ D oxazosin 0.3)uM D oxazosin 1 .5fjM 80 ▼ c D oxazosin 7.5)U.M o V *co 0 Doxazosin 30/uM — E 60 COc o L. 40 - X u> 20 - 0 0.25 0.5 1.0 2.0 4.0 Collagen Concentration (yug/ml) -111- Chapter 4; Drugs' efTects on platelet aggregation induced by single agonists Figure 4.3 Platelet aggregation induced by A D P (0.25-16jiM ) in PRP, in the absence (control) and presence o f naftopidil (A ) and doxazosin (B). D ata are also presented in the appendix. T able 14. (n=8) A 100 r o Control • Naftopidil 0.4//M V Naftopidil 2/l/M T Naftopidil 10//hl 80 - □ Naftopidil 40//M 60 - _C 4 0 - O) 20 - 0 0.25 0.5 1.0 2.0 4.0 8.0 16.0 ADP Concentration (/^M) 100 O Control 0 Doxazosin 0.3//hi V D oxazosin 1 80 ▼ Doxazosin 7.5//M c □ Doxazosin 30//M o V) — E 60 oo c o _ c 40 O) —I 20 0.25 0.5 1.0 2.0 4.0 8.0 ADP Concentration (/i-M) -112 Chapter 4: Drugs’ effects on platelet aggregation induced by single agonists Figure 4.4 Platelet aggregation induced by 5-H T (lO jaM ) in PR P, in the absence (control) and presence o f naftopidil (A ) and doxazosin (B ). D ata are also presented in the appendix. Table 15. (n=8) A 1 4 12 10 8 6 4 2 0 Control Naftopidil B 1 4 c o (/) (/) E 00c o I— *_1 Control - 113 - Chapter 4; Drugs’ effects un platelet aggregation Induced by single agonists 4.5. Discussion In this study naftopidil and doxazosin were tested at concentrations similar to the plasma concentrations observed therapeutically, having taken into account their extensive binding to plasma proteins. Thus, the lowest concentrations used were almost certainly considerably lower than the reported free drug concentrations, and the highest concentrations were equivalent to the reported free drug concentrations. The results obtained with the higher concentrations are therefore probably more reflective of the in vivo situation. Naftopidil and platelet aggregation Naftopidil, inhibited 5-HT-induced platelet aggregation and produced a small shift of the adrenaline dose-response curve only at the highest concentration used, i.e. 40 |iM. The observation that the inhibition of adrenaline- and 5-HT-induced aggregation did not occur at lower concentrations of naftopidil may indicate that the action of its higher concentration is produced by competition with the agonists which act on platelet a2-adrenoceptors and 5-HT^ receptors which are, respectively, adrenaline and 5-HT (Steer and Atlas 1982, De Clerk et al 1984). In addition, an inhibitory action by naftopidil on further points of the receptor-coupling mechanisms involved in aggregation, e.g. calcium mobilization and thromboxane A 2 generation are also perhaps indicated. When aggregation was induced by ADP and collagen, naftopidil failed to produce shifts of their dose-response curves, contrasting with the inhibitory action seen on aggregation induced by 5-HT or adrenaline.This may indicate that the anti-platelet effects of naftopidil are specific for 5-HT2 and (%2-adrenoceptors. -114- Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists Doxazosin and platelet aggregation Doxazosin, even at the highest concentrations used, failed to inhibit platelet aggregation induced by any of the agonists used. This contrasts with the report by Hernandez et al (1991b) that doxazosin produced strong inhibition of ADP-, adrenaline- and collagen -induced aggregation. This discrepancy may be reflective of the different vehicles used in preparing the drug solutions. In preparing doxazosin for the present study distilled water was used, whereas, methanol, at a final concentration of 3%, was used in the experiments reported by Hernandez et al (1991b). In preliminary experiments for this study, methanol at this concentration was found to inhibit platelet aggregation induced by adrenaline 2pM (49.1±1.7% inhibition, P<0.01; n=3), and there is little doubt that the interpretation of platelet aggregatory data can be compromised by the use of organic solvents for solubilizing drugs, as some exhibit anti-platelet activity themselves. The dose-response curve approach to studying anti-platelet actions of drugs In general, the effects of antihypertensive drugs on platelet aggregation in vitro have been studied using single agonists at one or two concentrations (Hjemdahl et al 1991). This, however, may have important implications for the results obtained as the aggregatory responses seen with low concentrations of agonists like adrenaline and ADP are different fi“om those obtained at higher concentrations. Monophasic aggregation induced by sub-threshold concentrations of adrenaline is not accompanied by the secretion of platelet granular contents or by thromboxane A 2 generation and reflects only the initial events in the agonist-receptor interaction (Siess 1989). Therefore, the potential effects of drugs on transduction mechanisms, e.g. TXA 2 formation and granular release, will not be evident with subthreshold concentrations of agonist. On the other hand, using high agonist concentrations may mask any competitive anti-platelet actions of drugs. -115 Chapter 4: Drugs’ effects on platelet aggregation induced by single agonists The relevance of the platelet agonists tested to hypertension-related cardiovascular disease Commonly, when antihypertensive drugs are studied for their effects on platelet aggregation agonists are used regardless of their relevance to hypertension-related cardiovascular disease. In the present study, however, the platelet agonists used are believed to play an important role in thrombosis and atherosclerosis. Thus, 5-HT, although a weak agonist by itself, was tested because it may have an important role in thrombosis through its potentiation of the effects of other platelet agonists (De Clerk et al 1988, McAulifife et al 1993). ADP, released by activated platelets or from damaged endothelium, may also be important with respect to the growth of mural thrombi on exposed sub-endothelial collagen (Bom and Kratzer 1984, Wagner and Hubbell 1992). Fibrillar collagen exposed at sites of vascular injury serves as a primary activator of platelet aggregation (Chesebro et al 1992), whilst, adrenaline, a substance of recognised importance in hypertension, may stimulate platelets by itself or potentiate the effects of other platelet agonists. Thus, in hypertension, platelets may be stimulated not only by raised plasma adrenaline concentrations but also by agonists present at sites of vascular injury, i.e. collagen and ADP, or by factors released by platelets as a result of increased haemodynamic stress (Marglit and livine 1992). - 116 Chapter 4: Drugs’ efTects on platelet aggregation induced by single agonists 4.6 Conclusions This study demonstrated that naftopidil, at concentrations comparable to its plasma therapeutic levels, taking into account its binding to plasma proteins, has the ability to slightly and selectively inhibit platelet aggregation induced by adrenaline and 5-HT but not that induced by collagen or ADP. This incomplete inhibition of platelet aggregation produced by naftopidil may be advantageous because the drug may have anti-thrombotic potential exclusive of the unfavourable impairment of the haemostatic mechanism caused by complete and prolonged platelet inhibition seen with other drugs. No inhibitory action by doxazosin could be demonstrated on aggregation induced by any of the agonists used. This lack of efifect, which contrasts with that reported for doxazosin by Hernandez et al (1991b), points to the importance of considering the possible anti-platelet effects of drug vehicles when studying the actions of drugs on platelet function. Moreover, drug effects may not be revealed in the presence of plasma proteins which bind drugs thereby reducing their effective free concentrations. Using high concentrations of platelet agonists may also mask any competitive drug effects. Therefore, different experimental approaches are required when studying the actions of drugs on platelet activity in order to reveal effects masked using some procedures. -117- Chapter 5 A Study of the effects of naftopidil and doxazosin on in vitro platelet aggregation induced by synergistic interactions between adrenaline and other agonists - 118 - Chapter 5: Drugs’ efTects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.1. Introduction Platelet activation is tightly regulated under physiological conditions but increased in pathological conditions such as thrombosis, atherosclerosis, diabetes and hypertension (Kjeldsen et al 1989, Sowers et al 1993). Many substances including hormones, autacoids and neurotransmitters act synergistically to stimulate or inhibit platelet activation (Ware et al 1987). Therefore, it could be assumed that substances which potentiate platelet function would aid in haemostasis or contribute to thrombosis at sites of endothelial injury. The ability of adrenaline to induce platelet aggregation in vitro has been interpreted as indicating that platelet activation by catecholamines in vivo may be regarded as a possible link between stress and cardiovascular disease (Kjeldsen et al 1991). However, the micro molar concentrations of adrenaline used routinely in vitro are not found normally in vivo. The presence of multiple platelet agonists at the sites of vascular injury, however, such as collagen and ADP or in circulating blood such as vasopressin, 5-HT or vasoactive peptides may lead to the potentiation of adrenaline- induced platelet aggregation, especially in conditions such as primary hypertension in which raised plasma adrenaline concentrations have been reported (Kjeldsen et al 1991). A variety of vasoactive peptides, stored and released from perivascular nerves or other tissues (endothelium, myocardium, smooth muscles), have marked effects on the cardiovascular system and may play a role in platelet/vessel interactions in vivo. Such peptides include endothelin, atrial natriuretic peptide, and Neuropeptide Y. - 119- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.1.1. Endothelin Endothelin (ET) is a 21 amino acid peptide which was initially isolated from conditioned medium of cultured porcine aortic endothelial cells (Yanagisawa et al 1988). The existence of three structurally and pharmacologically distinct isoforms of ET designated ET-1, ET-2 and ET-3, was predicted from the finding in man, rat and pig of three distinct endothelin related genes (Inoue et al 1989). Endothelin has been found to be released from endothelial and non-endothelial cells and to exist at relatively high levels in the central and peripheral nervous system (Kramer et al 1992). Endothelial cells, however, appear to produce exclusively ET-1, indicating the importance of ET-1 in the regulation of vascular tone (Masaki et al 1994). ET-1 does not appear to be stored intracellularly in most tissues. The expression of endothelin mRNA and the release of ET is stimulated by various factors including thrombin, adrenaline, angiotensin n, arginine vasopressin and transforming growth factor 6-1 (Lüscher et al 1992, Masaki et al 1991). Pre-pro-endothelin, which is composed of 212 amino acids, is generated and cleaved in the cytoplasm by specific endoproteases to form pro-endothelin-1, a 38-amino acid precursor (big endothelin) (Yanagisawa et al 1988). Big endothelin-1 is probably secreted from endothelial cells and converted to ET- 1 on the endothelial surface by the action of a membrane-bound endothelin-converting enzyme (Masaki et all 991). ET-1 is stable in plasma but can be enzymatically degraded by neutral endopeptidase, however, the biological significance of this process is unclear (Haynes and Webb 1993). ET is the most potent endogenous vasoconstrictor yet identified. Intravenous administration of ET to animals elicits an initial decrease in systemic blood pressure ___ Chapter 5: Drugs’ effects on aggregation Induced by synergistic interactions between adrenaline & other agonists followed by a prolonged pressor response associated with marked vasoconstriction (Yanagisawa et al 1988). The depressor and pressor responses to ET are mediated by different mechanisms (Le Monnier de Gouville et al 1990). ET-induced vasodilation is thought to involve contributions from endothelium-derived relaxing factor (EDRF), recently identified as nitric oxide (NO) (Lusher and Tanner 1993), and prostacyclin, released by ET-1 from endothelial cells (Masaki et al 1991), but this is still a matter of controversy. ETs act not only on vascular but also on non-vascular systems. Additional responses to ET include bronchoconstriction, stimulation of aldosterone secretion, promotion of cellular proliferation or cellular hypertrophy, and stimulation of atrial natriuretic peptide secretion (Masaki et al 1994, Hirata 1989). The diverse responses to ETs are mediated by specific receptors distributed in blood vessels, heart, adrenal gland, kidneys and brain (Davenport et al 1989). Two endothelin receptor subtypes designated ET^ and ETg have been characterised (Masaki et al 1994). Endothelial cells express ETg receptors which mediate vasodilation (Masaki et al 1994). The contraction of vascular smooth muscle may be mediated by either ET^ or ETg receptors (Warner et al 1994). Both types of ET receptors are coupled to phospholipase C and phosphoinositide turnover mediated via a pertussis toxin-insensitive G-protein (Masaki et al 1994). The reported concentrations of immunoreactive endothelin in venous plasma vary between 0.25 and 20 pg/ml (0.1- 8 pM) (Haynes and Webb 1993). The peripheral circulating levels of the peptide, however, are approximately tenfold lower than those which cause vascular contraction in vitro or in vivo, although concentrations at sites of vascular spasm may be greater (Haynes and Webb 1993). Moreover, low levels of ET-1 - 121 - C hapter 5: Drugs’ efTects on aggregation induced by synergistic interactions between adrenaline & other agonists may amplify the constrictor effects of other circulating hormones or autacoids (Balligand and Godfraind 1994). The circulating levels of ET are normal, or only marginally elevated in most patients with primary hypertension (vanhoutte 1993). However, elevated immunoreactive ET-1 levels have been reported in patients with vasopastic angina, acute myocardial infarction, renal failure (Haynes and Webb 1993) and atherosclerosis (Lerman et al 1991). Accordingly, ET has been implicated in the pathogenesis of coronary ischaemic syndrome, especially vasospasm (Kurihara et al 1989). 5.1.2. Neuropeptide Y (NPY) NPY, a 36-amino acid peptide, is a potent vasoconstrictor and occurs in many sympathetic nerves, co-localised with catecholamines, and in adrenal medullary chromaffin cells (Hughes 1994). NPY is also synthesized at the megakaryocyte level, and is stored and released by platelets (Myers et al 1988). In several species, including humans, intravenous infusion of NPY increases blood pressure and peripheral resistance associated with a reduction in heart rate and cardiac output (Pemow 1988). These effects of NPY are mediated by two NPY receptor subtypes, post-junctional Y^ and pre-junctional Y 2 subtypes (Wahlstedt et al 1986). Post- junctionally, NPY may act as a co-transmitter, potentiating the pressor effects of noradrenaline, whereas pre-junctionally, NPY inhibits sympathetic neuronal release of noradrenaline (Westfall et al 1990), resulting probably in a fall in heart rate and cardiac output. Both NPY receptor subtypes (Yj and % ) are linked to the pertussis toxin- sensitive G protein. Activation of NPY receptors in many tissues has been shown to ___ Chapter 5: Drugs’ effects on aggregation Induced by synergistic interactions between adrenaline & other agonists inhibit cAMP generation and to release calcium from intracellular stores by IP3- dependent or IP^-independent pathways (Hughes 1994). In humans, circulating levels of NPY, probably released from sympathetic neurons, are in the range of 10-80 pmol/1. Elevated levels of plasma NPY have been reported in patients with phaeochromocytomas, neuroblastomas and ganglioneuroblastomas (Hughes 1994), and in hypertensive patients (Erlinge 1992). However, there is limited evidence for the involvement of NPY in human and animal hypertension. 5.1.3. Atrial natriuretic peptide (ANP) A regulatory function of the cardiac atrium in the control of the diuretic and natriuretic actions of the kidney has been known since the 1950s (Henry et al 1956). The discovery in the early 1980s that rat atrial, but not ventricular, extracts caused a potent and rapid natriuretic-diuretic response clearly demonstrated that an atrial natriuretic factor is mainly responsible for the regulatory function of the heart on the kidney (de Bold et al 1981). This atrial natriuretic factor was extracted from rat atria and from other species, including human atrial tissue, and was identified as a peptide now known as atrial natriuretic peptide or factor (ANP, ANF) (Ballerman and Brenner 1985). Other tissues such as brain, pituitary, adrenal medulla and lung also contain ANP in small quantities (Inagami 1989). In the heart, ANP is stored within secretory granules predominantly in the pro-ANP form (126 amino acids) which is converted, perhaps at the plasma membrane, to the active circulating form (a-ANP, 28 amino acids) during the secretion process (Inagami 1989). The primary factors influencing ANP secretion seem to be atrial stretch (Au et ___ Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists al 1990), intra-atrial pressure and heart rate (Ledsome and King 1991). However, ANP can also be released in tissue cultures suggesting the involvement of receptor-mediated stimulation of secretion (Raskoaho et al 1991). The reported concentrations of plasma ANP in normal subjects are in the range of 10-60 pg/ml (approximately 4-20 pm) (Richards 1987). About two-fold to four-fold higher plasma levels of ANP have been demonstrated in patients with congestive heart failure who show apparent resistance to the biological effects of ANP indicating that elevated levels have some prognostic value in this condition (lervasi et al 1994). Raised plasma levels of ANP (about 1.5 fold to 3.0 fold) have also been repeatedly reported in essential hypertension, probably reflecting increased secretion of ANP due to either raised central venous pressure and/or abnormalities in cardiac structure or function (Sagnella and MacGregor 1994). Under normal physiological conditions ANP stimulates diuresis and natriuresis, and reduces vascular tone induced by vasoconstrictors such as noradrenaline and angiotensin H. The effects of ANP are mediated by specific receptors, coupled to guanylate cyclase, which occur in several target tissues e.g. renal, vascular and adrenal tissues (Inagami 1989). It has been demonstrated that in hypertensive patients, the induction of prolonged physiological increases in circulating ANP are associated with reductions in plasma sodium levels and blood pressure without major compensatory activation of the sympathetic and renin-angiotensin systems (Cusson et al 1990). These demonstrations indicate that essential hypertension is not associated with refractoriness to the actions of ANP and provide a basis for ANP as a potentially important therapeutic factor (Sagnella and MacGregor 1994). -124- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.2. Aim of the Study The aim of the study presented in this chapter was to examine the previously reported synergistic interactions between the platelet agonists ADP, collagen and 5-HT and adrenaline in induction of platelet aggregation (Huang and Detwiler 1981, De Clerk et al 1988) (see section 5.1.), to study any excitatory or inhibitory actions of the vasoactive peptides, endothelin, NPY and ANP, on adrenaline-induced platelet aggregation and to examine the effects of naftopidil and doxazosin on the synergistic interactions between adrenaline and other agonists. 