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General discussion and concluding remarks

C h a p te r 8; General discussion and concluding rem arks

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 o f 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)).

C h a p te r 8: General discussion and concluding rem arks

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

C h ap ter 8: G eneral discussion and concluding rem arha

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

C h ap ter 8: G eneral discussion and concluding rem arks

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

C h a p te r 8: G eneral discussion and concluding rem arks

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 TXA2 was inhibited by naftopidil but not doxazosin. Nifedipine also inhibited coUagen-induced TxÀ2 generation comparable in extent to that produced by naftopidil.

C h ap ter 8: G eneral discussion and concluding rem arks

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, TXA2 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

C h a p te r 8: G eneral discussion and concluding rem arks

transduction mechanisms which trigger the release of arachidonic acid and stimulate concomitant TXA2 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 tt2-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 a2-adrenoceptors and receptor-operated calcium channels