Elsevier

Biochemical Pharmacology

Volume 60, Issue 9, 1 November 2000, Pages 1267-1277
Biochemical Pharmacology

The stimulatory effects of cationic amphiphilic drugs on human platelets treated with thrombin

https://doi.org/10.1016/S0006-2952(00)00445-7Get rights and content

Abstract

The actions of eight cationic amphiphilic drugs on human platelets displayed three different effects according to drug concentration ranges. At lower concentrations (below ∼25 μM), the drugs stimulated secretory responses induced by 0.2 U/mL of thrombin, while at concentrations in the 25–50 μM range they inhibited these responses. Above 50–100 μM, the drugs caused permeabilization of the platelet plasma membrane as measured by leakage of cytoplasmic adenine nucleotides. The effects of these agents on phosphoinositide metabolism were monitored in platelets prelabeled with 32P-inorganic phosphate, such that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylinositol (PI), were labeled to equilibrium. In unstimulated platelets, the level of labeled PA decreased slightly (about 25%), with corresponding increases in PIP2 labeling up to drug concentrations of about 50 μM. In contrast to the relatively small changes in PI and PIP2, the levels of labeled PIP, precursor to PIP2, increased 2- to 4-fold in both resting and thrombin-treated platelets from 5 μM up to about 50–100 μM of drugs and remained elevated throughout the permeabilization concentrations. [32P]PA increased 20-fold over control upon thrombin activation and 5–30 μM of drugs caused [32P]PA to increase 30–37 times over that seen in control, resting platelets; the concentration of drugs that potentiated thrombin-induced [32P]PA elevation corresponded to that causing the potentiation of platelet secretion. Higher drug concentrations decreased [32P]PA elevation. [32P]PIP2 levels increased about 25% in response to thrombin treatment alone; low concentrations of drugs led to another 25% elevation. A significant decrease in [32P]PIP2 was seen above 30 μM, corresponding to inhibition of platelet secretion. Concentrations of 5–30 μM of several psychoactive agents, both neuroleptics and antidepressants, potentiated the thrombin-induced activation of platelets as measured by dense granule secretion and increased turnover of phosphoinositides. Remarkably, all of the drugs increased the levels of PIP even in resting platelets, indicating that they have common effects apart from the specific receptor interactions currently attributed to them. These common effects, e.g. an increase in membrane fluidity such as is known to be caused by amphipathic agents, may be in part responsible for the observed overlapping psychotrophic effects of tricyclic antidepressants and phenothiazines.

Section snippets

Platelet isolation

Blood was obtained from regular donors at the blood bank (Haukeland Hospital, Bergen, Norway), who claimed to have taken no medication for the previous ten days. Thirty-six milliliters of blood was mixed with 6 mL “acid citrate dextrose” (86.7 mM trisodium citrate, 73.3 mM citric acid, 100 mM dextrose) as anticoagulant, and then centrifuged at 535 g for 6 min at ambient temperature. Platelet-rich plasma was collected and centrifuged again at 1200 × g for 10 min (ambient temperature) to pellet

Results

The effects of cationic amphiphilic drugs on the parameters determined in the present study varied considerably among platelets from different blood donors. However, the parameters measured always varied with the concentration of a given drug in the same pattern among the donors. The results are therefore presented as single experiments, each of which has been performed with platelets from at least 3 donors.

Effects on cell permeabilization and secretion

The effects of varying concentrations of several psychoactive cationic amphiphilic drugs on membrane integrity, secretion, and phosphoinositide metabolism in resting and thrombin-activated platelets are described in this study. We found that our earlier observations showing that CPZ and TFZ caused permeabilization and loss of small molecular weight cell components at concentrations over 50 μM in both resting and thrombin-activated platelets 7, 18 were also true for all of the other psychoactive

Conclusions

The major finding we report here is that several psychoactive drugs, both neuroleptics and antidepressants, all exhibited a stimulatory effect at low drug concentrations (<30 μM) and an inhibitory effect at higher concentrations (>50 μM). The most obvious, common feature of these agents is that they are amphipathic enough to cross the blood–brain barrier. Such small molecular weight, lipid-soluble substances would easily distribute into the plasma membrane and intracellular membranes and not be

Acknowledgements

This work was supported by the Norwegian Research Council for Science, the Blix Fund, the EU Biomed 2 program contract no. BMH4-CT-97 2609, and the L. F. Saugstad Foundation.

References (39)

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