5.3. Study Design The excitatory effects of subthreshold concentrations of ADP, collagen and 5- HT were examined on platelet aggregation induced by sub-threshold concentrations of adrenaline (0.03, 0.06, 1.25, 0.25, 0.5 and IjiM), whereas the effects of endothelin, NPY and ANP were examined using sub- and supra-threshold concentrations of adrenaline (0.25, 0.5, 1, 2, 4, 8 , 16pM) in order to detect any excitatory or inhibitory actions produced by the peptides. PRP samples (270pl), prepared as described in chapter 2 (2.2.1),with platelet counts adjusted to 200x 10^/L, were pre-incubated for 2 minutes at 37 C, aggregation then being induced by sub-threshold concentrations of adrenaline (0.03, 0.06, 0.125, 0.25, 0.5 and IpM) alone or by adrenaline in the presence of ADP (0.5pM), collagen (0.125pg/ml) or 5-HT (2.5pM), which were added 15 seconds before the addition of adrenaline. The effects of the vasoactive peptides were studied by incubating PRP samples with endothelin (InM and IpM), NPY (0.1 pM and IpM) or ANP (1 nM and 10 nM) -125- Chapter 5: Drugs’ efiects on aggregation induced by synergistic interactions between adrenaline & other agonists for one minute, following one minute pre-incubation at 37°C, and aggregation was induced by a logarithmic range of adrenaline concentrations (0.25-16 pM). The effects of the peptides themselves on platelet aggregation were tested by incubating the peptides alone in PRP for 5 minutes. The effects of naftopidil and doxazosin on potentiated adrenaline-induced aggregation were investigated by pre-incubating PRP samples for one min at 37 °C followed by 1 min incubation with increasing concentrations of naftopidil (0.4, 2, 10 and 40pM) or doxazosin (0.3, 1.5, 7.5 and 30 pM), whereupon platelet aggregation was induced by sub-threshold concentrations of adrenaline in the presence of a potentiating agent. In all ejqjeriments, PRP samples pre-incubated with distilled water (naftopidil and doxazosin vehicle) served as controls and platelet aggregation was measured as described in chapter 2 (2.2.4). -126- Chapter 5; Drugs’ efiects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.4. Results 5.4.1. The effects of ADP, collagen and 5-HT on adrenaline-induced platelet aggregation A marked potentiation of the aggregatory responses to sub-threshold concentrations of adrenaline resulted from the prior addition of subthreshold concentrations of ADP, collagen or 5-HT ( Figure 5.1, Appendix, Table 17) with the steep portion of the potentiated dose-response curves being seen between 0.125 and 0.5 pM concentrations of adrenaline. 5.4.2. The effects of naftopidil and doxazosin on the potentiation by ADP, collagen and 5-HT of adrenaline-induced platelet aggregation. Naftopidil and, to a lesser extent, doxazosin inhibited adrenaline-induced aggregation potentiated by ADP ( Figure 5.2, Appendix, Table 18), collagen ( Figure 5.3, Appendix, Table 19) or 5-HT ( Figure 5.4, Appendix, Table 20) in a dose-dependent manner. The inhibitions produced by the two drugs of the potentiated aggregatory responses to 0.25 pM adrenaline are presented in table 5.1. The maximum inhibition produced by 40 pM naftopidil was 70.9±7.8% (95 Cl from 52.5% to 89.3%; P<0.001,n=8) and was seen when adrenaline (0.25pM) was used in combination with collagen. On the other hand, the maximum inhibition produced by doxazosin was 42.2% ±11.3 (95% Cl from 15.5% to 68.9%; P<0.01) again occurring when adrenaline (0.5pM) was used in combination with collagen. -127- Chapter 5; Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists Figure 5.1 Platelet aggregator>' responses to subthreshold concentrations of adrenaline (0.03- l|iM) potentiated by ADP (0.5pM) (A), collagen (0.125pg/ml) (B) and 5-HT (2.5pM) (C). Data are also presented in the appendix. Table 17.( n= 8 , except at IpM adrenaline where n=4). O Adrenaline alone c- 100 o # ADP a lo n e V Adrenaline+ADP ^ 80 E g 6 0 o 4 0 O) 20 c 100 O Adrenaline alone o 0 Collagen alone ^ 80 V Adrenaline+Collagen E g 60 p I— 4 0 D) 20 c O Adrenaline alone # 5-HT alone V Adrenaline+5-HT D) 0.03 0.06 0.125 0.25 0.5 Adrenaline Concentration (juM) - 128- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists Figure 5.2 Effect of naftopidil (A ) and doxazosin (B ) on the potentiation by A D P (0.5pM ) o f platelet aggregation induced by subthreshold concentrations o f adrenaline (0 .0 3 -IpM ). D ata are also presented in the A ppendix, T able 18. (*: P< 0.05 com pared w ith co n tro l, n=8, except at Ip M where n=5). . 0 A d renaline alone ADP a lo n e 100 r # V Adrenaline+ADP(control) T Naftopidil 0.4^M □ N aftopidil 2/J.M 80 - A N aftopidil 10/xM ■ Naftopidil 40yU,M 60 x: 4 0 - cn 20 - 0 0.03 0.06 0.125 0.25 0.5 1.0 Adrenaline Concentration (ytzM) B O Adrenaline alone 100 ^ ADP a lo n e O Adrenaline +ADP (control) □ D oxazosin 0.3/xM 80 - ▼ D ox azo sin 1 .5juM c V D oxazosin 7,5/uM o V) 0 Doxazosin in E 60 - (/) c o X 40 - O) 20 - 0 0.03 0.06 0.125 0.25 0.5 1.0 Adrenaline Concentration (/zM) -129 Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists Figure 5.3 Effect of naftopidil (A) and doxazosin (B) on the potentiation by collagen (0.125pg/ml) of platelet aggregation induced by subthreshold concentrations of adrenaline (0.03- l|iM). Collagen had no aggregatory effect when applied on its own. Data are also presented in the Appendix, Tablai9. (*: P<0.05,**: P<0.01 compared with control, n =8 except at IpM where n=4). 100 - O Adrenaline clone # Adrenaline+Collagen (Control) V Naftopidil 0.4/xM 80 ~ ▼ Naftopidil □ Naftopidil 10yixM ■ Naftopidil 40yuM 60 - 40 - 20 - 0 0.03 0.06 0.125 0.25 0.5 Adrenaline Concentration (/zM) B 100 O Adrenaline alone 0 Adrenaline+Collagen (Control) V D o x azo sin 0.3/xM 80 - ▼ D oxazosin 1.5/xM □ D o x azo sin 7.5/i.M ■ D o x azo sin 30/.iM 60 - 40 - O) l_i 20 - 0 0.03 0.06 0.125 0.25 0.5 Adrenaline Concentration (/.z-M) -130- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists F igure 5.4 Effect of naftopidil (A ) and doxazosin (B ) on the potentiation by 5-H T (2.5pM ) o f platelet aggregation induced by subthreshold concentrations o f adrenaline. D ata are also presented in the A ppendix, T able 20. (*: P< 0.05 com pared w ith control, n=8, except at IpM w h e r e n = 5 ) A 100 r O Adrenaline alone # 5-HT alone V Adrenallne+5-HT (Control) ▼ Naftopidil 0.4^M 8 0 - □ Naftopidil IjiU ■ Naftopidil 10/xM A Naftopidil 40/uM 6 0 - 4 0 - !_j 20 - 0.03 0.06 0.125 0.25 0.5 Adrenaline Concentration (/xM) B 100 O Adrenaline clone ^ 5—HT alone V Adrenaline+5—HT (Control) T D oxazosin 0.3yUM 8 0 - □ D oxazosin 1 .5yU.M ■ D oxazosin 7.5yU.M A D oxazosin 30^iM 6 0 - SI 4 0 - 0 5 20 - 0 0.03 0.06 0.125 0.25 0.5 Adrenaline Concentration (yuM) -131- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists Table 5.1 % inhibition by naftopidil and doxazosin of the aggregatory responses to adrenaline (0.25 pM) potentiated by ADP, 5-HT or collagen. Potentiating Naftopidil Naftopidil Doxazosin Doxazosin agent 40 pM 10 pM 30 pM 7.5 pM ADP 58.3 38.4 24.6 18.4 (36.8 to 79.8) (17.4 to 59.4) (3.2 to 46.0) (0.7 to 36.1) (0.5 fiM) PO.OOl PO.Ol PO.05 PO.05 5-HT 58.9 20.4 28.0 0.9 (40.0 to 77.8) (-5.4 to 46.2) (4.1 to 51.9) (-11.5 to 13.4) (2.5 fiM) PO.OOl P=NS PO.05 no inhibition Collagen 70.9 20.3 28.9 -1.9 (52.5 to 89.3) (-2.7 to 43.2) (10.0 to 47.8) (-30.7 to 26.8) (0.125 lig/mi) PO.OOl P=NS PO.Ol no inhibition Results are expressed as means and 95% confidence intervals (n= 8 ). NS= not statistically significant. - 132- Chapter 5: Drugs’ efiects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.4.3. The effects of the vasoactive peptides endothelin, NPY and ANP on adrenaline-induced platelet aggregation The three vasoactive peptides did not induce platelet aggregation on their own and had no effects, either excitatory or inhibitory, on adrenaline-induced platelet aggregation. This was apparent from the absence of any significant shift in the adrenaline dose-response curves when PRP samples were pre-incubated with endothelin 1 ( Figure 5.5, Appendix, Table 21), NPY (Figure 5.6, Appendix, Table 22) and ANP ( Figure 5.3, Appendix, Table 23). As the vasoactive peptides were found not to influence adrenaline-induced platelet aggregation, experiments with naftopidil or doxazosin were not performed. Figure 5.5 Effect of endothelin 1 (ET-1) on adrenaline-induced platelet aggregation.. Data are also presented in the appendix. Table 21. (n= 6). 100 O Control # E ndothelin 1nM V Endothelin lyuM 80 60 40 U) 20 0 0.25 0.5 1.0 2.0 4.0 8.0 16.0 Adrenaline Concentration -133- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists Figure 5.6 Effect of neuropeptide Y (NPY) on adrenaline-induced platelet aggregation. Data are also presented in the appendix. Table 22. (n = 6). 100 O Control • NPY O.lyuM V NPY lyLtM 80 c o *C/3(/) E 60 v\ c a h- - 40 O) _j 20 0.25 0.5 1.0 2.0 4.0 8 .0 16.0 Adrenaline Concentration (ytiM) Figure 5.7 Effect of atrial natriuretic peptide (ANP) on adrenaline-induced platelet aggregation. Data are also presented in the appendix. Table 23. (n = 6). 100 O Control # ANP 1nM V ANP lOnM 80 60 40 O) L i 20 0 0.25 0.5 1.0 2.0 4.0 8.0 16.0 Adrenaline Concentration (/uM) - 134- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.5. Discussion 5.5.1. The potentiation by ADP, collagen and 5-HT of adrenaline-induced aggregation The synergistic interactions between platelet agonists in the induction of platelet aggregation in vitro is a well known phenomenon (Huang and Detwiler 1981, Petty and Scrutton 1993). However, previous studies of such interactions have invariably involved using adrenaline as the potentiator of aggregation induced by other agonists, rather than as the primary agonist (De Clerk et al 1988, Vanags et al 1992). In the present study, however, a range of adrenaline concentrations were used with fixed concentrations of other agonists serving as potentiating agents. Thus, ADP, collagen and 5-HT, which are believed to play an important role in the processes of thrombosis and atherosclerosis, potentiated the responses to adrenaline.This potentiation may correspond to the situation occurring in essential hypertension where increases in plasma adrenaline levels have been reported together with increased platelet aggregability (Nyrop and Zweifler 1988, Mehta and Mehta 1981, Kjeldsen et al 1989, 1991, Winther et al 1992). In hypertension, platelets may be stimulated not only by raised plasma adrenaline concentrations but also by agonists released by platelets, e.g. 5-HT, as a result of increased haemodynamic stress or by agonists present at sites of vascular injury, i.e. collagen and ADP (Margalit and Livine 1992). Precise information regarding the mechanism(s) by which synergistic interactions of platelet agonists occur is not available (Kroll and Schafer 1989), however proposed mechanisms include phosphoinositide mobilisation (Steen et al 1988, Bushfield et al 1987), protein kinase C activation (Siess and Lapetina 1989), and arachidonic acid release and metabolism (Ardlie et al 1985). Evidence indicates that increases in [Ca^^jj caused by one agonist may prime platelets for subsequent stimulation by other agonists. - 135- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists 5.5.2. Utilization of combinations of platelet agonists in drug studies Very few pharmacological studies have utilized combinations of platelet agonists when examining the effects of drugs on platelet function (De clerk et al 1988, Vanags et al 1992, Zucker et al 1993). However, by using combinations of agonists to examine drug actions it is believed that conditions are applied which more closely mimic those occurring physiologically, as in vivo platelets are exposed to more than one agonist, at any one time, and which act together to exert their physiological actions in haemostasis or their pathological effects in thrombosis and atherosclerosis ( De Clerk et al 1988, Ardlie et al 1985). Moreover, potential drug effects on the magnified responses to low concentrations of agonists may be detectable, as the masking effects of high concentrations of platelet agonists are avoided. 5.5.3. The inhibition by naftopidil and doxazosin of the potentiated responses to adrenaline The synergistic interactions between adrenaline and ADP, collagen or 5-HT were inhibited by naftopidil and, to a lesser extent, doxazosin. Therefore, the present study, taken together with the study described in chapter 4, demonstrates that applying different experimental approaches to the study of the action of drugs on platelet activity may be necessary to reveal otherwise masked effects. When the effects of doxazosin on aggregation induced by adrenaline alone were studied no inhibitory action was observed. With aggregation induced by sub-threshold concentrations of adrenaline in combination with sub-threshold concentrations of ADP, collagen or 5-HT, however, doxazosin produced dose-dependent inhibition. __ Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists As regards the mechanisms whereby naftopidil and doxazosin produce their inhibitory effects on the potentiated adrenaline responses, there are two possibilities. First, naftopidil and doxazosin may possess competitive (% 2-&drenoceptor antagonistic activity as evidenced by the inhibitory effects observed with the lower concentrations of agonists (potentiators and adrenaline) used for experiments involving potentiation of adrenaline-induced aggregation, which is overwhelmed when higher concentrations of individual agonists are used. Second, the two drugs may influence the mechanisms by which adrenaline responses are potentiated by other agonists, which may differ fi'om those by which platelets are activated by higher concentrations of adrenaline alone. 5.5.4. The vasoactive peptides and platelet aggregation The results presented in this chapter regarding the effects of vasoactive peptides on platelet aggregation indicate that ET-1, NPY and ANP do not induce platelet aggregation by themselves or influence aggregation induced by adrenaline. Endothelin The inability of ET-1 to stimulate human platelet aggregation has been reported previously (Astarie-Dequker et al 1992, Pietraszek et al 1992, Battistini et al 1990, Matsumoto et al 1990, Ohlstein et al 1990). The reported effects of ET-1 on human platelet aggregation induced by adrenaline, however, are equivocal. Thus, Ohlstein et al (1990) showed that endothelin did not influence adrenaline-induced aggregation whereas Matsumoto et al (1990) reported that ET-1 potentiated platelet aggregation induced by sub-threshold concentrations of adrenaline, although this was demonstrated in only 52.2% of the subjects studied, and no significant potentiation of adrenaline- induced changes of other parameters such as [Ca^^j, PHi and membrane potential could ___ Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists be detected. Also, it was shown that ET-1, when pre-incubated with PRP for 3 min, inhibited the serotonergic amplification of adrenaline-induced aggregation, the mechanism (s) underlying these findings however, being to be apparently complicated (Pietraszek et al 1992). ET-1 has been shown to inhibit ex vivo and in vivo platelet aggregation in animals (Herman et al 1993) but was attributed to the ability of ET-1 to induce the release of prostacyclin in amounts sufiBcient to inhibit platelet aggregation (Herman et al 1993). In conclusion, it appears that human platelets are not affected directly by ET-1 although there may be an indirect interaction in vivo. NPY It was reported that porcine NPY inhibited in vitro adrenaline-induced aggregation of human platelets (Dewar et al 1989) and accordingly it was speculated that NPY released fi’om platelets during haemostasis might act to prevent the inappropriate spread of aggregation while promoting local vasoconstriction (Dewar et al 1989). However, this could not be confirmed by other investigators (Myers et al 1991) when the effects of human NPY (InM and lOpM) on adrenaline-induced aggregation was studied in both PRP and gel-filtered human platelets. The primary role of NPY, therefore, seems to be in enhancing vasoconstriction. ANP The inability of ANP to induce human platelet aggregation has been previously reported (De Caterina et al 1985, Loeb and Gear 1988). Because ANP possesses vasorelaxant activities, it was hypothesized that it may resemble other vasodilators in -138- Chapter 5: Drugs’ effects on aggregation induced by synergistic interactions between adrenaline & other agonists their inhibitory actions on platelet function. However, using the optical aggregatory technique, De Caterina et al (1985) have reported that ANP (24- and 25- amino acids) concentrations between 4 and 400pM did not influence human platelet aggregation or thromboxane generation induced by adrenaline. On the other hand, Loeb and Gear (1988), using a quenched-flow technique coupled to single-particle counting, have reported that rat ANP (1-28 amino acids) potentiated adrenaline-induced aggregation with a maximal potentiating effect occurring between 10 to 100 pM. This effect may be due to an ANP-induced increase in platelet cGMP levels since ANP receptors has been found to be closely associated with guanylate cyclase (Inagami 1989). In the same study, however, human ANP was found not to interfere with adrenahne-induced aggregation. Human platelets are known to possess binding sites for ANP (Schiffrin et al 1991) but their physiological or functional roles are not yet clear. 5.6. Conclusions This study demonstrated that a marked potentiation of aggregation induced by subthreshold concentrations of adrenaline resulted from the prior addition of low concentrations of ADP, collagen or 5-HT. The vasoactive peptides ET-1, NPY and ANP did not influence adrenaline-induced platelet aggregation. The potentiated responses to adrenaline were inhibited in a dose-dependent manner by naftopidil and, to a lesser extent, doxazosin which suggests that the two drugs may have anti-thrombotic potential. - 139- CHAPTER 6 A study of the effects of naftopidil and doxazosin in comparison with nifedipine on platelet signal transduction mechanisms - 140- Chapter 6: Drugs’ efTects on platelet signal tranduction mechanisms 6.1. Effects of naftopidil, doxazosin and nifedipine on stimulus-induced calcium mobilization 6.1.1. Introduction Calcium plays a central role in stimulus-response coupling mechanisms in secretory and contractile cells including platelets (Siess 1989, Rink and Sage 1990, Heemskerk and Sage 1994). The storage sites for Ca^^ within platelets include the dense tubular system, dense granules, mitochondria and a plasma membrane-associated Ca^^ pool (Siess 1989). Calcium indicators The cytosolic calcium ion concentration, [Ca^^Jj, in platelets can be measured by the use of fluorescent indicators such as quin- 2, fura-2 or indo- 1, or by the use of the Ca^^-sensitive 20KDa photoprotein aequorin (Johnson et al 1985). Aequorin can be introduced into human platelets although only a small proportion of the added protein ( less than 0.05%) is incorporated, and its localization within platelets is unknown (Johnson et al 1985, Yamaguchi et al 1986). Aequorin and quin-2 appear to differ in their regional intra platelet distribution (Johnson et al 1989, Lages and Weiss 1989) as the calcium signals elicited by platelet agonists differ both qualitatively and quantitatively in aequorin- and quin- 2-loaded platelets. With aequorin, [Ca^^]^ changes are correlated with aggregation, and peak [Ca^^Ji after platelet stimulation is 2-lOpM (Johnson et al 1985). Quin-2-induced signals, however, do not always correlate with functional platelet responses (Ware et al 1986) and the fluorescence of the quin- 2-calcium complex is very insensitive to changes in the 1-lOpM range (Tsien et al 1982). Resting [Ca^^]i measured by fluorescent -141- Chapter 6: Drugs' efTects on platelet signal tranduction mechanisms indicators tend to be in the range of 70-80nM (Rink and Sage 1990) which is about 10,000 fold less than the concentration of free Ca^^ in plasma (ImM). Estimates of resting [Ca“^]j in aequorin-loaded platelets have been much higher (2-4pM) than measurements with quin-2 or fura-2 (Johnson et al 1985, Ware et al 1989). Calcium mobilization Many platelet agonists produce an increase in [Ca^^]j which causes platelet activation (Siess 1989). The increase in can be caused either by Ca^^ influx from the extracellular medium via Ca^^ channels or by liberation from one or more intracellular storage pools. The dense tubular system and the plasma membrane are the most likely sites of release of Ca^^ after platelet activation, whereas mitochondria, as in most cell types, are not a significant source of releasable Ca^"^. Most of the Ca^^ stored in dense granules occurs in an insoluble matrix together with biogenic amines and ATP, and is released into the extracellular medium as a consequence of exocytosis. In the absence of extracellular Ca^"" a rise in the cytosolic Ca^\ as indicated by an increase in quin -2 or fura -2 fluorescence or aequorin luminescence, is caused by the release of Ca^^ from intracellular stores. Thrombin, collagen, PAF, arachidonic acid, U44619 (endoperoxide analogue), vasopressin, ionomycin and A23187 have all been reported to cause a rise in cytosolic Ca^^ in the absence of extracellular (Siess 1989). Adrenaline, on the other hand, induces an aequorin-detectable Ca^^ rise only in the presence of extracellular Ca^^ (Ware et al 1986). influx The contribution of Ca^^ influx to the rise in platelet [Ca^^Ji has been determined by measuring stimulus-induced increases in [Ca^^]j in the presence and absence of - 142- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms extracellular Ca^\ Thrombin, collagen, ADP, PAF, U46619 and vasopressin produce a greater rise in [Ca^^]j in the presence of external Ca“^ than in its absence (Siess 1989). Platelets possess receptor-operated Ca^^ channels in their plasma membranes which open as a direct consequence of agonist-receptor binding. In general, Ca~^ influx through the receptor-operated channels probably accounts for the observation that several neurotransmitters can stimulate transmembraneous calcium influx in the absence of changes in membrane potential (van Zwieten et al 1987). With respect to the receptors that trigger the receptor-operated Ca^" channels, «-adrenoceptors, and especially those of the « 2-subtype, are considered to be of vital importance. This is because of the general susceptibility of (% 2-adrenoceptor-mediated vasoconstriction to impairment by blockade of the transmembraneous calcium influx caused by calcium antagonists (van Zwieten et al 1987). The participation of voltage-operated Ca^^ channels in platelet Cà^ influx is controversial. Thus, some studies have suggested that platelets, in contrast to cardiac and smooth muscle cells, do not have high threshold (L) or low threshold (T) channels and have no identifiable high affinity dihydropyridine binding sites (Ogawa and Ono 1989, Doyle and Ruegs 1983, Rink and Sage 1990). However, the existence of a voltage-operated, dihydropyridine-sensitive calcium channel (L-type) has been suggested by others (Pales et al 1991). The type and nature of the receptor-operated Ca^"" channels in the platelet plasma membrane are unknown. The membrane glycoprotein Ilb/nia complex may be involved in Ca^"' influx in resting and stimulated platelets (Rink and Sage 1990), since monoclonal antibodies against the glycoprotein Ilb/IIIa complex reduce agonist-induced Ca^^ influx - 143- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms (Fowling and Hardisty 1985). Moreover, a decrease in the number of glycoprotein nb/HIa molecules expressed on platelets that were unable to aggregate normally, either due to the congenital bleeding disorder Glanzmann's thrombasthenia or to experimental manipulation, was found to be associated with decreased platelet Ca^^ uptake (Johnston and Heptinstall 1988). Also, when incorporated into liposomes, the isolated glycoprotein Ilb/IIIa complex could act as a Ca^^ channel (Rybak et al 1988). Currently little is known about the role of inositol phosphates in mediating calcium influx directly across the platelet plasma membrane, but inositol triphosphate has been found to release Ca"" from membrane vesicles enriched with plasma membrane (Rengasamy and Feinberg 1988). 6.1.2. Aim of the Study The aim of the study was to investigate the effects of naftopidil and doxazosin on the collagen- and adrenaline- induced increase in platelet [Ca^"];. The calcium antagonist nifedipine was used as a positive control, but its application was limited to experiments in which collagen was used because of the inhibitory effects of DMSO (nifedipine vehicle) observed on adrenaline-induced responses. Thus, although DMSO did not inhibit collagen-induced platelet responses, it interfered with adrenaline-induced ones, this observation being in agreement with that of Lehuu and Curtis (1987) who found that the inhibitory effects of DMSO were greater for adrenaline—induced responses than for collagen. 6.1.3. Study design The effects of naftopidil, doxazosin and nifedipine on the increase in platelet [Ca^^Ji induced by collagen or adrenaline were examined in platelets obtained from - 144- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms healthy non-smoking male subjects aged between 28-54 years, none of whom had consumed any aspirin-like drugs for at least 2 weeks before the study. Platelets were washed and loaded with aequorin as described in Chapter. 2 (2.2.2). For control incubations samples of platelet suspension (800pi) were mixed with fibrinogen suspension ( 200pi, 0.06% final concentration) and incubated for 1 min at 37° C followed by a further min incubation in the presence of the drug vehicle (lOpl), whereupon stimulation was achieved by the addition of collagen (5 pg/ml) or adrenaline (16pM). The effects of the drugs were studied in stimulated platelet samples pre- incubated for 1 min with naftopidil (0.4, 2, 10 and 40pM), doxazosin (0.3, 1.5, 7.5 and 30pM) or nifedipine (0.28, 1.4, 7 and 28pM). As previously described for naftopidil and doxazosin, concentrations of nifedipine were tested equivalent to the reported therapeutic plasma fi’ee drug concentration (Kleinbloesem et al 1984), and 5 times, 25 times and 100 times that concentration, keeping in mind that some binding of the drug to proteins, including Bovine serum albumin (used in washed platelet preparation), present in the platelet suspension may have taken place. Platelet aggregation and the increase in [Ca^^]j as indicated by a rise in aequorin luminescence were recorded simultaneously as described in Chapter. 2 (2.2.5). The possibility of quenching of the calcium signal by the nifedipine solutions which are yellow in colour was ruled out by calibrating the apparatus, as described in Chapter. 2 (2.2.5), in the presence and absence of nifedipine. 145- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms 6.1.4. Results 6.1.4.1. Collagen- and adrenaline-induced increase in [Ca^^]| and platelet aggregation Collagen (5gg/ml) induced a rise in [Ca“^]i as indicated by a transient aequorin signal which coincided with the onset of aggregation (Figure 6 .1), the increase in platelet Ca^^ concentration in control samples being 4.97±0.63 pM (n=10). Like collagen, although to a lesser extent, adrenaline (16pM) induced a transient rise in [Ca^^]j. This rise, however, occurred after the onset of platelet aggregation (Figure 6.2), coinciding most likely with granular release and TXA 2 synthesis, and therefore differing from the collagen-induced response. The first small signal of calcium rise, which is indicated in figure 6.2, was observed in only one experiment and may represent a rise in intra platelet calcium induced by adrenaline itself additional to the Ca^^ mobilisation produced by adrenaline through aggregation. The measured increase in [Ca^^Ji induced by adrenaline in control samples was 3.60±0.96pM (n= 6). 6.1.4.2. The effect of naftopidil on the collagen- and adrenaline-induced increase in [Ca^^], and platelet aggregation. Naftopidil produced a concentration-dependent inhibition of the collagen-induced rise in [Ca^^j as shown in Figure 6.3 (Appendix, Table 24) , statistically significant inhibitions being obtained with 2pm (12.6±5.9%, 95% Cl from -2.7 to 27.9%, P< 0.05, n=6), lOpM (13.8±3.8%, 95% Cl from 4.4 to 23.2%, P<0.05, n=7) and 40pm (22.9±4.0%, 95% Cl from 13.2 to 32.6%, P<0.05, n=7) concentrations of the drug. Collagen-induced platelet aggregation, however, was not concomitantly inhibited by naftopidil ( Figure 6.3, Appendix, Table 24), except at 40pM when a statistically significant inhibition of aggregation (23.3±11.7%, 95% Cl from -5.4 to 51.9%, P<0.05 n=7) was observed. - 146 - Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms The adrenaline-induced increase in [Ca^^]j was also inhibited in a dose-dependent manner by naftopidil ( Figure 6.4, Appendix, Table 25) with an estimated IC 50 (drug concentration which produces 50% inhibition of the control adrenaline-induced [Ca^^]j increase) of 4 .8 pM (Figure 6.5, Appendix, Table 26), statistically significant inhibitions being obtained with lOpM (70.6±9.5%, 95% Cl fi'om 46.2 to 95.2%, P<0.05; n= 6) and 40pM (100±0.0%, 95% Cl fi'om 100 to 100%, P<0.001, n= 6), whereas the inhibition obtained with 2 pM naftopidil (22.3±7.3%, 95% Cl fi'om 3.4 to 41.1, n= 6) was not statistically significant. Unlike the results obtained with collagen the inhibition by naftopidil of the rise in [Ca“"]j was consistently parallelled by an inhibition of platelet aggregation (Figure 6.4, Appendix, Table 25), statistically significant inhibition being obtained at lOpM (83.4±2.9%, 95% Cl fi'om 75.8 to 90.9%, P<0.05; n= 6) and 40pM (100±0.0%, 95% Cl fi'om 100 to 100%, P<0.05; n= 6). The inhibition of platelet aggregation produced by naftopidil at 2 pM (25.7±11.4%, 95% Cl fi'om -3.5 to 55.0, n=6) was not statistically significant. The estimated IC 50 for naftopidil with respect to adrenaline-induced platelet aggregation was 3.8 pM (Figure 6.6, Appendix, Table 26). -147- Chapter 6: Drugs' efTects on platelet signal tranduction mechanisms Figure 6.1 Representative traces of platelet aggregation and the aequorin-indicated Ca^^ signal induced by collagen (5 pg/ml). Addition of collagen is indicated by the arrow. 6 minutes Figure 6.2 Representative traces of platelet aggregation and the aequorin-indicated Ca^^ signal induced by adrenaline (16pM). Addition of adrenaline is indicated by the arrow. The first small signal of Ca^^ rise, which is indicated by the star, was observed only in the traces of one experiment (see section 6 .1.4 .1). - 148- Chapter 6: Drugs’ efTects on platelet signal tranduction mechanisms F igure 6.3 Effect of N aftopidil on C ollagen (5pg/m l)-induced [Ca'^]; increase and platelet aggregation in w ashed platelets. T he data are derived from the appendix. T able 24a, and also presented in the appendix. T able 24b. (*: P< 0.05 com pared w ith control) 120 - c cl 00 o 'o & c "oCD CD CD < I Naftopidi Naftopidi Naftopidil Naftopidil 0.4/.rM 10/xM AOfÆ (N = 6) (N = 6) (N = 7) (N = 7) Figure 6.4 E ffect o f N aftopidil on adrenaline (16pM )-induced [Ca^"]j increase and platelet aggregation in w ashed platelets. T he data are derived from the appendix. T able 25a, and also presented in the appendix. T able 25b. (*: P< 0.05 com pared w ith control) ^ 1 2 0 1 2 0 o c o o o 100 1 0 0 'o 8 0 5 c o 60 "o o> D 0 u 40 L_ 40 CD CD < 20 * * ■ ■ 0 Naftopidil Naftopidil Naftopidil Naftopidil 0.4/-Z.M 2/jM 10fiU 40/^M (N = 6) (N = 6) (N = 6) (N = 6) - 149- Chapter 6: Drugs’ eflects on platelet signal tranduction mechanisms Figure 6.5 Inhibitory effect of naftopidil on adrenaline-induced [Ca^^J^ increase. Data are also presented in the appendix. Table 26. ( n= 6). 100 0) w o u0) u 80 c CN+ 60 □ o "o 40 c o 20 -0 .5 0.0 0.5 1.0 1.5 Naftopidil Concentration (Log /xM) Figure 6.6 Inhibitory effect of naftopidil on adrenaline-induced platelet aggregation. Data are also presented in the appendix. Table 26. ( n= 6). o CD 0) 80 CD O % S 60 CL 40 c o 20 c -0 .5 0.0 0.5 1.0 1.5 Naftopidil Concentration (Log yiiM) - 150- Chapter 6: Drugs' effects on platelet signal tranduction mechanisms 6.I.4.3. The effect of doxazosin on the collagen- and adrenaline-induced increase in [Ca^^]; and platelet aggregation Doxazosin, like naftopidil, produced a concentration-dependent inhibition of the collagen-induced rise in [Ca"^j ( Figure 6.7, Appendix, Table 27 ), statistically significant inhibitions being obtained at 1.5pM (8.6±3.5%, 95% Cl fi'om -0.4 to 17.7%, P<0.05, n=6), 7.5fiM (15.0±3.8%, 95% Cl from 5.1 to 24.9, P<0.05; n= 6) and 30pM (17.4±2.5%, 95% Cl from 11.5 to 23.4%, P<0.05; n= 8 ). Unlike naftopidil, however, inhibition of the rise in [Ca^^J^ by doxazosin was not mirrored by a statistically significant inhibition of platelet aggregation ( Figure 6.7, Appendix, Table 27). The adrenaline-induced increase in [Ca^""]i was also inhibited by doxazosin in a concentration-dependent manner as shown in Figure 6.8 (Appendix, Table 28), although a statistically significant inhibition was only obtained at 30pM (37.6±13.7%, 95% Cl fi'om 2.4 to 72.8, P<0.05, n= 6). The effects on the rise in [Ca^""]i were reflected by statistically significant inhibitions of aggregation by doxazosin at 7.5pM (35.8±12.6%, 95% Cl fi'om 3.4 to 68.2%, P<0.05; n= 6) and 30pM (49.6±17.2%, 95% Cl fi'om 5.3 to 93.9, P<0.05; n= 6), ( Figure 6.8 , Appendix, Table 28). 151- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms Figure 6.7 Effect o f doxazosin on C ollagen (5|ug/m l)-induced [C a“‘]j increase and platelet aggregation in w ashed platelets. T he data are derived from the appendix. T able 27a, and also presented in the appendix, table 27b. *;P<0.05 ^ 1 2 0 r- n 1 2 0 P C 100 lO O o 80 - 6 0 o - 1 4 0 (u Doxazosin Doxazosin Doxazosin Doxazosin 0 . 3 / . r M 1 .5 /^ M 7 . 5 / . i M 30AiM (N = 6) (N = 6) (N = 6) (N = 8) Figure 6.8 Effect o f doxazosin on adrenaline (16pM )-induced [Ca"*]; increase and platelet aggregation in w ashed platelets. T he data are derived from the appendix. T able 28a, and also presented in the appendix, T able 28b. *P<0.05 120 c 100 T I o Xwl o 80 - 6 0 - o 4 0 - u 20 - 0 Doxazosin Doxazosin Doxazosin Doxazosin 0.5/âM 1 .5/./M 7.5/LiM 30/iM (N = 6) (N = 6) (N = 6) (N = 6) - 152 - Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms 6.1.4.4. The effect of nifedipine on the collagen-induced increase in [Ca^"^]i and platelet aggregation T he expected concentration-dependent inhibition by nifedipine o f the collagen- induced rise in [C a^'jjw as obtained and is show n in Figure 6.9 (A ppendix, T able 29). Statistically significant inhibitions w ere seen w ith 1.4pM (14.5± 4.3% , 95% C l from 4.0 to 25.0% , P<0.05, n=7), 7pM (19.3±3.5% , 95% C l fi'om 10.8 to 27.7, P<0.05; n=7) and 28pM (47.8±2.7% , 95% C l fi'om 41.7 to 54.1% , P<0.05; n=9) nifedipine. A s for naftopidil, the inhibition o f th e rise in [C a“*]i w as parallelled by a statistically significant inhibition o f platelet aggregation (Figure 6.9, A ppendix, Table 29), inhibitions of 24.0±9.2% ( 95% C l fi'om 1.4 to 46.6% , P<0.05, n=7), 40.1±11.4% ( 95% C l from 12.2 to 67.9% , P<0.05; n=7) and 55.1±9.2% ( 95% C l from 33.9 to 76.3% , P<0.05, n=9) being recorded for 1.4pM , 7pM and 28pM nifedipine, respectively. Figure 6.9 E ffect o f nifedipine on C ollagen (5pg/m l)-induced [C a“^]j increase and platelet aggregation in w ashed platelets. T he data are derived from the appendix. T able 29a and are also presented in the appendix. Table 29b. (*: P< 0.05 com pared w ith c o n t r o l ) C' 120 o 120 .o 5 100 o 80 Nifedipine Nifedipine Nifedipine Nifedipine 0.28/uM 1.4/zM 7yuM 28uM (N = 7) (N = 7) (N = 7) (N = 9) - 153 - Chapter 6: Drugs' effects on platelet signal tranduction mechanisms 6.1.5. Discussion In this study the reactivity of aequorin-loaded platelets was, on the whole, satisfactory although low reactivity was observed with a few preparations. This may be due to partial dissociation of the GP Hb-IIIa complex, caused by EOT A in the washing solution (Shattil et al 1985), and/or the damaging effects of DMSO used during aequorin loading. Adrenaline-induced responses in washed platelets In the present study, using washed and aequorin-loaded platelet preparations, adrenaline (16pm) induced almost maximal aggregation accompanied by increases in [Ca^^j. Previous studies performed with washed or gel filtered platelets have, however, yielded conflicting results regarding adrenaline-induced aggregation (Lanza et al 1988, Steen et al 1993). According to Lanza et al (1988) the aggregation of washed platelets in response to adrenaline is indicative of platelet pre-activation, since adrenaline alone does not induce modifications of the morphology, metabolism or function of intact and fimctional washed human platelets, but interacts with (X2-adrenoceptors and potentiates biochemical and aggregatory responses induced by other platelet agonists. Moreover, pre-activation of platelets by the permeabilization procedure used in this study to load aequorin has been suggested as a possible cause of the increase in [Ca^^j observed after stimulation by adrenaline (Lanza et al 1988). Platelet pre-activation, and hence adrenaline-induced aggregation, in the present study may be assumed to occur due to the absence of a stimulator of adenylate cyclase e.g. PGI 2 or PGEj, which are commonly included in platelet washing solutions to avoid mechanical activation during centrifugation or resuspension, and/or the presence of trace amounts of extra-cellular ADP generated by lysis of contaminating ADP-rich erythrocytes, or by lysis, secretion or leakage from platelets. 154- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms Inhibition of adrenaline-induced responses by naftopidil and doxazosin The observed adrenaline-induced aggregation and increase in [Ca*"]i in the washed platelets, whether mediated solely by a 2-&(lrenoceptors or by the activation of alternative receptors by other agonists, was dose-dependently inhibited by naftopidil and, to a lesser extent, doxazosin, with complete inhibition being produced by naftopidil (40pM). The inhibitory actions of both drugs on adrenaline-induced platelet aggregation in washed platelets, which contrasts with the slight inhibition produced by naftopidil and lack of inhibition by doxazosin in PRP (as presented in Chapter 4), are strong indications of the influence of plasma protein binding in reducing the available free concentrations of the drugs and, therefore, masking of the inhibitory effects of the drugs when using PRP. The extent of drug binding to albumin in washed platelets, in comparison with that occurring in plasma, is probably small as the concentration of albumin added to the platelet washing medium is Ig/L, which is much less than that normally present in plasma, i.e. 35-55g/L. Adrenaline is known to be the only activator of human platelets which is fully dependent on the presence of external Ca^"^ for the induction of platelet responses (Owen et al 1981) and, in general, activation of a-adrenoceptors by adrenaline triggers calcium influx through receptor-operated calcium channels (van Zweiten et al 1987). Accordingly, the inhibitory actions of naftopidil and doxazosin on the adrenaline-induced increase in [Ca^^Jj, and hence platelet aggregation, may be mediated by a blockade of Ca^^ influx, which may involve (%2-&drGnoceptors, receptor-operated calcium channels and/or voltage-operational calcium channels. In fact naftopidil has been reported to inhibit Ca^"' entry into vascular and cardiac muscle and this was suggested to be via L- type calcium channels (Himmel et al 1991, Grundk et al 1991). Moreover, the thrombin- induced increase in human platelet [Ca^^fi has been reported to be decreased by naftopidil (Lijnen 1992). - 155- Chapter 6: Drugs’ effects on platelet signal trandnction mechanisms The concomitant inhibition by naftopidil of the rise in platelet [Ca^^]j and platelet aggregation is in keeping with the concept of adrenaline-induced aggregation being associated with Ca^^ influx and an increase in [Ca"^]|. Inhibition of collagen-induced responses by naftopidil and doxazosin. Naftopidil and doxazosin reduced slightly and to a comparable extent the elevation in [Ca^^]j induced by collagen but was not parallelled by an inhibition of platelet aggregation, except at the highest concentration of naftopidil tested. It has been suggested that collagen-induced platelet activation, mediated by activation of phospholipase C, is relatively insensitive to increases in [Ca^^]j but is dependent on cyclooxygenase products (Lapetina and Siess 1983). Therefore, the rise in [Ca^^]j as measured by aequorin, is not the sole determinant of collagen-induced platelet responses (Ware et al 1986) and, accordingly, the inhibition of this rise may not interfere with collagen-induced responses. The reduction by naftopidil and doxazosin of the collagen-induced rise in [Ca^^]i may involve blockade of Ca“^ influx, as it is known that external Ca^^ contributes to the aequorin-indicated rise in [Ca^^jj induced by collagen (Ware et al, 1986). Inhibition of collagen-induced responses by nifedipine The inhibitory effects of nifedipine on platelet function have been previously reported for in vitro (Johnson 1981, Kiyomoto et al 1983, Takahara et al 1985, Wally et al 1989) and ex vivo studies in normal subjects (Winther et al 1991) and hypertensive patients (O'smialowska et al 1990, Belousov and Kushnaryov 1991). Nifedipine has also been reported to inhibit the platelet synthesis of TxA 2, both in vitro and in vivo (Davi et al 1982). In some studies, however, nifedipine was found to have little or no effect on -156- Chapter 6: Drugs* effects on platelet signal tranduction mechanisms platelet aggregability (Vinge et al 1988), and when administered to healthy subjects did not affect significantly platelet aggregation or TxAj formation. Moreover, in vivo platelet function as indicated by measurement of plasma p-TG levels after nifedipine therapy was not significantly altered (Islim et al 1992) or even increased (Takahara et al 1985, Mundal et al 1993). However, the elevated platelet calcium concentrations observed in disorders related to thrombosis were found to be normalized by nifedipine therapy (Ahn et al 1987). In the present study, nifedipine inhibited in a dose-dependent manner the collagen-induced rise in [Ca^^j and was parallelled by a comparable inhibition of platelet aggregation. This is probably consistent with the blockade by nifedipine of a dihydropridine-sensitive transmembraneous calcium channel with consequent inhibition of Ca^^-dependent signal transduction mechanisms involved in platelet aggregation, e.g. thromboxane Aj synthesis (Davi et al 1982). - 157- C'hiipter 6: Drugs' efTects on platelet signal tranduction mechanisms 6.2. Effects of naftopidil and doxazosin on stimulus-induced thromboxane A 2 generation 6.2.1. Introduction Thromboxane Aj (TxA ) is the principal metabolite of arachidonic acid in platelets. Arachidonic acid is released from phospholipids, mainly phosphatidyl choline, by the action of phospholipase A 2 (PLA2) in response to a variety of stimuli including growth factors, hormones, neurotransmitters, and mechanical disruption. The release of arachidonic acid is the first and rate-limiting step in the synthesis of TXA 2 (see Chapter 1, Section 1.3.2). The platelet synthesis of TXA 2 from arachidonic acid is followed by immediate extrusion of TXA 2 into the extracellular medium, A^ereupon it binds to specific receptors on the plasma membrane of other platelets or vascular smooth muscle cells and exerts, respectively, powerful pro-aggregatory and vasoconstrictive actions (Kroll and Schafer 1989). Its actions on non-activated and activated platelets are respectively, as a powerful positive signal and as a feedback promoter leading to amplification of platelet activation (Armstrong et al 1983). TXA2 is extremely unstable (half-life 30 sec) and is hydrolysed rapidly to the stable inactive metabolite thromboxane B 2 (TXB2) (Oates et al 1988). In vivo, therefore, the actions of TXA 2 are largely limited to the micro environment of its release. The amounts of TXA 2 produced by stimulated platelets are determined by the regulation of PLA 2 and/or the enzymes involved in the synthesis of 2TxA from arachidonic acid. Therefore, drug effects on these enzymes are of primary importance in determining the synthesis of TXA 2 and other arachidonic acid metabolites. Aspirin, for - 1 5 8 - Chapter 6: Drugs* efTects on platelet signal tranduction mechanisms example, irreversibly inactivates platelet cyclo-oxygenase by acétylation, leading to a decrease in TXA2 synthesis (Vane 1971). Other substances, for example ridogrel (Weber et al 1992), inhibit TXA 2 synthetase and hence decrease the synthesis of TXA 2, and may result in the re-direction of the metabolism of cyclic endoperoxides to PGEj which also possesses pro-aggregatory activity (Defreyn et al 1982). 6.2.2. Aim of the Study The aim of the study presented in this section was to investigate the effects of naftopidil and doxazosin on the collagen- and adrenaline-induced generation of TxA 2in platelets. The calcium antagonist nifedipine was also tested in order to investigate the relationship between Ca^^ mobilization and TXA 2 generation but, as described in section 6.1.2. its application was limited to incubations in which collagen was used as the platelet agonist. 6.2.3. Study Design Samples of washed platelets loaded with aequorin were stimulated, as described in section 6.1.3, by adrenaline (16pM) in the absence or presence of naftopidil (lOpM and 40|iM) or doxazosin (7.5|iM and 30pM), and by collagen (5pg/ml) in the absence or presence of naftopidil (40pM), doxazosin (30pM) or nifedipine (28 pM). The levels of TXB2, the stable metabolite of 'ÇxA, were determined in supernatants derived from washed platelet preparations as described in chapter 2 (2.2.8 ). - 159 C hapter 6: Drugs’ efTects on platelet signal tranduction mechanisms 6.2.4. Results 6.2.4.1. TxB; generation and platelet aggregation induced by collagen and adrenaline. Collagen (S^ig/ml) increased TXB 2 from a basal level of 7.69±1.28 to 96.34±13.37 ng/ml/10* platelets (n=10, p<0.001), and adrenaline (16pM) increased TXB2 from a basal level of 2.44±0.61 to 8.02±1.08 ng/ml/10* platelets (n= 6, p<0.01). The two agonists, however, produced comparable extents of aggregation, being 78.8±7.9% (n= 6) for adrenaline and 85.0±3.8 % (n=10) for collagen. The basal levels of TXB2 were subtracted from the stimulated levels when comparing the effects of the drugs on TXB 2 generation. 6.2.4.2. The effect of naftopidil on the adrenaline- and collagen-induced generation of TxBj Naftopidil (lOpM and 40pM) produced a statistically significant inhibition of the adrenaline-induced generation of TXB 2 ( Figure 6.10, Appendix, Table 30). Thus, TXB 2 levels were inhibited by 42.5±10.3% ( 95% Cl from 15.9 to 69.0%; P<0.05, n= 6) and 81.8±7.5% (95% Cl from 62.6 to 101.1%; p<0.05, n= 6) with lOpM and 40pM naftopidil, respectively, with an estimated IC 50 (drug concentration which produces 50% inhibition of the control level of TXB 2 ) of 12.6pM. The collagen-induced generation of TXB 2 was also significantly inhibited by 40|iM naftopidil, inhibition being 59.5±9.2% (95% Cl from 33.9 to 85.0; p< 0.01, n=5) (data are derived from the appendix. Table 31). - 160 - Chapter 6: Drugs* efiects on platelet signal tranduction mechanisms 6.2.4.S. The effect of doxazosin on adrenaline- and collagen-induced generation of TxBj In the presence of doxazosin (7.5pM and 30pM), the adrenaline-induced generation of TxBj was slightly inhibited ( Figure 6.11, Appendix, Table 30) but did not reach statistical significance, % inhibitions for 7.5pM and 30pM being 16.4±% (95% Cl fi’om -10.5 to 43.5%, n=4) and 15.3±% (95% Cl fi’om -43.1 to 73.7%, n= 6), respectively. Moreover, an inhibition of 28.8±11.9% (Cl -4.2% to 61.8 %, n=5) (data are derived fi’om the appendix. Table 32) by doxazosin (30pM) of the collagen-induced generation of TxB 2 was observed but again did not achieve statistical significance. 6.2.4.4. The effect of nifedipine on the collagen-induced generation of TxBj Nifedipine (28pM) inhibited significantly the collagen-induced generation of TxB2, inhibition being 53.7±11.3% (95% Cl from 24.6 to 82.91, p< 0.01, n=6) (data are derived fi’om the appendix. Table 33). - 161 Chapter 6: Drugs’ efTects on platelet signal tranduction mechanisms Figure 6.10 E ffect o f naftopidil on adrenaline-induced TXB 2 generation. D ata are derived from the appendix. T able 30a, and are also presented in the appendix, T able 30b. gQ _ *: P<0.05, n=6 5 0 o uo 4 0 m m CD X 20 ■I Naftopidil Naftopidil 10^NI 40/xM Figure 6.11 Effect o f doxazosin on adrenaline-induced TXB 2 generation. D ata are derived from the appendix. T able 30a and are also presented in the appendix, T able 30b. *; P< 0.05, n=& 120 - 100 c 8 0 E uo *4— o 6 0 ÛQ X 4 0 20 Doxazosin Doxazosin 7.5/xM 30mM n = 4 n = 6 - 162 - Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms 6.2.5. Discussion The levels of TXB2 determined in supernatants derived from unstimulated platelets were normally low, whereas higher levels of TXB 2 were detected on platelet stimulation with collagen and adrenaline indicating that the washed platelet preparations were in a satisfactory condition in their unstimulated state and responded normally to agonists. The levels of TXA2, as reflected by the concentrations of its stable metabolite TXB2, generated on platelet stimulation with collagen were higher than those produced with adrenaline, providing confirmation of the classification of collagen and adrenaline as strong and weak agonists, respectively. The differences as regards the levels of TXA 2 generated by these agonists were, however, not reflected by differences regarding the extents of platelet aggregation produced, the two agonists producing comparable extents of aggregation. This can be explained by the fact that some platelet agonists do not possess an absolute requirement for TXA 2 for the activation of platelets (Kroll and Schafer 1989). Thus, strong platelet agonists such as collagen or thrombin, although potent stimulators of TXA 2 generation, can bypass the arachidonic acid pathway and stimulate platelet activation and aggregation directly, whereas weak agonists, such as adrenaline, need the reinforcing effect of TXA 2 generation to cause platelet activation and produce maximal aggregation. The release of arachidonic acid by the action of PLA 2, which is a Ca^^- dependent enzyme, is the first and rate-limiting step in the synthesis of TxA. 2. Therefore, the rapid rise in cytoplasmic Ca^^ induced by platelet agonists would activate PLAg and trigger arachidonic acid release and TXA2 production (Font et al 1992), whereas the inhibition of [Ca^^Ji mobilization by calcium channel blockers or specific receptor antagonists would be expected to result in the inhibition of TXA 2 production. -163- C hapter 6: Drugs’ efTects on platelet signal tranduction mechanisms Effects of naftopidil and doxazosin on the adrenaline-induced generation of TxA^ It was observed in this study that the adrenaline-induced generation of TXA 2 was significantly inhibited by naftopidil, whereas the extent of inhibition produced by doxazosin was low and statistically insignificant. An explanation for this difference in the effects of the two drugs may found by examining the data presented in section 6.1.4, which shows that naftopidil was more potent than doxazosin in inhibiting the adrenaline- induced increase in [Ca^^]j and, therefore, was presumably more effective in blocking the signal transduction mechanisms which trigger the release of arachidonic acid and the concomitant increase in TXA 2 production. Another explanation, however, may involve an effect of naftopidil on further steps in the cascade of TXA 2 synthesis, e.g on enzymes such as cyclooxygenase or TXA 2 synthase. Effects of naftopidil, doxazosin and nifedipine on the collagen-induced generation of TxAj When collagen was used to stimulate aggregation, naftopidil was comparable to nifedipine in its inhibitory action on TXB 2 generation, whereas the inhibitory action of doxazosin was again not statistically significant. Despite this, doxazosin and nifedipine produced similar extents of inhibition of the collagen-induced increase in [Ca^^J^ The extent of inhibition of TXA 2 generation produced by naftopidil was, however, more than that exerted on the rise in [Ca^^]^ which suggests again, as discussed above, that other mechanisms, in addition to calcium blockade, may be involved in the inhibitory action of naftopidil on TXA 2 generation. -164- Chapter 6: Drugs’ effects on platelet signal tranduction mechanisms 6.3. Effect of naftopidil and doxazosin on platelet cAMP 6.3.1. Introduction Elevation of platelet cAMP results in inhibition of most platelet responses, both morphologic and metabolic, to agonists. This elevation can be produced by stimulation of adenylate cyclase or by inhibition of phosphodiesterase. Stimulation of adenylate cyclase by agonists such as adenosine and PGI 2 can be receptor-mediated, or via direct action on the enzyme by substances such as forskolin (Siess 1989). The mechanisms by which cAMP elevation inhibits platelet function may involve activation of cAMP-dependent protein kinases (Brass et al 1993), which activate calcium pumps to stimulate dense tubular uptake of Ca^^ (Tao et al 1992) and phosphorylate myosin light chain kinase making platelets less sensitive to activation (Hathaway et al 1981). 6.3.2. Aim of Study The aim of the study was to investigate the effects of naftopidil and doxazosin on platelet cAMP content under resting conditions and on adrenaline stimulation. 6.3.3. Study Design To examine the effects of the drugs on platelet basal cAMP levels, washed platelets (0.4ml, platelet count: 300xl0^/L), prepared as described in Chapter 2 (2.2.2), were mixed with a fibrinogen suspension ( 0.1ml, 0.06% final concentration) and incubated for 1 min at 37°C before the addition of naftopidil (40pM) or doxazosin (30pM), whereupon incubation was continued for a further 1 min. The effects of the Chapter 6: Drugs’ eflects on platelet signal tranduction mechanisms drugs on adrenaline-induced alterations in cAMP levels were tested by incubating platelets with naftopidil (40pM) or doxazosin (30pM) for 1 min before the addition of adrenaline (16pM) and incubation continued for 2 to 6 min. All incubations were terminated by adding ice-cold ethanol (0.5 ml, 65%, final concentration) and cAMP was extracted and its concentrations measured as described in Chapter 2 (2.2.7). 6.3.4. Results Neither naftopidil (40pM) nor doxazosin (30pM) affected significantly the basal level of platelet cAMP ( Figure 6.12, Appendix, Table 34). Two minutes after the addition of adrenaline (16pM) no changes could be detected in cAMP levels between control samples (minus drugs) or samples incubated with naftopidil or doxazosin. However, 6 minutes after the addition of adrenaline the cAMP levels declined significantly compared to the control, % decline being 36.6±8.1(P<0.05, n=4). Both naftopidil and doxazosin partially prevented the adrenaline-induced decline in cAMP ( Figure 6.13, Appendix, Table 34). Thus the adrenaline-induced declines of cAMP were 14.4±20% (P=not significant, n=4) and 23.5±7.9% (P=not significant, n=4) in presence of naftopidil and doxazosin, respectively. - 166 - Chapter 6; Drugs’ eflects on platelet signal tranduction mechanisms Figure 6.12 E ffect o f naftopidil and doxazosin on basal levels o f platelet cA M P . D ata are also presented in the appendix. T able 34. (n=6) 15 r i/) (D D CL O E CL & 0 Basal level +Naftopidil 4-Doxazosin 40/uM 3 0 / x M Figure 6.13 Effect o f naftopidil and doxazosin on platelet cA M P levels in presence o f adrenaline. D ata are also presented in the appendix. T able 35. (*; P < 0.05 com pared w ith control, n=6) Basal cAMP KXM cAMP in presence of Adrenaline 15 r Y/A Naftopidil 40/.aM I 11 Doxazosin 30/zM (/) "o £ Cl 00 10 - O o E CL 5 h CL 2 < o 2 6 Incubation time (minutes) - 167 - Chapter 6: Drugs’ efTects on platelet signal tranduction mechanisms 6.3.5. Discussion A decrease in cAMP levels as a consequence of adrenaline stimulation in platelets is not well established (Huang and Detwiler 1986). However, in the present study, a statistically significant decrease in platelet cAMP was detected after the incubation of platelets with adrenaline for 6 minutes, although a decrease was not detected after 2 minutes incubation. Platelet aggregation usually occurs within one minute of the addition of adrenaline, confirming that adrenaline-induced platelet aggregation and the decrease in platelet cAMP are separate and independent events. It is known, however, that both events are mediated by a single type of a 2-adrenoceptor (Siess 1989). Therefore, the blockade of platelet a 2-adrenoceptors would result in the prevention of both events, the extent to which this occurred depending upon the number of (% 2- receptors occupied and the relative numbers of a 2-adrenoceptors required to elicit each event. The EC50 values for the inhibition of platelet adenylate cyclase and the induction of primary aggregation have been reported to be 0.54pM and 1.2pM respectively (Siess 1989). It has also been reported that only 10% of platelet 02-adrenoceptors are required to be occupied by adrenaline to elicit 50% inhibition of adenylate cyclase (Siess 1989). This is reflected in a familial platelet (X2-adrenoceptor defect where impaired platelet aggregation but normal reductions in cAMP levels induced by adrenaline have been reported (Siess 1989). Therefore, it appears that fewer platelet 062-adrenoceptors are required for adrenaline-induced inhibition of adenylate cyclase than for aggregation. This suggests that the partial prevention by naftopidil and doxazosin of the adrenaline-induced inhibition of cAMP may be explained by the presence of a reserve of platelet 062- adrenoceptors which are unoccupied by the drugs. - 168 - Chapter 6; Drugs’ efTects on platelet signal tranduction mechanisms 6.4. Conclusions The calcium channel blocker nifedipine produced a dose-dependent inhibition of the collagen-induced rise in platelet Ca^^ which was mirrored by an inhibition of TXA 2 generation and platelet aggregation. Like nifedipine, naftopidil and doxazosin also inhibited the collagen-induced [Ca^^]j increase albeit to a lesser extent than nifedipine. The Ca^^-dependent platelet responses, i.e. TXA 2 generation and platelet aggregation, induced by collagen in washed platelets were significantly inhibited by naftopidil but not by doxazosin. Naftopidil and doxazosin inhibited to different extents the [Ca^^], increase induced by adrenaline. Thus, the maximal inhibitions produced by naftopidil and doxazosin were respectively 100±0.0% and 37.6±13.7%. TXA 2 generation induced by adrenaline was also significantly inhibited by naftopidil but not by doxazosin. In conclusion, the results suggest that both naftopidil and doxazosin possess some blocking activity against membranous calcium channels which may mediate the partial inhibitory actions of the drugs against the collagen-induced platelet calcium increase. In addition, an antagonism by naftopidil and, to a lesser extent, doxazosin of platelet o( 2-adrenoceptors would prevent Ca^^ influx through receptor-operated calcium channels resulting in the antagonism of adrenaline-induced responses which are fully dependent on platelet Ca^ influx (Owen et al 1981). - 169- CHAPTER 7 A study of the effects of naftopidil, doxazosin and nifedipine on stimulus-induced platelet granular release -170- Chapter 7 : Platelet granular release 7.1. Introduction The release of platelet factors from dense granules, «-granules and lysosomes in response to agonists occurs by exocytosis (Holmsen 1985), and involves the contraction of actomyosin within the platelet cytoplasm and of microfilaments which surround the secretory granules (Gerrard and White 1976). This results in the centralization of the granules, their fusion with the channels of the surface-connected canalicular system and the subsequent release of their contents through the channel pores to the extracellular medium (White and Krumwiede 1987). Actomyosin contraction in platelets is believed to be regulated by phosphorylation of myosin light chain proteins (Gerrard et al 1989). The platelet release reaction is dependent on the agonist concentration and strength (Mustard and Packham 1976). Thus, secretion of acid hydrolases from lysosomes requires high concentrations of strong agonists such as thrombin or collagen, while secretion of the contents of «-granules and dense granules is observed with all platelet agonists (Siess 1989). Apparently, the contents of the different granules are secreted at different rates and are not always released in parallel under the same conditions of platelet activation. Thus, it is proposed that the process of secretory exocytosis involves first the release of dense granules (release I), followed by the «- granules (release II) (Kaplan et al 1979, Holmsen et al 1982, Holt and Niewiarowski 1985). Different mechanisms are involved in initiating release from the different granules. Thus, it has been shown that «-granule secretion in response to ADP is dependent on Na'^/H^ exchange but is independent of arachidonic acid metabolism, _ 1 7 1 _ Chapter 7: Platelet granular release whereas arachidonate metabolites, i.e. endoperoxides and TxAz, are involved in mediating dense granular secretion (Kinder et al 1993). The various platelet agonists require different platelet cyclo-oxygenase activity with respect to the induction of granular release and irreversible platelet aggregation. The weak agonists adrenaline and ADP require both cyclo-oxygenase activity and primary aggregation to induce secretion and secondary irreversible aggregation (Charo et al 1977). Aggregation and secretion induced by high concentrations of thrombin or collagen (strong agonists) are not diminished by cyclo-oxygenase blockers (Charo et al 1977, Siess 1989). Responses induced by low concentrations of thrombin and collagen, however, are reported to be mediated by platelet endoperoxides and TxAj (Siess et al 1983). The release reaction constitutes a mechanism whereby platelet responses are amplified. Substances released fi’om dense granules, such as serotonin (5- hydroxytryptamine, 5-HT) and ADP, synergise with each other, as well as with the primary platelet stimulus or with agonists present in the circulation, to rapidly form irreversible platelet aggregates (De Clerk and De ChafiFoy De Courcelles 1989) or to stabilise preformed aggregates (Charo et al 1977). In addition, the release of «-granule components results in the occurrence of high concentrations of the adhesive proteins (fibrinogen, fibronectin, thrombospondin and vWF) at the platelet surface, and growth factors, such as platelet derived growth factor (PDGF), that appear to be important in modulating tissue regeneration and remodelling during wound healing (Niewiarowski 1994). 172- Chapter 7: Platelet granular release Uptake and release of 5-HT by platelets Human platelets accumulate, store and release 5-HT. Accumulation of 5-HT by platelets is believed to involve two distinct transport systems. The first, a transporter located in the plasma membrane, carries 5-HT through the plasma membrane into the cytoplasm. This transporter is Na^-dependent and coupled to a ouabain-sensitive Na^-K^ ATPase (Hawiger 1992), and is inhibited by tricyclic antidepressants such as imipramine. The second transporter is located in the dense granule membrane and requires an electrochemical proton gradient, generated by a H^-pumping ATPase, to exchange cytoplasmic 5-HT for one or more intra granular protons (Fishkes and Rudnick 1982). The transport of 5-HT into the dense granules is unaffected by ouabain but can be inhibited phannacologically e.g. by reserpine and tetrabenazine (Da Prada and Pletscher 1969). The structures of the plasma membrane (Blakely et al 1991) and granular 5-HT transporters (Liu et al 1992) have been elucidated by molecular cloning and found to be related to a class of transport proteins with 12 transmembrane domains. Despite the clear structural differences between the cloned granular transporters and the family of Na^- dependent plasma membrane transporters, the overall structural homology between them remains striking. In addition, tetrabenazine displaceable [^H] ketanserin binding to the 5-HT transporter has been characterized in human platelet plasma membranes (Leysen et al 1991) as well as in isolated dense granule membranes (Chatteijee and Anderson 1993). The extent of accumulation of 5-HT in the granule compartment is believed to depend on the rate of transport fi'om the cytosol into the granule and the cytosohc metabolism of the amine by enzymes such as monoamine oxidase and phenylsulphotransferase (Launay et al 1994). __ Chapter 7; Platelet granular release Specificity of the platelet 5-HT transporter The specificity of the plasma membrane transporter for 5-HT is not certain, whilst the granular transporter is rather non-specific and possesses broad selectivity for amines, in addition to 5-HT, such as adrenaline, noradrenaline, dopamine and histamine (Rudnick 1986) which are also accumulated and stored in platelets (Bom et al 1958, Da Prada and Picotti 1979, McCulloch et al 1987, Tomlinson et al 1989, Ratge et al 1991), and released fi'om platelets in response to aggregating agents (Abrams and Solomon 1969, Bom and Smith 1970, Smith et al 1986). The nature and specificity of the system involved in dopamine uptake into platelets is unclear, since some studies have indicated that it enters platelets by low affinity uptake via the 5-HT transport system (Gordon and Olverman 1978). Other studies, however, have indicated the presence of a specific uptake system for this amine (Dean and Copolov 1992). No studies on the mechanisms of transport for other catecholamines have been reported. Platelet catecholamine concentrations reflect those in plasma (Smith et al 1985, Rosen et al 1987, Tomlinson et al 1989) but do not show changes with acute elevations in catecholamines plasma levels (McCulloch et al 1987). Chronic sympathetic overactivity, as occurs in pheochromocytoma (Zweifler and Julius 1982) and prolonged exercise over a period of 6-8 days (Chamberlain et al 1990), does, however, appear to raise platelet catecholamines suggesting that a persistent elevation of catecholamines in plasma is a prerequisite for their transport into platelets. This is consistent with a low affinity of catecholamines for the platelet transport system and indicates that their movement across the platelet membrane is largely by passive diffiision (Da Prada and Picotti 1979). -174- Chapter 7: Platelet granular release Platelet storage compartments of 5-HT In normal platelets, 5-HT is found in both releasable (dense-granule storage compartment) and non-releasable pools (thrombin-resistant compartment, pool I) (Launay et al 1994). Under experimental conditions predisposing to the loss of 5-HT from the dense granules, such as flooding the system with 5-HT or leakage during prolonged incubation (Launay et al 1994), another 5-HT pool (pool II) becomes evident (Costa et al 1977, 1981) which represents 5-HT that has been lost from the dense granules into the cytoplasm. The non-releasable pool may be associated with platelet proteins, e.g. the so called 5-HT-binding proteins (Liu et al 1992) and actin, (Small and Wurtman 1985), which have the ability to bind 5-HT. Based on the kinetic constants established for the movement of 5-HT between the various 5-HT compartments, it has been proposed that 5-HT enters dense granules mainly from the extracellular medium rather than via uptake into pool I, both compartments having direct access to extracellular 5-HT (Launay et al 1994). This hypothesis is supported by the finding that dense granules are anchored to the platelet plasma membrane in the absence of any secretory stimulation (Moriomoto et al 1990). Thus, extracellular 5-HT could cross the fused cytoplasmic and vesicular membranes by means of “specific channels” (Launay et al 1994). PDGF- origin and release by platelets PDGF is a cationic dimeric protein with a molecular weight of 28-35KDa. The two polypeptides of the dimer are designated A and B, and are products of distinct genes so that PDGF can be either homodimeric (PDGF-AA, PDGF-BB) or heterodimeric (PDGF-AB) (Ostman et al 1992). PDGF was originally isolated from platelets but is now known to be expressed by several normal and transformed cells (Khachigian and 175- Chapter 7: Platelet granular release Chesterman 1993). In platelets, PDGF exists in extremely small quantities (3.0-10.0 ng/10^ platelets) (Singh et al 1982, Huang et al 1983) and may have its origin in the megakaryocytes (Castro-Malaspina et al 1981). Until recently, it was believed that PDGF, purified fi’om human platelets, consists of about 70% PDGF-AB and 30% PDGF-BB (Heldin and Westermark 1989, Hart et al 1990). More recently, however, it has been concluded that these results reflect an artefact of the purification procedures employed, and that the predominant form of PDGF in human platelets is in fact PDGF- AA (Soma et al 1992). PDGF is normally stored in the platelet «-granules and is released to the extracellular medium on platelet activation, PDGF-BB being poorly secreted whilst PDGF-AA is readily secreted (Huang et al 1983). Thus, PDGF-AA may act on secreting or neighbouring cells by autocrine or paracrine mechanisms whereas PDGF-BB may act only as an autocrine regulator of cell growth (Spom 1980, Huang et al 1983). Inhibition of the platelet release reaction The platelet release reaction is reduced by inhibition of the signal transduction mechanisms involved in platelet activation. Inhibitors of the release reaction include substances which elevate intra platelet cAMP (prostaglandins E^, Dj and I 2; adenosine, papaverine, dipyridamole; methykanthines), calmodulin inhibitors, calcium antagonists, local anaesthetics and specific platelet receptor antagonists (Holmsen 1994). Inhibition of arachidonate liberation, cyclo-oxygenase, or thromboxane synthetase also inhibits the platelet release reaction (Weiss 1989). Aspirin, apart fi*om blocking platelet cyclo- oxygenase inhibits granule centralization and myosin light chain phosphorylation induced by adrenaline and low concentrations of collagen or arachidonic acid but not that induced by thrombin, TXA 2, lysophophatidic acid and A23187 (Gerrard et al 1989). -176- Chapter 7: Platelet granular release 7.2. Aim of the study The aim of the study presented in this chapter was to investigate the effects of naftopidil and doxazosin on the collagen- and adrenaline- induced release of 5-HT, an index of dense granule release, and PDGF, a marker of a-granule release. Nifedipine was also tested but, as previously mentioned in Chapter 6 (6.1.2), only in incubations in which platelets were stimulated with collagen. 7.3. Study design The effects of the three drugs, i.e. naflopidil, doxazosin and nifedipine, on platelet granular release induced by collagen and adrenaline were examined in platelets obtained from healthy non-smoking male subjects aged 30-54 years, none of whom had consumed any aspirin-like drugs for at least 2 weeks before the study. Samples of washed platelets loaded with aequorin were prepared and stimulated as described in Chapter 6 (6.1.3.) by collagen (5pg/ml) or adrenaline (16pM).The effects of the drugs were studied in stimulated platelet samples pre-incubated for 1 min with naflopidil (0.4, 2,10, and 40pM), doxazosin (0.3,1, 7.5, and 30pM) or nifedipine (0.28, 1.4. 7 and 28pM). Samples of platelet suspension, pre-incubated with distilled water and stimulated with agonists, served as controls, and unstimulated samples were used to determine the basal levels of the released products. Platelet aggregation, measured as described in Chapter 2 (2.2.5), was allowed to continue for 5 minutes after which platelet suspensions were removed rapidly and centrifuged at 10,000g for 4 minutes at 4°C to obtain the supernatants containing the released products. The released 5-HT was measured as described in Chapter 2 (2.2.6), and released PDGF as described in Chapter 2 (2.2.10). 177- Chapter 7: Platelet granular release 7.4. Results 7.4.1. 5-HT release induced by collagen and adrenaline The platelet aggregation induced by collagen (5pg/ml) was accompanied by a statistically significant release of 5-HT fi'om a basal level of 13.88Ü.39 to 188.67±26.37 pmol/10* platelets (P<0.001, n=9). Adrenaline-induced platelet aggregation was also accompanied by a statistically significant release of 5-HT in all subjects with one exception, the results obtained with this subject therefore being excluded fi’om the statistical analysis. Thus, adrenaline- induced 5-HT release increased from a basal level of 11.00±1.46 to 110.60±29.9 pmol/10* platelets (P<0.02, n=6). In investigating the effects of the drugs on stimulated 5-HT release, basal levels of 5-HT release were subtracted from the stimulated levels in both control and test samples. 7.4.2. The effects of naftopidil on collagen- and adrenaline- induced 5-HT release. The collagen-induced release of 5-HT that accompanied platelet aggregation was increased by naftopidil in a dose-dependent manner (Figure 7.1, Appendix, Table 36), statistically significant increases of 71.6±17% (p<0.01, n=6), 89.1±16.8% (P<0.0, n=7) and 69.7±15.4% (P<0.01, n=6) occurring with concentrations of 2pM, lOpM and 40pM, respectively. Adrenaline-induced 5-HT release was also increased by the lowest concentration of naftopidil tested (0.4pM) albeit non-significantly. However, at lOpM and 40pM naftopidil dose-dependently inhibited 5-HT release ( Figure 7.2, Appendix, Table 37), significant inhibition being obtained at 40|iM naftopidil (58.7±15.7%, p<0.05; n=5). It should be pointed out, however, that when the platelet aggregation data was examined, the 5-HT level was found not to have returned to its basal level with 40pM naftopidil whereas aggregation was inhibited by 100±0.0% (n=5, P<0.05) (see also Figure 6.4; chapter 6). - 178- Chapter 7; Platelet granular release Figure 7.1 Effect of naftopidil on collagen (5fig/m l) - induced 5-H T release (% control). D ata show n are derived from the appendix. T able 36a, and are also presented in the appendix. T able 36b . (*: P < 0.05 com pared w ith control) 200 ~D c (D o O u 1 6 0 D -o o C 120 II 8 0 O (D O ^ 40 lO Naftopidil Naftopidil Naftopidil Naftopidil 0.4/.iM 2/dW 1 G /.iM A-OfÆ (N = 4) ( N = 6 ) ( N = 7 ) ( N = 6 ) Figure 7.2 Effect o f naftopidil on adrenaline (16pM ) - induced 5-H T release (% control). D ata show n are derived from the appendix. T able 37a, and are also presented in the appendix. Table 37b . (*P < 0.05 com pared w ith control) o L_ ■D c 0> o O Ü 1 6 0 3 T5 o C 120 1 5 (D c 03 (n o o 8 0 c 0 ) (D 0 "O < 1— X 40 1 LO No topidil N a f opidil Naf opidil Naf opidil 0.4/xM 2/.iM lO^W ( N = 5 ) ( N = 5 ) (N = 5) ( N = 5 ) - 179- Chapter 7: Platelet granular release 7.4.3. Effect of doxazosin on collagen- and adrenaline- induced 5-HT release. Doxazosin produced a dose-dependent increase in collagen-induced 5-HT release (Figure 7.3, Appendix, Table 38), statistically significant increases occurring with 7.5pM (81.7±19.5%, P<0.05, n=6) and 30pM (78.4±23.3%, p<0.05, n=6) doxazosin Adrenaline-induced 5-HT release was also increased in the presence of doxazosin but did not reach statistical significance (Figure 7.4, Appendix, Table 39) 7.4.4. Effect of nifedipine on collagen-induced 5-HT release Collagen-induced 5-HT release was inhibited by nifedipine in a dose-dependent manner ( Figure 7. 5, Appendix, Table 40). The inhibitions of 5-HT release at 0.28jiM, 1.4pM, 7pM and 28pM nifedipine were, respectively, 17.3±11.2% (95% Cl fi'om -10.2 to 44.7, P=not significant; n=6), 14.9±8.6% (95% Cl fi'om -14.0 to 43.9, P=not significant; n=5), 38.8±11.8% (95% Cl fi'om 11.3 to 66.2, P<0.05; n=6) and 61.2±8.5 (95% Cl firom 34.9 to 87.5, P<0.001; n=7). - 180- Chapter 7: Platelet granular release Figure 7.3 E ffect o f doxazosin on collagen (Sgg/m l) - induced 5-H T release (% control). D ata show n are derived from the appendix. T able 38a, and are presented also in the appendix. T able 38b. (* P < 0.05 com pared w ithe control) 200 o ■o § 160 CD u 3 “O c 20 CD c DCO (U CD 8 0 O) CD _□ "5 I— CJ X 40 ui Doxazosin Doxazosin Doxazosin Doxazosin 0.3/^lvl 1.5/j.M 7.5/.(M 3 0 / i . M (N = 3) (N = 5) (N = 6) ( N = 6 ) Figure 7.4 Effect of doxazosin on adrenaline (16pM )-induced 5-H T release (% control). D ata show n are derived from the appendix. T able 39a, and are also presented in the appendix. T able 39b. ^ 4 0 0 O “O L_ CD "c U o 3 o 3 0 0 -Q C 5 (D 0) C C/1 o 200 o CD c CD (D i_ -Q 1— < X 100 1 m Doxazosin Doxazosin Doxazosin Doxazosin 0.3^lv1 1.5/iM 7.5/xM 30/.iM (N = 6) (N = 5) (N = 6) (N = 5) - 181 - Chapter ?: Platelet granular release Figure 7.5 Effect o f nifedipine on collagen (Spg/m l) - induced 5-H T release (% control). D ata show n are derived from the appendix. T able 40a, and are also presented in the appendix. T able 40b . (*:P < 0.05 com pared w ith control) 100 80 ■Q c (D o 0 o 3 TJ o _C 60 1 5 c 0> 000) CD o ) 0 Nifedipine Nifedipine Nifedipine Nifedipine 0 . 28)16 1.4/2M 7 / / M 28^W ( N = 6 ) (N = 5) (N = 6) (N = y ) - 182- Chapter 7: Platelet granular rdease 7.4.5. PDGF release induced by collagen and adrenaline Collagen increased the release of PDGF from a basal level of 1.17±0.39 to 4.25±0.51 ng/10* platelets ( ?<0.001, n=7), whilst adrenaline increased it from a basal level of 1.08± 0.19 to 5.37±1.02 ng/10* platelets ( P <0.01, n=7). In investigating the effects of the drugs on stimulated PDGF release, basal levels of PDGF release were subtracted from the stimulated levels in both control and test samples. 7.4.6. Effect of naftopidil on collagen and adrenaline- induced release of PDGF Naftopidil at 40pM had no effect on the collagen induced release of PDGF, levels of PDGF in control and test samples being respectively 3.46±0.2 and 3.11±0.54 ng/10* platelets (n=6, data are derived from the Appendix Table 41). However, both concentrations of naftopidil tested, i.e. lOpM and 40pM, produced a dose-related inhibition of adrenaline- induced PDGF release (Figure 7.6, Appendix, Table 42). Thus, in the presence of 40pM naftopidil the levels of PDGF decreased to values of 0.91±0.17 ng/10* platelets (n=7) which were lower than the levels obtained with unstimulated platelets (1.08±0.19 ng/10* platelets, n=7) and which resulted in the inhibition by naftopidil of adrenaline-induced release of PDGF reaching 125.7±16.3% (95% Cl from 87.0 to 164.3, P<0.001; n=7). Naftopidil at a concentration of lOpM also inhibited significantly the release of PDGF, inhibition being 82.9±13.7% (95% Cl from 42.7 to 123.2%, P<0.01; n=6). - 183- Chapter 7: Platelet granular release 7.4.7. Effect of doxazosin on collagen- and adrenaline- induced release of PDGF Doxazosin (30pM) like naftopidil (40pM) had no effect on collagen-induced PDGF release, PDGF levels in control and test samples being respectively 2.98±0.37 and 2.34±0.80 ng/10* platelets (n=6, data are derived from the Appendix, Table 43). However, like naftopidil, doxazosin also produced dose-related inhibition of the release of PDGF induced by adrenaline (Figure 7.7, Appendix, Table 44), a statistically significant inhibition of 70.3±31.5% (95% Cl from 6.4 to 134.2, P<0.05; n=6) being obtained with 30pM doxazosin . 7.4.8. Effect of nifedipine on collagen-induced release of PDGF Nifedipine (28 pM), like naftopidil and doxazosin, did not inhibit the collagen- induced release of PDGF, levels of PDGF in control and test samples being respectively 3.10±0.23 and 2.69±0.4 ng/10* platelets (n=6, data are derived from the Appendix, Table 45). -184- Chapter 7: Platelet granular release Figure 7.6 Effect o f naftopidil on adrenaline (16pM ) - induced PD G F release (% control). D ata show n are derived from the appendix. T able 42a, and are also presented in the appendix. T able 42b. (*: P < 0.05 com pared w ithe control, n = 6). * 20 c o *0 -20 o Û Û_ -4 0 Naftopidil Naftopidil 1 O y L ^ M 40yLxM Figure 7.7 Effect of doxazosin on adrenaline (16pM ) - induced PD G F release (% control). D ata show n are derived from the appendix. T able 42a, and are also presented in the appendix. T able 42b. (*: P < 0.05 com pared w ithe control, n = 6). 80 r 60 c o o o 40 o c, 20 CL Doxazosin Doxazosin 7.5/./.M 30/iM - 185 - Chapter 7: Platelet granular release 7.5. Discussion Drug effects on coilagen-induced 5-HT release The data presented in this chapter indicate that naftopidil and doxazosin increase collagen-induced 5-HT release whilst nifedipine decreases it. The inhibition exerted by nifedipine was expected and is consistent with its inhibitory actions on calcium mobilization and platelet aggregation (see chapter 6). However, the enhancement of collagen-induced 5-HT release observed when platelets were pre-incubated with naftopidil and doxazosin was not expected and may indicate that both drugs inhibit platelet 5-HT re-uptake. This hypothesis is based on the assumption that a proportion of the 5-HT released on stimulation is, under normal circumstances, taken back up by platelets via the plasma membrane 5-HT transporter and this can be blocked by uptake inhibitors. As known uptake inhibitors were not included in the incubations it might be concluded that naftopidil and doxazosin exert this effect. Indeed, naftopidil has been reported to inhibit ^-5-HT uptake by human platelets (Breidert et al 1993). Moreover, urapidil, a naftopidil analogue, has also been reported to inhibit 5-HT uptake (Storck and Kirsten, 1990). It may be suggested that the inhibition of 5-HT uptake/re-uptake by both naftopidil and urapidil is due to their 5-HTi^ receptor agonism, since (+)8-0H-DPAT (8-hydroxy-2 (di-N-propylamine) tetraline which possesses 5-HTi^ agonistic activity has been shown to bind to the platelet 5-HT transporter and to inhibit 5-HT uptake (leni and Meyerson 1988). Thus, naftopidil may act via similar mechanisms to influence 5-HT binding and reuptake. - 186 - Chapter 7; Platelet granular release Influence of naftopidil and doxazosin on adrenaline-induced 5-HT release Adrenaline-induced platelet responses are mediated by a 2-adrenoceptors (Grant and Scrutton 1979). The inhibitory effects of naftopidil and doxazosin on adrenaline- induced aggregation are therefore indicative of the antagonistic effects of these drugs on platelet (%2-adrenoceptors and/or other mechanisms associated with adrenaline-induced responses. The variable, albeit statistically non-significant, increases in adrenaline-induced 5-HT release produced by doxazosin are inconsistent with the drug’s effect on collagen- induced 5-HT release. However, the different patterns of effects produced by naftopidil on adrenaline-induced 5-HT release, i.e. increases and decreases in 5-HT release with, respectively, low and high concentrations of naftopidil, probably reflects the different mechanisms of action of adrenaline and collagen in inducing 5-HT release and may also throw light on some aspects of these mechanisms. Hence, adrenaline-induced release of 5-HT would be expected to occur as a result of aggregation. The observation that the total inhibition by naftopidil of adrenaline-induced aggregation was not associated with total inhibition of 5-HT release, however, indicates that a proportion of 5-HT release induced by adrenaline is exclusive of its effects on platelet aggregation. This points to the possibility that a proportion of the 5-HT release by adrenaline may not be as a result of aggregation. 187- Chapter 7: Platelet granular release Hypothetical mechanism for adrenaline-induced 5-HT release and platelet aggregation It is possible that adrenaline-induced 5-HT release is not produced as a result of aggregation but rather by another mechanism e.g. leakage of 5-HT from the dense granules due to the flooding of the system with adrenaline. Thus, platelets can take up adrenaline and accumulate it in their dense granules (Rudnick 1986), this process possibly causing 5-HT release through its displacement in the dense granule by adrenaline. This hypothesis is based on the finding that certain amphetamine derivatives, including p-chloramphetamine (PCA) and 3, 4-methylenedioxymethamphetamine (MDMA), may act as substrates for the platelet plasma 5-HT transporter, competing with 5-HT and causing 5-HT release apparently by a process of exchange (Rudnick and Wall 1992, 1993). Human platelets resemble sympathetic neurons (Abrams and Solomon 1969, Bom and Smith 1970) sharing similar uptake mechanisms for free amines in vitro. The biogenic amine transporters and not exocytosis are thought to mediate amphetamine-induced release of catecholamines and 5-HT from nerve terminals (Rudnick and Wall 1992), this process being sensitive to transport inhibitors and Ca^^ independent. The entry of adrenaline into platelets may occur by passive difrusion (Da Prada and Picotti 1979), e.g. when the system is saturated with adrenaline, or through a specific uptake system, possibly the 5-HT transporter itself (Rudnick 1986). It can be postulated, therefore, that the entry of adrenaline into platelets, through whatever mechanism, will induce 5-HT release by exchange and when a certain threshold level of -188 - Chapter 7: Platelet granular release 5-HT in the medium is reached, a synergistic interaction between adrenaline bound to (%2-receptors and 5-HT bound to 5-HT;-receptors will induce aggregation resulting in further release. The blockade of the 5-HT transporter, as indicated by the effect of naftopidil on collagen-induced 5-HT release, would result in reduced platelet uptake of adrenaline and hence its exchange with granular 5-HT, resulting in dose-dependent inhibition of 5-HT release and consequent inhibition of the synergistic interaction between adrenaline and 5-HT in the induction of platelet aggregation and further monoamine release. Support for this hypothesis can be derived from the finding of Cerrito et al (1993) that the amount of 5-HT released determines the extent of adrenaline-induced aggregation. Thus, in this study adrenaline failed to produce platelet aggregation in PRP when the released products of platelets were prevented fi'om further interaction with platelets in the suspension by dint of a perfusion method (Cerrito et al 1993). Inhibition of PDGF release a-granular release, as represented by platelet PDGF efflux was dose-dependently inhibited by naftopidil with total inhibition being obtained at 40 jiM. This effect parallels the inhibitory effect produced by naftopidil on adrenaline-induced calcium mobilization, TxA2 formation and platelet aggregation. Doxazosin also, in parallel with its small inhibitory effects on these parameters, inhibited the adrenaline-induced release of PDGF. The collagen-induced release of PDGF was not affected by naftopidil, doxazosin or even nifedipine indicating that platelet a-granular release may not be dependent on the platelet Ca^^ increase or TxA .2 formation, both of which were inhibited by nifedipine and naftopidil. ___ Chapter 7: Platelet granular release 7.6. Conclusions The present study showed that naftopidil and doxazosin increase collagen- induced 5-HT release possibly indicating that these drugs possess 5-HT transporter blocking activity which might result in the prevention of the reuptake of 5-HT released on platelet stimulation. The calcium channel blocker nifedipine, however, decreased 5- HT release, in parallel with its inhibitory influence on calcium mobilization, TxAz formation and platelet aggregation (see chapter 6), providing confirmatory evidence that arachidonic acid metabolites, i.e. TXA 2 and endoperoxides are involved in mediating dense granule secretion (Kinder et al 1993). A hypothesis regarding a possible mechanism for adrenaline-induced platelet aggregation and 5-HT release was presented. This hypothesis was derived fi’om the observation that naftopidil appeared to inhibit adrenaline-induced 5-HT release. Thus, the blockade of the 5-HT transporter by naftopidil may also affect adrenaline uptake resulting in the inhibition of 5-HT release and platelet aggregation. Naftopidil and doxazosin inhibited the adrenaline-induced release of PDGF indicating that their interference with a 2-adrenoceptor-coupling mechanisms is involved in their platelet actions. PDGF may play a significant role in the pathogenesis of atherosclerosis by stimulating the proliferation of arterial smooth muscle cells (Ross 1993). Therefore, the inhibitory effects produced by naftopidil and doxazosin, which are, after all, antihypertensive drugs, may be useful clinically in reducing the risk of coronary heart disease associated with hypertension. The lack of any inhibitory effect of naftopidil and doxazosin on collagen-induced PDGF release indicates that different receptor- __ Chapter 7: Platelet granular release coupling mechanisms are involved in adrenaline- and collagen-induced release of PDGF. Nifedipine in contrast with its inhibitory action on dense granular release, expressed as 5-HT release, had no effect on a-granular release. This observation supports the concept that different mechanisms appear to be involved in mediating release from different granules (Kinder et al 1993). -191- Chapter 8 General discussion and concluding remarks -192- Chapter 8; General discussion and concluding remarks The studies presented in this thesis were aimed at testing the effects of the adrenoceptor antagonists antihypertensive drugs naftopidil and doxazosin on human platelet aggregatory and secretory processes in vitro, and at investigating the possible mechanisms of any antiplatelet actions detected for the two drugs. In some of these studies the calcium channel blocker nifedipine was included for comparative purposes. The presented studies included also an examination of the effects of the vasoactive peptides, endothelin (ET), neuropeptide Y (NPY) and atrial natriuretic peptide (ANP), on adrenaline-induced platelet aggregation 8.1 Stability of in vitro platelet responses It was necessary, before starting the drug studies, to consider carefully the experimental conditions to be employed in order to ensure optimal platelet stability as failure to do so might have lead to the generation of spurious results. Therefore, platelet sensitivity to the weak agonists ADP and adrenaline was determined after the storage of PRP under various conditions (see chapter 3). The results of this study indicated that in order to stabilize the PRP pH and ensure optimal platelet stability, PRP samples should be stored at room temperature in the absence of air and tested within four hours of sampling. It was also found that platelet count adjustment influenced platelet responsiveness (see chapter. 3). Nevertheless, it was concluded that standardization of platelet counts is necessary when studying the actions of drugs on platelet responses, to standardize the ratio of ligand to receptor (for further discussion see chapter 3 (3.5)). -193- Chapter 8: General discussion and concluding remarks 8.2. The effect of the vasoactive peptides on adrenaline-induced platelet aggregation The vasoactive peptides ET-1, NPY and ANP were found not to induce platelet aggregation by themselves or influence aggregation induced by adrenaline (see chapter 5). 8.3 Comparative studies on the effects of naftopidil, doxazosin and nifedipine on platelet aggregation The effects of naflopidil and doxazosin on platelet aggregation were investigated using three experimental approaches. The first approach was to study the effects of the drugs on platelet aggregation induced in PRP by single platelet agonists up to supra threshold concentrations (see chapter 4), whilst the second involved stimulating aggregation in PRP using a range of subthreshold concentrations of adrenaline in the presence of subthreshold concentrations of other agonists (see chapter 5). The third approach entailed studying the effects of the drugs on aggregation in washed platelet preparations induced by supra threshold concentrations of collagen and adrenaline, which are strong and weak agonists respectively (see chapter 6). These different experimental approaches were found necessary in order to reveal fully the effects of the drugs on platelet aggregation. Thus, when aggregation was induced in PRP by single agonists, naflopidil failed to produce shifts of ADP and collagen dose-response curves, while slight inhibitory actions were seen on aggregation induced by 5-HT and adrenaline. However, marked inhibitory effects of naftopidil were observed on aggregation induced by subthreshold concentrations of adrenaline in combination with other agonists. The adrenaline-induced aggregation of washed platelets was also inhibited dose-dependently by naftopidil, with total inhibition being obtained at the - 194- Chapter 8: General discussion and concluding remarha maximal concentration of naftopidil tested, i.e. 40 pM. This same concentration also produced a slight inhibition of collagen- induced platelet aggregation. The results obtained with doxazosin also indicated the necessity of using different experimental approaches in studying the effects of drugs on platelet aggregation. Thus, doxazosin failed to inhibit aggregation induced by individual agonists, i.e. collagen, adrenaline, ADP and 5-HT, but produced dose-dependent inhibition of platelet aggregation induced by sub-threshold concentrations of adrenaline in combination with ADP, collagen or 5-HT. When the effects of doxazosin on washed platelets were examined inhibition of adrenaline-induced aggregation also was observed. Collagen-induced aggregation, on the other hand, was not inhibited by doxazosin. In comparison with naftopidil and doxazosin, nifedipine produced a significant decrease of collagen-induced platelet aggregation in washed platelets. The effect of nifedipine on adrenaline-induced responses was not studied because of the inhibitory effect of its vehicle, DMSO, on platelet aggregation. 8.4 Plasma protein binding and the masking of drug effects The marked inhibitory effect of naftopidil, which reached 100% at 40 pM, on adrenaline-induced platelet aggregation in washed platelets, in contrast with the slight inhibition observed in PRP, is a strong indication of the influence of plasma protein binding in reducing the available effective free concentrations of a drug and, therefore, masking its inhibitory effects (for more discussion see Chapter 6 (6.1.5)). Therefore, it is concluded that the concentrations of drugs to be tested in platelet studies involving PRP should be carefully considered by taking into account established therapeutic plasma concentrations and extents of binding to plasma protein. -195- Chapter 8: General discussion and concluding remarks 8.5 Influence of drug vehicles on platelet aggregation The failure of doxazosin to inhibit platelet aggregation in PRP contrasts with a previous report (Hernandez et al 1991b) that doxazosin inhibited potently platelet aggregation induced by various agonists, including collagen, adrenaline and ADP. This discrepancy may be reflective of the different vehicles used in preparing drug solutions. In the present study distilled water was used whereas Hernandez et al (1991b) used methanol at a final concentration of 3% which was found in preliminary experiments to inhibit adrenaline-induced aggregation. 8.6 Effects of naftopidil and doxazosin in comparison with nifedipine on platelet 5-HT release Collagen-induced 5-HT release was increased in the presence of naftopidil and doxazosin perhaps indicating that the platelet reuptake system for 5-HT is inhibited by these drugs. No such indication was found for nifedipine which, by contrast, inhibited 5-HT release. It may be suggested that the blockade of 5-HT uptake by naftopidil is due to its 5-HTia agonistic activity (see Chapter 7 (7.5)), however, no such activity has been reported for doxazosin. The different ways in which naftopidil affected the adrenaline-induced release of 5-HT and platelet aggregation in washed platelets may indicate that a proportion of 5- HT release occurs exclusive of platelet aggregation. This observation gave rise to a hypothesis regarding the mode of action of adrenaline in the induction of platelet aggregation and release (see Chapter 7 (7.5.)). This hypothesis considers the possibility -196- Chapter 8: General discussion and concluding remarks that adrenaline is taken up by platelets and accumulates in the dense granules causing 5- HT release via an exchange mechanism. When a certain threshold level of 5-HT in the medium is reached a synergistic interaction between adrenaline bound to « 2" receptors and 5-HT bound to 5 -HT2 receptors will induce aggregation and T?^ formation, resulting in further release. This hypothesis may help in resolving the controversy regarding the agonistic effects of adrenaline on platelet aggregation, some investigators believing that the platelet-stimulatory effects of adrenaline through ccj-adrenoceptors requires simultaneous activation by other agonists (Lanza et al 1988, Steen et al 1993). 8.7 Comparative studies of the effects of naftopidil, doxazosin and nifedipine on platelet signal transduction mechanisms Naftopidil and doxazosin were found to inhibit calcium mobilization induced by collagen and adrenaline indicating that these drugs possess calcium channel blocking activity. In comparison with naftopidil and doxazosin, nifedipine, as expected, produced a more marked inhibition of collagen-induced calcium mobilization. Adrenaline- and collagen- induced generation of TXA 2 was inhibited by naftopidil but not doxazosin. Nifedipine also inhibited coUagen-induced TxÀ 2 generation comparable in extent to that produced by naftopidil. Naftopidil and doxazosin did not affect basal platelet cAMP levels but prevented its adrenaline-induced decline indicating that the drugs interfere with adrenaline- mediated receptor-coupling mechanisms. Chapter 8: General discussion and concluding remarks 8.8 Effect of naftopidil and doxazosin on a-granular release In agreement with their inhibitory action on adrenaline-induced platelet aggregation in washed platelets naftopidil and, to a lesser extent, doxazosin inhibited the release of PDGF, a marker of a-granular release. Collagen-induced release of PDGF, however, was not inhibited by naftopidil, doxazosin or nifedipine. Thus, the statistically significant inhibition of collagen-induced calcium mobilization, platelet aggregation and TxA2 formation observed with naftopidil was not reflected by an inhibition of PDGF release confirming that collagen, a strong platelet agonist, can bypass the arachidonic acid pathway and stimulate platelet activation directly. Nifedipine, which was used as a positive control, also had no effect on PDGF release in spite of its inhibitory action on collagen-induced calcium mobilization, TxAj formation, platelet aggregation and 5-HT release confirming that different mechanisms are involved in initiating release fi’om a and dense granules, with a-granule secretion being independent of arachidonic acid metabolism (Kinder et al 1993). 8.9 Summary Naftopidil was found to suppress all adrenaline-induced responses in washed platelets, i.e. calcium mobilization, TXA 2 formation, platelet aggregation, dense granule release and a-granule release. Doxazosin also inhibited adrenaline-induced calcium mobilization, a-granule release and platelet aggregation, but to a lesser extent than naftopidil, and did not decrease TXA2 formation or dense granule release. Therefore, it can be suggested that naftopidil, at the concentrations used, is more effective in blocking the signal -198- Chapter 8: General discussion and concluding remarks transduction mechanisms which trigger the release of arachidonic acid and stimulate concomitant TXA 2 production. This may explain the significant inhibition produced by naftopidil on the synergistic interactions between adrenaline and other platelet agonists in the induction of platelet aggregation (see chapter 5), as this interaction has been suggested to involve calcium mobilization (Steen et al 1988) and arachidonic acid release and metabolism. The inhibitory effects of naftopidil and doxazosin on adrenaline-induced calcium mobilization, platelet aggregation and a-granule release may involve the antagonism of tt 2-adrenoceptors which mediate adrenaline-induced responses. Moreover, the inhibitory effects of naftopidil on 5-HT-induced platelet aggregation in PRP (see chapter 4) suggest that naftopidil may possess 5 -HT2 antagonistic activity. If adrenaline-induced platelet responses are mediated by synergistic interactions between adrenaline and 5-HT as is hypothesized, the multiple inhibitory effects of naftopidil on 02-adrenoceptors and 5-HT2 receptors may explain the ability of naftopidil to inhibit completely adrenaline-induced calcium mobilization, a-granule release and platelet aggregation. Doxazosin, at the concentrations used, was not shown to inhibit 5-HT-induced platelet aggregation which suggests that doxazosin does not have antagonistic activity at 5 -HT2 receptors. This may explain the lower potency of doxazosin, compared with naftopidil, in inhibiting adrenaline-induced responses. The partial inhibitory effects of doxazosin seen on adrenaline-induced platelet responses may be mediated by blocking a 2-adrenoceptors and receptor-operated calcium channels The suggested antagonistic actions of naftopidil on a 2~adrenoceptors and 5 -HT2 -199- C hapter 8: General discussion and concluding remarks receptors and the observed inhibitory efifects of naftopidil on adrenaline-induced calcium mobilization may be mechanisms by which naftopidil produces marked inhibitory effects on adrenaline-induced TxA^ formation. However, when the effects of naftopidil and doxazosin were tested on collagen ( a strong agonist)-induced Ca^^ mobilization and TxA2 formation, both drugs inhibited calcium mobilization but only naftopidil inhibited TxA2 formation. The inhibition by naftopidil of collagen-induced TXA 2 formation may indicate, therefore, direct effects of the drug on specific sites of the TXA 2 synthetic cascade and this requires further investigation. The steps in the TXA 2 synthetic cascade which may be affected by naftopidil include arachidonic acid release from platelet phospholipids, which is stimulated by calcium-dependent PLA 2, and cyclooxygenase and/or TXA2 synthetase actions In conclusion, the results fi’om the in vitro studies on platelet flmction, presented in this thesis, suggest that the «i-adrenoceptor antagonists’ antihypertensive drugs, naftopidil and doxazosin, may possess antiplatelet effects. The importance of platelet overactivity in thrombosis and cardiovascular disease has been emphasised in recent years (see Chapter 1 (1.6.5.2)). Therefore, clinical studies may be required for further investigation of the antithrombotic potential of naftopidil and doxazosin. -200- Publications Pertaining to the thesis 1. Alarayyed, N.A., Smith, C.C.T., Betteridge, D.J., Prichard, B.N.C. Comparative study of the effects of naftopidil and doxazosin on platelet aggregation in vitro. Cardiovascular Drugs and therapy. 1993, 7(supple 2): 460 ( abstract). 2. Alarayyed, N.A., Cooper, M B., Smith, C.C.T., Prichard, B.N.C. In vitro effect of the a^-adrenergic receptor blockers naftopidil and doxazosin on calcium mobilisation in platelets. Br.J.Clin.Pharmacol. 1994. 37:472P-473P (abstract). 3. Alarayyed, N.A., Graham, B.R., Ferdous A.H., Betteridge, D.J., Prichard, B.N.C., Smith, C.C.T. An examination of some factors which influence the stability of in vitro platelet responses. Platelets. 1994.5:317-324. 4. Alarayyed, N.A., Graham, B.R., Prichard, B.N.C., Smith, C.C.T. The potentiation of adrenaline-induced in vitro platelet aggregation by ADP, collagen, and serotonin and its inhibition by naftopidil and doxazosin in normal human subjects. Br. J. Clin. Pharmac. 1995. 39:369-374. -201- Appendix: Tables -202- Table 1. Changes with time ( hour post-equilibration) of platelet aggregation (% light transmission) induced by the ADP threshold concentration (3.00±0.44fiM) in diluted PRP (platelet counts = 200x10^/L) stored in the presence or absence of air. (n=6). Time 0 2 4 6 PRP air-free Mean 74.20 69.00 57.70 45.80 SD 10.70 14.40 12.90 20.30 SEM 4.40 5.90 5.30 8.30 PRP with air Mean 70.00 42.40 34.90 SD 9.70 10.80 6.50 SEM 4.00 4.40 2.60 Table 2. Changes with time ( hour post-equilibration) of platelet aggregation (% light transmission) induced by the adrenaline threshold concentration (1.5±0.32pM) in diluted PRP (platelet count = 200x10^/L) stored in the presence or absence of air. (n=6). Time 0 2 4 6 PRP air-free Mean 50.90 25.10 22.90 14.90 SD 26.90 11.50 13.70 9.70 SEM 11.00 4.70 5.60 4.00 PRP with air Mean 22.00 22.10 13.40 SD 9.70 12.60 6.80 SEM 4.00 5.10 2.80 -203- Table 3. Changes with time ( hour post-equilibration) of platelet aggregation (% light transmission) induced by the ADP threshold concentration (2.67±0.67 pM) in diluted PRP (platelet count = 200x10^/L) stored at 22X, 13X and 4X. (n=3). Time 0 1 2 3 4 5 6 22X Mean 79.50 76.80 69.20 66.70 66.90 63.30 5130 SD 4.70 6.30 12.00 8.90 14.10 17.70 6.90 SEM 2.70 3.60 6.90 5.20 8.20 10.20 4.00 13°C Mean 74.40 79.90 72.30 81.50 74.80 84.80 SD 5.00 4.60 11.40 7.00 1.20 16.00 SEM 2.90 2.70 6.60 4.00 0.70 9.20 4!C Mean 76.30 73.80 70.40 68.90 76.30 78.70 SD 4.20 7.00 9.60 4.00 5.50 13.20 SEM 2.40 4.00 5.50 2.30 3.20 7.60 Table 4. Changes with time ( Hour post-equilibration) of platelet aggregation ( % light transmission) induced by the adrenaline threshold concentration (1.67±0.33pM) in diluted PRP (platelet count = 200x10^/L) stored at 22°C, 13°C and 4°C. (n=3). Time 0 1 2 3 4 5 6 22*C Mean 78.50 70.30 55.60 48.40 24.40 24.20 13.00 SD 10.40 13.90 29.20 34.40 19.50 23.30 11.20 SEM 6.00 8.00 16.90 19.90 11.30 13.50 6.50 13°C Mean 74.20 69.30 84.20 78.80 85.10 89.10 SD 10.70 12.70 1.80 5.30 7.20 10.60 SEM 6.20 7.30 1.00 3.00 4.20 6.10 4!C Mean 75.60 76.00 84.20 74.30 87.70 88.50 SD 9.20 9.00 11.30 7.80 12.30 10.50 SEM 5.30 5.20 6.50 4.50 7.00 6.00 -204- Table 5. Changes with time ( Hour post-equilibration) of platelet aggregation ( % light transmission) induced by the ADP threshold concentration (2.67±0.67 pM) in diluted (platelet count = 200x10^/L) PRP stored at 22T and 37°C. (n=3). Time 0 1 2 3 4 5 6 22°C Mean 78.60 76.40 74.20 74.40 66.20 59.30 54.20 SD 3.70 5.90 7.90 2.30 13.90 4.30 1.50 SEM 2.10 3.40 4.50 1.30 8.00 2.50 0.90 3TC Mean 54.00 56.80 46.80 41.10 41.00 41.10 SD 10.80 9.90 9.80 8.10 8.60 2.80 SEM 6.20 5.70 5.70 4.70 5.00 1.60 Table 6. Changes with time ( Hour post-equilibration) of platelet aggregation ( % light transmission) induced by the adrenaline threshold concentration (1.67±0.67 pM) in diluted PRP (platelet count = 200x10^/L) stored at 22X and 37°C. (n=3). Time 0 1 2 3 4 5 6 22°C Mean 79.30 78.20 77.00 74.20 63.40 23.30 25.60 SD 5.30 2.70 8.50 11.40 27.70 15.70 15.80 SEM 3.00 1.60 4.90 6.60 16.00 9.10 9.10 37°C Mean 12.50 11.20 6.80 3.60 0.00 0.00 SD 3.30 7.00 6.00 6.20 0.00 0.00 SEM 1.90 4.00 3.50 3.60 0.00 0.00 205- Table 7. Platelet aggregation (% light transmission) induced by threshold concentrations of ADP (3.00±0.44gM and 2.03±0.4gM) in diluted PRP (platelet count=200xl0^/L) and undiluted PRP (mean platelet count=469xlO^/L; range 296-659x10^/L). (n=6). ADP 0.25 0.5 1 2 4 8 16 _ (m i______Diluted PRP Mean 5.30 12.40 26.50 58.90 74.60 82.60 84.80 SD 1.60 4.20 7.60 16.00 9.30 5.30 5.30 SEM 0.70 1.70 3.10 6.50 3.80 2.20 2.20 Undiluted PRP Mean 6.70 14.90 36.20 69.20 77.90 77.90 81.00 SD 1.80 2.60 19.40 15.10 8.80 5.30 4.20 SEM 0.70 1.10 7.90 6.20 3.60 2.20 1.70 Table 8. Platelet aggregation (% light transmission) induced by threshold concentration of adrenaline (1.50±0.32pM and 0.65±0.17jiM) in diluted PRP (platelet count = 200x10^/L) and undiluted PRP (mean platelet count=469xlO^/L; range 296-659x10^/L). (n=6). Adrenaline 0.25 0.5 1 2 4 8 16 „M ® ______Diluted PRP Mean 7.50 28.50 33.70 66.60 70.70 71.70 73.00 SD 5.90 30.90 34.70 16.90 9.00 10.20 5.80 SEM 2.40 12.60 14.20 6.90 3.70 4.20 2.40 Undiluted PRP Mean 28.60 46.40 72.20 74.90 73.70 75.10 77.10 SD 38.20 33.30 5.40 4.30 3.80 5.10 4.30 SEM 1560 13.60 2.20 1.80 1.60 2.10 1.80 - 206 - Table 9. Changes with time (hour post-equilibration) of platelet aggregation (% light transmission) induced by the ADP threshold concentration (3.00±0.44|iM and 2.03±0.4pM) in diluted PRP (platelet count=200xl0^/L) and undiluted PRP (mean platelet count=469xlO^/L; Time 0 1 2 3 4 5 6 Diluted PRP Mean 70.60 65.00 57.00 52.30 50.50 45.00 41.80 SD 6.30 8.70 11.70 18.70 13.50 12.50 11.20 SEM 2.60 3.50 4.80 7.60 5.50 5.10 4.60 Undiluted PRP Mean 70.20 55.40 43.80 32.30 29.60 24.50 23.90 SD 10.00 23.00 21.60 12.00 13.40 11.30 14.20 SEM 4.10 9.40 8.80 4.90 5.50 4.60 5.80 Table 10. Changes with time (hour post-equilibration) of platelet aggregation (% light transmission) induced by the adrenaline threshold concentration (1.50±0.32pM and 0.65±0.17pM) in diluted (platelet count = 200x10^/L) and undiluted PRP (mean platelet count=469xlO^/L; range 296-659x10"/L). (n=6). Time { hour) 0 1 2 3 4 5 6 Diluted PRP Mean 64.80 41.60 34.50 19.10 11.90 8.10 8.40 SD 11.80 23.70 27.80 9.20 7.70 2.80 5.20 SEM 4.50 9.00 10.50 3.80 2.90 1.10 2.00 Undiluted PRP Mean 73.30 72.00 54.70 43.80 39.00 11.60 4.80 SD 4.90 7.10 35.30 32.10 38.70 16.70 3.20 SEM 2.00 2.90 14.40 13.10 15.80 6.80 1.30 207- Table 11. Platelet aggregation (% light transmission) induced by the ADP threshold concentration (3.00±0.44gM and 2.03±0.4|iM) in diluted PRP (platelet count=200xl0^/L) and undiluted PRP (mean platelet count=469xlO^/L; range 296-659x10^/L) after 24 hours of PRP storage ( Day 1) in the absence and presence of adrenaline (2pM).( platelet counts were determined at Ohour).(n = 6). Time (day) 0 1 1+adrenaline Diluted PRP Mean 73.00 17.90 53.00 SD 7.80 9.80 21.60 SEM 3.20 4.00 8.80 Undiluted PRP Mean 72.50 7.10 29.00 SD 12.30 5.00 14.70 SEM 5.00 2.00 6.00 -208- Table 12a. Effect of naftopidil (Naf) and Doxazosin (Dox) on adrenaline-induced platelet aggregation (% light transmission).(n= 8). Adrenaline 0.25 0.5 1 2 4 8 16 (pM) Control Mean 25.70 34.80 47.20 66.70 81.40 7850 74.80 SD 24.50 28.30 30.50 10.90 12.50 7.80 9.10 SEM 8.70 10.00 10.80 3.90 4.40 2.80 3.20 +Nafm.4pMl Mean 16.90 28.90 43.70 66.70 79.90 85.00 75.70 SD 11.00 24.40 19.90 16.60 10.60 13.30 16.20 SEM 3.90 8.60 7.00 5.90 3.70 4.70 5.70 +Nafr2pMl Mean 17.70 35.20 41.60 71.40 79.90 80.00 73.90 SD 12.50 25.90 26.30 14.00 20.30 17.00 17.20 SEM 4.40 9.20 9.30 4.90 7.20 6.00 6.10 +Naf riOpMl Mean 15.50 33.70 41.40 63.60 71.70 73.90 78.50 SD 10.10 29.30 26.30 14.00 15.60 7.40 14.90 SEM 3.60 10.40 9.30 4.90 5.50 2.60 5.30 +Dox r0.3pMl Mean 21.20 32.30 56.60 73.40 74.40 77.90 79.00 SD 17.00 28.40 25.20 6.70 14.30 13.80 16.00 SEM 6.00 10.00 8.90 2.40 5.10 4.90 5.70 +Dox n.5pM) Mean 22.00 30.80 48.90 71.00 71.20 74.30 76.00 SD 21.50 28.20 23.10 18.40 14.70 13.30 10.90 SEM 7.60 10.00 8.20 6.50 5.20 4.70 3.90 +Dox (7.5pM) Mean 23.30 31.00 45.10 68.40 74.70 78.50 76.90 SD 21.90 29.00 26.90 11.00 11.80 11.00 12.00 SEM 7.70 10.20 9.50 3.90 4.20 3.90 4.20 -209- Table 12b. Effect o f naftopidil (40pM) and Doxazosin (30|iM) on adrenaline- induced platelet aggregation (% light transmission). (n= 8). Adrenaline 0.25 0.5 1 2 4 8 16 ______Control Mean 18.60 30.60 38.60 65.80 83.70 83.30 84.60 SD 15.80 31.10 31.10 18.40 9.00 8.80 10.70 SEM 5.60 11.00 11.00 6.50 3.10 3.80 +Naf MOpMl Mean 12.30 18.10 34.10 47.20 74.70 70.70 73.70 SD 9.20 22.00 26.60 25.20 16.60 12.80 12.40 SEM 3.20 7.80 9.40 8.90 5.90 4.50 4.40 +Doxf30plVD Mean 12.20 24.60 36.80 68.40 77.70 80.00 80.80 SD 9.20 22.40 24.90 18.10 9.10 9.50 15.70 SEM 3.20 7.90 8.80 6.40 3.20 3.40 5.50 -210- Table 1j . Effect of naftopidil (Naf) and Doxazosin (Dox) on collagen-induced platelet aggregation (% light transmission). (n= 8). Collagen 0.25 0.5 1 2 4 8 JÙriE/Ml______Control Mean 1.80 23.30 62.00 85.70 88.10 92.20 SD 2.80 22.00 23.40 15.70 9.70 6.80 SEM 1.00 7.80 8.30 5.50 3.40 2.40 +Naf f0.4plVn Mean 1.30 19.80 60.40 88.20 86.50 88.90 SD 2.40 22.90 29.40 5.30 6.30 6.60 SEM 0.80 8.10 10.40 1.90 2.20 2.30 +Naf (2pM) Mean 1.80 21.70 58.30 84.90 86.40 90.00 SD 3.60 24.80 26.00 9.00 8.00 7.30 SEM 1.30 8.80 9.20 3.20 2.80 2.60 +Naf nOpMl Mean 1.60 24.40 60.40 85.70 88.40 90.20 SD 2.80 28.40 27.00 9.20 7.80 10.70 SEM 1.00 10.00 9.50 3.30 2.80 3.80 4-Naf MOpMl Mean 1.30 18.40 54.10 78.60 90.00 88.00 SD 2.60 21.30 27.00 13.60 6.20 7.70 SEM 0.90 7.50 9.50 4.80 2.20 2.70 +Dox f0.3pMl Mean 1 23.5 60.4 88.9 91.6 93.2 SD 2 28.4 25.9 11.6 8.3 7.3 SEM 0.7 10 9.2 4.1 2.90 2.6 +Dox n.SpMl Mean 1.50 18.30 64.50 80.60 91.50 88.70 SD 2.70 17.40 23.20 13.80 8.10 8.40 SEM 1.00 6.10 8.20 4.90 2.90 3.00 +Dox f7.5pMl Mean 1.20 15.80 56.30 84.40 89.70 90.80 SD 2.30 15.50 28.50 12.30 7.20 7.30 SEM 0.80 5.50 10.10 4.30 2.50 2.60 +Dox GOfiM) Mean 1.80 10.50 58.80 85.40 88.30 93.50 SD 3.30 10.50 28.70 8.90 9.60 4.40 SEM 1.20 3.70 10.10 3.10 3.40 1.60 -211- Table 14. Effect of naftopidil (Naf) and Doxazosin (Dox) on ADP-induced platelet aggregation (% light transmission). (n= 8). ADP (jiMl 0.25 0.5 1 2 4 8 16 Control Mean 7.80 16.20 37.70 70.20 83.70 91.00 87.70 SD 5.00 6.30 18.60 22.90 9.80 8.60 6.60 SEM 1.80 2.20 6.60 8.10 3.50 3.00 2.30 +Naf f0.4p]VD Mean 5.90 14.00 35.70 63.70 79.80 89.60 91.30 SD 2.50 6.30 16.20 18.70 11.50 8.70 6.70 SEM 0.90 2.20 5.70 6.60 4.10 3.10 2.40 +Naf Mean 6.20 15.50 29.80 65.10 86.00 92.00 90.30 SD 2.70 6.70 13.30 19.30 8.30 6.40 8.40 SEM 1.00 2.40 4.70 6.80 2.90 2.30 3.00 +Naf nOpM'l Mean 6.60 15.00 36.50 70.60 79.90 88.00 88.40 SD 2.40 7.30 17.80 18.70 8.60 6.30 6.00 SEM 0.80 2.60 6.30 6.60 3.00 2.20 2.10 +Naf f40pM'l Mean 6.30 15.00 32.20 68.50 81.10 89.50 90.40 SD 2.20 6.00 12.90 17.40 9.30 6.30 5.00 SEM 0.80 2.10 4.60 6.10 3.30 2.20 1.80 +Dox f0.3p]VD Mean 6.60 14.50 36.90 68.90 81.90 89.40 91.30 SD 3.20 6.00 18.40 18.70 14.30 9.20 8.20 SEM 1.10 2.10 6.50 6.60 5.10 3.30 2.90 +Dox n.5pM) Mean 6.10 12.90 31.60 69.20 84.70 89.60 91.80 SD 2.70 5.50 17.00 18.40 8.60 9.30 6.00 SEM 1.00 1.90 6.50 6.60 3.00 3.30 2.10 +Dox (7.5pMl Mean 6.60 13.70 36.20 70.00 75.60 88.70 90.10 SD 3.10 6.80 17.50 15.70 14.70 10.00 7.70 SEM 1.10 2.40 6.20 5.50 5.20 3.50 2.70 +Dox OOpMl Mean 8.40 16.20 34.20 64.20 78.40 88.40 88.50 SD 7.90 6.40 20.00 16.70 13.00 5.60 7.60 SEM 2.80 2.30 7.10 5.90 4.60 2.00 2.70 -212 Table 15. Effect of naftopidil (Naf) on 5-HT(10pM)- induced platelet aggregation (% light transmission). (n= 8). Control +Naf +Naf +Naf + Naf ------——------___ IPA^M i______i?jiMl__. _ÜQ mM)___ Mean 10.80 10.50 10.90 11.10 8.40 SD 3.90 3.00 4.00 4.40 3.60 SEM 1.40 1.10 1.40 1.60 1.30 Table 16. Effect of Doxazosin (Dox) on 5-HT(10pM)- induced platelet aggregation (% light transmission). (n=8). Control +Dox +Dox +Dox +Dox (p.3_pMl (1.5_pMl C30gM) Mean 10.80 11.50 10.90 9.90 10.70 SD 3.90 4.90 4.80 3.70 3.90 SEM 1.40 1.70 1.70 1.30 1.40 Table 17. Adrenaline-induced platelet aggregation (% light transmission) potentiated by ADP, Collagen and 5-HT. Aggregation induced by ; ADP (O.SjiM) alone; (Mean: 12.7, SD: 3.3, SEM: 1.2), Collagen (0.5pg/ml) alone: (Mean:0.5, SD:1.4, SEM:0.5 ), 5-HT (2.5pM) alone: (Mean: 8.4, SD: 3.9, SEM: 1.4).(n = 8, except at adrenaline IpM where n = 4) Adrenaline (jiM) 0.03 0.06 0.125 0.25 0.5 1 Adrenaline alone Mean 0 2.9 9.8 8.9 17.4 12.0 SD 0 4.7 9.4 6.5 10.8 3.3 SEM 0 1.7 3.3 2.3 3.8 1.7 Adrenaline+ADP Mean 18.5 42.9 66.4 77.6 87.4 93.3 SD 22.3 25 25.4 13.6 12.5 9.7 SEM 7.9 8.8 9.00 4.8 4.4 4.9 Adrenaline+Collagen Mean 2.3 39.3 59.1 69.4 82.3 87.0 SD 4.4 30.5 34.8 26.0 15.7 9.0 SEM 1.6 10.8 12.3 9.2 5.5 4.5 Adrenaline+5-HT Mean 8.3 17.6 37.2 74.7 77.3 88.9 SD 4.2 4.4 20.9 11.2 14.9 15.8 SEM 1.5 1.6 7.4 4 5.3 7.9 -213- Table 18 Effect of naftopidil (Naf) and doxazosin (Dox) on adrenaline-induced aggregation ( % light transmission) potentiated by ADP (O.SpM). ADP- ______induced aggregation: Mean: 9.1 SD; 3.2; SEM:1.1 (n=8) ______ Adrenaline (pMi 0.03 0.06 0.125 0.25 0.5 1 Adrenaline alone Mean 0.00 2.20 5.80 8.50 13.60 15.30 SC 0.00 3.00 4.50 4.60 10.40 9.80 SEM 0.00 1.10 1.60 1.60 3.70 4.40 Adrenaline+ADP Mean 18.50 32.20 54.30 71.90 70.90 76.80 SD 22.30 27.50 32.10 15.80 17.20 8.50 SEM 7.90 9.70 11.30 5.60 6.10 3.80 +Naf f0.4pM) Mean 20.20 24.10 43.30 64.80 70.30 74.20 SD 25.50 23.70 24.60 20.30 16.60 16.60 SEM 9.00 8.40 8.70 7.20 5.90 7.40 +Naf (2pM) Mean 19.50 29.40 35.50 56.50 72.30 71.60 SD 23.30 32.80 20.80 24.20 21.20 12.00 SEM 8.20 11.60 7.40 8.50 7.50 5.40 + NafnOpMl Mean 19.10 22.60 37.90 46.00 68.40 75.00 SD 28.80 26.90 31.50 23.30 23.60 8.30 SEM 10.20 9.50 11.10 8.20 8.30 3.70 +Naf MOpMl Mean 11.90 15.30 18.10 31.20 38.00 44.30 SD 7.20 13.30 9.90 , 25.40 16.30 26.90 SEM 2.50 4.70 3.50 9.00 5.80 12.00 +Dox fO.SpM) Mean 21.40 28.00 38.60 60.10 77.50 75.80 SD 23.90 29.00 29.20 19.50 21.80 12.40 SEM 8.40 10.20 10.30 6.90 7.70 5.60 +Dox (l.SpM) Mean 14.40 28.80 41.30 69.40 74.00 67.80 SD 11.00 28.30 32.20 16.50 14.20 18.00 SEM 3.90 10.00 11.40 5.80 5.00 8.00 +Dox r7.5pMD Mean 23.40 29.00 43.50 59.90 73.60 73.40 SD 28.30 31.40 32.40 23.00 25.40 10.50 SEM 10.00 11.10 11.50 8.10 9.00 4.70 +Dox nOpM) Mean 18.90 21.50 32.00 53.80 68.10 63.00 SD 22.40 25.80 32.80 23.70 18.20 26.00 SEM 7.90 9.10 11.60 8.40 6.40 11.60 -214- Table 19. Effect o f N aftopidil (N af) and D oxazosin (D ox) on adrenaline-induced aggregation (% light transm ission) potentiated by collagen (0 .125pg/m l). A d r e n a l i n e 0.03 0.06 0.125 0.25 0.5 1 ( u M ) A d r e n a l i n e Mean 0.00 1.40 5 . 4 0 8 . 8 0 15.70 17.90 SD 0.00 2.70 4.20 4.50 9.60 10.40 SEM 0.00 0.90 1.50 1.60 3.40 5.20 + C o l l a g e n Mean 2.30 19.00 24.00 47.50 7 5 . 3 0 64.10 SD 4.40 35.30 34.10 2 9 . 7 0 20.70 27.00 SEM 1.60 12.50 12.10 10.50 7.30 13.50 4-N af ro .4|i]v n Mean 1.50 18.80 24.50 48.80 57.40 68.50 SD 2.80 35.70 32.10 26.60 22.40 27.90 SEM 1.00 12.60 11.30 9.40 7.90 14.00 + N a f (2p]Vn Mean 1.90 19.00 19.40 39.90 62.70 57.00 SD 3.50 35.20 30.20 31.00 25.80 2 8 . 8 0 SEM 1.20 12.40 10.70 11.00 9.10 14.40 +N af riO pM 'l Mean 1.30 13.20 21.10 37.80 39.40 40.30 SD 2.60 29.90 30.70 29.10 27.30 30.50 SEM 0.90 10.60 10.80 10.30 9.70 15.20 + N a f M O p M l Mean 1.40 6.40 3.90 9.80 22.10 25.10 SD 2.70 16.00 4.10 6.30 21.30 26.90 SEM 0.90 5.70 1.40 2.20 7.50 13.40 +Dox rO.SpMl Mean 2.80 21.90 17.50 47.10 66.10 67.90 SD 5.70 40.50 25.90 30.40 26.10 31.00 SEM 2.00 14.30 9.20 10.70 9.20 15.50 +Dox(1.5p]VD Mean 1.20 14.80 14.60 45.50 55.90 64.50 SD 2.50 30.00 30.10 32.00 20.90 31.00 SEM 0.90 10.60 10.60 11.30 7.40 15.70 +Dox f T . S p M ) Mean 2.00 15.00 16.60 48.70 59.60 68.90 SD 3.90 30.60 25.70 31.70 2 8 . 4 0 27.70 SEM 1.40 10.80 9.10 11.20 10.00 13.80 +Dox nOpM'l Mean 2.10 8.20 7.60 35.90 41.60 62.50 SD 4.00 16.40 8 . 5 0 31.20 24.90 27.00 SEM 1.40 5.80 3.00 11.00 8.80 13.50 n 8 8 8 8 8 4 -215- Table 20. Effect of naftopidil (Naf) and doxazosin (dox) on adrenaline-induced platelet aggregation (% light transmission) potentiated by 5-HT. Aggregation induced by 5-ETT (2.5pM) alone:Mean: 8.3, SD: 4.1; SEM: ______1.5.(n = 8, except at IpM where n = 5). ______ Adrenaline (gM} 0.03 0.06 0.125 0.25 0.5 1 Adrenaline alone Mean 0.00 0.80 3.80 7.00 11.70 14.50 SD 0.00 2.20 3.30 4.00 5.80 11.60 SEM 0.00 0.80 1.20 1.40 2.00 5.20 Adrenaline+5-HT Mean 8.50 12.90 19.20 50.20 69.50 76.20 SD 3.80 5.70 4.50 28.60 10.90 5.90 SEM 1.30 2.00 1.60 10.10 3.90 2.60 -Hnaftopidiir0.4uM) Mean 7.60 9.20 13.80 51.90 70.40 83.00 SD 4.10 4.70 6.50 27.40 16.40 4.50 SEM 1.50 1.70 2.30 9.70 5.80 2.00 +naftopidil QpM) Mean 7.70 8.90 13.60 47.10 64.20 73.90 SD 4.20 3.80 5.50 26.60 18.70 17.80 SEM 1.50 1.30 1.90 9.40 6.60 7.90 -Hiaftopidil ( 10 pM) Mean 7.90 9.10 12.20 36.50 46.00 72.70 SD 4.50 4.80 5.10 21.50 20.00 20.30 SEM 1.60 1.70 1.80 7.60 7.10 9.10 +naftopidil r40fiM) Mean 6.10 6.20 8.50 14.90 29.00 36.90 SD 3.30 2.30 5.10 4.20 24.00 15.90 SEM 1.20 0.80 1.80 1.50 8.50 7.10 +Dox {'0.3fiM> Mean 8.00 13.50 14.50 52.70 58.80 82.40 SD 4.00 13.20 5.80 29.00 21.00 8.20 SEM 1.40 4.70 2.00 10.20 7.40 3.70 +Dox fl.SpMl Mean 8.40 9.70 15.60 50.40 60.50 83.80 SD 4.00 4.00 5.80 29.00 19.50 8.30 SEM 1.40 1.40 2.00 10.20 6.90 3.70 +Dox (7.5pM) Mean 8.40 8.80 14.00 50.70 58.40 73.60 SD 4.10 3.70 5.60 30.30 21.80 6.60 SEM 1.40 1.30 2.00 10.70 7.70 2.90 +Dox OOpM) Mean 8.20 9.50 13.40 33.10 49.10 68.00 SD 4.10 4.30 6.70 25.20 26.90 24.70 SEM 1.40 1.50 2.40 8.90 9.50 11.00 -216- Table 21. Adrenaline-induced platelet aggregation ( % light transmission) in PRP (platelet count = 200x10^/L), in the presence of endothelin. (n= 6 ). Adrenaline 0.25 0.5 1 2 4 8 16 cone {gM) Control Mean 27.30 45.20 62.60 73.70 75.60 81.60 80.20 SD 25.10 21.60 10.90 11.20 8.30 5.80 5.30 SEM 10.20 8.80 4.40 4.60 3.40 2.40 2.20 +Endothelin (InM) Mean 30.20 41.00 64.50 74.80 79.00 80.30 80.80 SD 25.70 17.60 9.80 13.90 6.90 3.80 2.30 SEM 10.50 7.20 4.00 5.70 2.80 1.60 0.90 +Endothelin (IpM) Mean 32.20 48.90 63.00 69.90 78.30 80.40 79.50 SD 22.70 27.60 15.00 10.60 8.00 3.70 4.80 SEM 9.30 11.30 6.10 4.00 3.30 1.50 2.00 Table 22. Adrenaline-induced platelet aggregation (% light transmission) in PRP (platelet count = 200x10^/L), in the presence of NPY. (n=6). Adrenaline 0.25 0.5 1 • 2 4 8 16 „ M £ L______Control Mean 33.20 48.30 70.00 76.00 77.40 80.10 79.50 SD 28.10 26.00 9.70 8.60 9.40 6.90 6.00 SEM 11.50 10.60 4.00 3.50 3.80 2.80 2.40 +NPY ro.ipivn Mean 31.70 52.60 69.30 71.20 78.40 79.20 82.90 SD 24.10 17.90 8.10 11.30 5.40 11.00 6.60 SEM 9.80 7.30 3.30 4.60 2.20 4.50 2.70 +NPYrip]vn Mean 35.10 52.00 65.20 75.10 76.40 79.10 79.00 SD 30.10 22.30 11.00 10.00 9.90 10.90 10.80 SEM 12.30 9.10 4.60 4.10 4.00 4.40 4.40 -217- Table 23. Adrenaline-induced platelet aggregation (% light transmission) in PRP (platelet count = 200x10^/L), in the presence of ANP. ( n= 6 ). Adrenaline 0.25 0.5 1 2 4 8 16 Control Mean 28.90 43.50 69.70 75.90 73.40 75.30 78.50 SD 24.20 21.90 10.80 10.20 12.00 15.20 5.70 SEM 9.90 8.90 4.40 4.20 4.90 6.20 2.30 +ANPrinlVn Mean 34.50 35.70 68.00 65.60 76.80 73.70 80.20 SD 23.50 13.20 13.60 10.30 7.50 15.20 8.50 SEM 9.60 5.40 5.54 4.20 3.00 6.20 3.50 +ANPriOn]Vn Mean 33.80 27.40 60.70 69.10 77.80 76.00 75.90 SD 26.40 7.20 14.50 14.70 11.20 15.80 7.20 SEM 10.80 2.90 5.90 6.00 4.60 6.40 2.90 - 218 - Table 24a. Effect of Naftopidil (Naf) on collagen (5pg/m)l-induced [Ca^^]i increase (pM) and platelet aggregation (% light transmission). Control Naf Naf Naf Naf 0.4jiM lOgM 40 pM Mean 4.97 5.13 4.16 4.16 4.08 SD 2.00 2.50 2.70 2.19 2.30 SEM 0.63 1.00 1.10 0.83 0.87 % light transmission Mean 74.60 61.60 71.50 63.00 64.90 SD 22.30 21.70 20.70 27.30 28.50 SEM 7.00 8.90 8.50 10.30 10.80 n 10 6 6 7 7 Table 24b. Effect of Naftopidil (Naf) on collagen (5pg/ml)-induced increase (% control) and platelet aggregation (% control). Naf N af Naf Naf 40pM 0.4pM 2jLiM lOpM [C4^1(pM) Mean 95.30 87.40 86.21 77.11 SD 6.57 14.55 10.18 10.55 SEM 2.68 5.94 3.84 3.90 Platelet aggregation Mean 85.92 99.00 86.00 76.72 SD 22.38 20.56 24.60 31.10 SEM 9.13 8.39 9.28 11.72 n 6 6 7 7 -219- Table 25a Effect of naftopidil (Naf) on Adrenaline (16pM)-induced [Ca^^Jj increase (|iM) and platelet aggregation (% light transmission). (n= 6 ). Control Naf N af N af N af (p.4jiM l OQmM) ____ ( 4 0 j ^ } ___ Mean 3.60 3.52 2.73 1.32 0.00 SD 2.34 2.38 1.86 1.21 0.00 SEM 0.96 0.97 0.76 0.49 0.00 % light transmission Mean 70.00 70.60 52.30 10.30 0.00 SD 27.90 35.00 31.10 4.60 0.00 SEM 11.40 14.30 12.70 1.90 0.00 Table 25b. Effect of naftopidil (Naf) on Adrenaline (16pM)-induced [Ca^^Ji increase (% control) and platelet aggregation (% control). (n=6). Naf Naf Naf Naf ______CIQmM)_____(4_QmM)_ tCa£l Mean 97.23 77.75 29.36 0.00 SD 7.84 17.96 23.35 0.00 SEM 3.20 7.30 9.50 0.00 Platelet aggregation Mean 95.69 74.27 16.63 0.00 SD 17.81 27.88 7.17 0.00 SEM 7.27 11.38 2.93 0.00 -220- Table 26. Percent inhibition by naftopidil (Naf) of Adrenaline (16pM)-induced [Ca^^]i increase and platelet aggregation. (n=6). Naf Naf Naf Naf ______— — — — — — — — — — — — — — — — — — __ _(4QgM)___ fCa^'li (% inhibition) Mean 2.77 22.25 70.65 100.00 SD 7.84 17.95 23.34 100.00 SEM 3.20 7.33 9.53 100.00 Platelet aggregation (% inhibition) Mean 4.31 25.74 83.37 100.00 SD 17.81 27.85 7.18 100.00 SEM 7.27 11.37 2.93 100.00 -221- Table 27a. Efifect of Doxazosin (Dox) on collagen (5gg/ml)-induced [Ca^^]j increase (gM) and platelet aggregation (% light transmission). Control Dox Dox Dox Dox (p.3_pMl (1.5 gM) C7.5iiMl (30gM) tCa^l fuM) Mean 4.97 5.15 3.83 4.28 4.23 SD 2.00 2.55 1.06 2.00 1.80 SEM 0.63 1.04 0.43 0.81 0.63 % light transmission Mean 74.60 60.80 76.70 74.60 63.50 SD 22.30 21.80 24.20 18.20 31.70 SEM 7.00 8.90 9.90 7.40 11.20 n 10 6 6 6 8 Table 27b. Efifect of Doxazosin (Dox) on collagen (5gg/ml)-induced [Ca^^]i increase (% control) and platelet aggregation (% control). Dox Dox Dox Dox ______0-5 gM) (].5ÿMl I30gM)__ [C a^ i Mean 99.41 91.35 85.00 82.56 SD 1.44 8.66 9.44 7.10 SEM 0.58 3.53 3.85 2.51 Platelet aggregation Mean 96.60 97.61 93.02 80.02 SD 21.24 10.14 17.34 34.86 SEM 8.70 4.14 7.00 12.30 n 6 6 6 8 222- Table 28a. Effect of Doxazosin (Dox) on adrenaline (16^M)-induced [Ca^^]j increase (|iM) and platelet aggregation (% light transmission). Control Dox Dox Dox Dox O.SjiM l.SjiM l.SjiM 30gM [Ca'li(nM ) Mean 3.49 3.35 3.34 2.86 2.26 SD 2.16 2.02 1.74 1.65 2.15 SEM 0.82 0.82 0.71 0.67 0.88 % light transmission Mean 67.10 73.50 69.20 48.30 35.60 SD 26.50 20.90 28.80 34.50 40.00 SEM 10.00 8.50 11.70 14.10 16.30 n 7 6 6 6 6 Table 28b. Effect of Doxazosin (Dox) on adrenaline (16pM)-induced increase (% control) and platelet aggregation (% control). Dox Dox Dox Dox O.SjiM l.SjiM l .S j iM 30gM i c e . 1 . Mean 99.92 97.85 75.54 62.36 SD 13.49 20.28 14.82 33.53 SEM 5.50 8.27 6.04 13.70 Platelet aggregation Mean 101.69 98.59 64.18 50.37 SD 16.32 12.34 30.85 42.26 SEM 6.70 5.03 12.60 17.24 n 6 6 6 6 -223- Table 29a. Efifect of nifedipine (Nif) on collagen (5pg/ml)-induced [Ca^^], increase (pM) and platelet aggregation (% light transmission). Control Nif Nif Nif Nif 0.28gM 1.4jiM T.OjiM 28pM [Ca^KwM) Mean 5.49 5.28 4.41 4.10 2.95 SD 2.57 3.26 2.64 1.97 1.43 SEM 0.77 1.23 1.00 0.75 0.48 % light transmission Mean 76.10 59.90 58.60 41.40 37.20 SD 21.70 25.10 32.40 29.30 28.30 SEM 6.50 9.50 12.20 11.10 9.40 n 11 7 7 7 9 Table 29b. Efifect of nifedipine (Nif) on collagen (5pg/ml)-induced [Ca^^]i increase (% control) and platelet aggregation (% control). Nif Nif Nif Nif 0.28jliM 1.4jiM T.OpM 28 pM [Cal1i Mean 94.23 85.49 80.66 52.09 SD 10.68 11.30 9.43 8.00 SEM 4.00 4.30 3.56 2.70 Platelet aggregation Mean 81.62 76.02 59.94 44.87 SD 21.14 24.52 30.28 27.67 SEM 8.00 9.20 11.40 9.20 n 7 7 7 9 -224- Table 30a. Efifect of naftopidil (naf) and doxazosin (dox) on the adrenaline (16|iM)- stimulated release of 1 x 62 (ng/ml/10* platelets Basai Stimulated +Naf +Naf +Dox +Dox TxB2 TxB2_ (iPl^Ml 7.5jiM 30gM Mean 2.44 8.02 5.76 3.28 8.10 7.14 SD 1.49 2.64 1.84 1.52 1.07 3.37 SEM 0.61 1.08 0.75 0.62 0.54 1.38 n 6 6 6 6 4 6 Table 30b Effect of naftopidil (naf) and doxazosin (dox) on the adrenaline (16pM)- stimulated release of TxB, (% control). +Naf +Naf +Dox +Dox (lOjiMl 7.5gM 30gM Mean 57.54 18.20 83.56 84.77 SD 25.31 18.39 16.95 55.53 SEM 10.33 7.51 8.50 22.70 n 6 6 4 6 Table 31. Effect of naftopidil (Naf) on the collagen (5 pg/ml)-stimulated release of Basai TxB]_ Stimulated TxBj2_ +Naf (40]iM) Mean 8.34 84.00 33.52 SD 5.08 44.97 12.94 SEM 2.27 20.10 5.79 -225- Table 32. Effect of doxazosin (Dox) on the collagen (5^g/ml)-stimulated release of TxBi (ng/ml/10* platelets). (n=5). Basal TxB^ Stimulated TxB-, +Dox (30gM) Mean 6.50 91.52 66.86 SD 2.32 40.80 39.37 SEM 1.04 18.21 17.57 Table 33. Effect of nifedipine (Nif) on collagen (5pg/ml)-stimulated release of TxB2 (ng/ml/10* platelet). (n=6). Basal TxB^ Stimulated TxB^ +Nif28gM Mean 5.67 89.04 47.92 SD 2.22 43.61 37.38 SEM 0.90 17.80 15.26 Table 34. Effect of Naftopidil (Naf) and Doxazosin (Dox) incubated for 2 min in washed platelets, on basal levels of platelet cAMP (pmol/ml/10* platelets). (n=6). Basal cAMP +Naf (40jiNr) + Dox {30pM)^ Mean 11.70 12.66 11.26 SD 5.07 6.28 5.03 SEM 2.07 2.56 2.05 - 226 - Table 35. Effect of naftopidil (Naf) and Doxazosin (Dox) on platelet cAMP (pmol/ml/10* platelets) in the presence of adrenaline (16pM). (n=4). Basal cAMP +Adrenaline + Adrenaline +Adrenaline +Naf (40 pM) +Dox(30pM) Incubation for 2 min Mean 9.32 9.75 10.03 10.44 SD 4.00 3.95 6.20 2.91 SEM 2.00 1.97 3.08 1.45 Incubation for 6 min Mean 10.30 6.75 8.16 7.75 SD 4.65 4.08 4.97 3.87 SEM 2.32 2.04 2.48 1.93 Table 36a. Effect of naftopidil (Naf) on the collagen (5pg/ml)-induced release of 5- HT (Pmol/ml/10* platelets). Basal Stimulated +Naf +Naf +Naf +Naf 5-HT 5-HT 0.4 yM lOpM 40 pM Mean 13.88 188.67 203.39 284.17 287.72 374.10 SD 4.18 79.11 126.57 142.68 128.29 147.13 SEM 1.39 26.37 63.28 58,24 48.59 60.05 n 9 9 4 6 7 6 Table 36b. Effect of naftopidil (Naf) on the collagen (5pg/ml)-induced release of 5- HT ( % control). +Naf +Naf +Naf +Naf 0.4 jgM lOgM 40pM Mean 107.27 171.57 189.13 169.68 SD 39.60 41.72 44.53 37.62 SEM 19.80 17.02 16.90 15.35 n 4 6 7 6 -227- Table 37a Effect of naftopidil (Naf) on the adrenaline (16pM)-induced release of 5-HT. (pmol/10* platelets). (n=5). Basal Stimulated +Naf + Naf + Naf + Naf 5-HT 5-HT 0.4jiM ^ M lOgM 40 pM Mean 12.48 128.54 172.37 149.14 80.69 47.37 SD 3.07 65.50 67.72 101.63 26.18 14.21 SEM 1.37 29.24 30.23 45.37 11.69 6.34 Table 37b Effect of naftopidil (Naf) on the adrenaline (16pM)-induced release of 5-HT ( % control). (n=5). +Naf + Naf + Naf + Naf 0.4jiM ^ M lOgM 40gM Mean 166.21 109.89 66.95 41.47 SD 112.38 38.44 28.22 35.15 SEM 50.16 17.16 12.60 15.69 Table 38a. Effect of doxazosin (Dox) on the collagen (5pg/ml)-induced release of 5-HT (pmol/ml/10* platelets). Basal Stimulated +Dox +Dox +Dox +Dox 5HT 5-HT 0.3jiM 1.5iiM 7.5jiM 30gM Mean 14.04 180.78 207.59 241.73 277.57 353.7 SD 04.45 80.70 50 86 102.26 82.16 149.11 SEM 01.57 28.51 29.40 41.74 33.53 60.86 n 8 8 3 6 6 6 Table 38b Effect of doxazosin (Dox) on collagen (5pg/ml)-induced release of 5-HT (% control). +Dox +Dox +Dox +Dox 0.3jiM 1.5jiM 7.5jiM 30gM Mean 117.27 146.67 181.66 178.35 SD 26.17 28.25 47.88 57.20 SEM 15.12 11.53 19.55 23.34 n 3 6 6 6 228- Table 39a. Effect of doxazosin (Dox) on the adrenaline (16^iM)-induced release of Basal Stimulated Dox +Dox +Dox +Dox 5-HT 5-HT 0.3jiM l.^ M 7.5jiM 30yM Mean 11.0 110.60 149.63 156.37 188.36 194.43 SD 3.57 73.25 84.29 38.63 69.44 110.96 SEM 1.46 29.90 34.40 17.24 28.34 49.54 n 6 6 6 5 6 5 Table 39b. Efifect of doxazosin (Dox) on the adrenaline (16|iM)-induced release of Dox +Dox +Dox +Dox 0.3jiM 1.5jiM 7.5jiM 30yAi Mean 180.77 147.34 293.74 158.51 SD 138.84 66.70 276.36 47.53 SEM 56.70 29.80 112.80 21.22 n 6 5 6 5 - 2 2 9 - Table 40a. Effect of nifedipine (Nif) on the collagen (5pg/ml)-induced release of 5- HT (pmol/10* platelets). Basal Stimulated +Nif +Nif +Nif +Nif 5-HT 5-HT 0.28_gM 1.4jiM TgM 28gM Mean 13.08 178.18 150.01 155.01 99.42 77.68 SD 3.77 78.84 60.64 59.80 54.00 54.87 SEM 1.33 27.75 24.75 26.70 22.04 20.78 n 8 8 6 5 6 7 Table 40b Effect of nifedipine (Nif) on the collagen (5pg/ml)-induced release of 5-HT (% control). +Nif +Nif +Nif +Nif 0.28jgM 1.4jiM TjLiM 28 gM Mean 82.75 85.04 61.27 38.81 SD 27.45 19.35 28.88 22.43 SEM 11.20 8.64 11.80 8.49 n 6 5 6 7 Table 41, Effect of naftopidil on collagen-induced release of PDGF (ng/ml/10* platelets). (n=6). Basal Stimulated +Naftopidil PDGF PDGF (40g&Q^ Mean 1.64 5.10 4.75 SD 0.94 1.03 1.58 SEM 0.38 0.42 0.64 -230- Table 42a. Effect of naftopidil on the adrenaline-induced release of PDGF (ng/ml/10* platelets) Basal Stimulated Naftopidil Naftopidil PDGF PDGF (40pM) (lOpM) Mean 1.08 5.44 0.91 2.60 SD 0.50 2.77 0.46 1.24 SEM 0.19 1.05 0.17 0.50 n 7 7 7 6 Table 42b. Effect of naftopidil on adrenaline-induced release of PDGF (% control). Naftopidil Naftopidil (40 pM) (lOpM) Mean -25.68 17.60 SD 42.96 33.48 SEM 16.27 12.68 n 7 6 -231- Table 43. Efifect of Doxazosin on the collagen-induced release of PDGF (ng/ml/10* Basal Stimulated Doxazosin PDGF PDGF ______L m m ____ Mean 1.22 4.41 3.67 SD 0.81 1.04 2.44 SEM 0.33 0.43 0.99 Table 44a. Effect of doxazosin on the adrenaline-induced release of PDGF (ng/ml/10* platelets) Basal Stimulated Doxazosin Doxazosin PDGF PDGF (30pM) (7.5pM) Mean 1.08 5.37 3.46 4.42 SD 0.50 2.69 2.81 1.75 SEM 0.19 1.02 1.15 0.78 n 7 7 6 5 Table 44b. Effect of doxazosin on the adrenaline-induced release of PDGF (% control). Doxazosin Doxazosin (30pM) (7.5pM) Mean 29.69 60.33 SD 77.29 26.86 SEM 31.54 11.99 n 6 5 -232- Table 45. Efifect of nifedipine on the collagen-induced release of PDGF (ng/ml/10* platelets). (n=6). Basal Stimulated Nifedipine PDGF PDGF (28pM) Mean 1.11 4.22 3.79 SD 0.83 1.04 1.58 SEM 0.34 0.43 0.65 -233- References Abrams, W.B., Solomon, H.M. 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