Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.     

Comparison of serotonin uptake by dense bodies inside and outside human platelets

ATP-dependent proteolysis in reticulocyte extracts is stimulated by ubiquitin, a polypeptide which is covalently conjugated to proteins. It has been proposed that ATP and ubiquitin act by repressing an inhibitor of an ATP-independent protease, rather than by conjugation to substrate proteins [Speiser, S. and Etlinger, J.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3577-3580]. We find that the inhibitor preparation used by these authors contains a positively required factor of the ATP-ubiquitin proteolytic system, which can be separated from two types of protease inhibitors by gel filtration chromatography. The following observations indicate that the "inhibitors" are endogenous protease substrates which compete with the labeled substrate: (a) inhibition is competitive with exogenous substrate; (b) inhibition is abolished by a preincubation of "inhibitor" with protease prior to the addition of labeled substrate. These findings are not consistent with the notion that the inhibitors play a regulatory role in the ATP-ubiquitin proteolytic pathway.     

Reduced serotonin uptake by spontaneously hypertensive rat platelets

Platelets of Spontaneous Hypertensive (SH) rats show a significant reduction in serotonin uptake, compared with that of rats with normal blood pressure. The difference was due to the V_max, which was about 40% of normals, while the K_m values were almost constant. The possible analogy between blood platelets and serotonergic nerve terminals in SH rats is considered in the evaluation of the results.     

Covalent complexes of albumin with serotonin, ketanserin and lysergic acid antagonize the activity of serotonin in human platelets

Covalent conjugates of bovine serum albumin (BSA) and 5-HT, ketanserin or d-lysergic acid were synthesized and characterized by polyacrylamide gel electrophoresis, whole blood clearance experiments in mice and aggregation studies with human platelets. Using the standard synthesis procedure, each mol of BSA bound 13.4 mol of [3H]5-HT. Derivatization did not cause significant protein aggregation as determined by electrophoresis. All three conjugates antagonized the ability of 5-HT to amplify aggregation caused by low concentrations of ADP. The antagonist activity of each conjugate was concentration dependent; 2.6 microM 5-HT-BSA completely inhibited the aggregation caused by 13 microM 5-HT. None of the BSA drug conjugates, including 5-HT-BSA, amplified platelet aggregation caused by ADP in the absence of 5-HT. Aggregation by ristocetin, collagen, epinephrine or ADP alone was not significantly affected by the conjugates. Whole blood elimination experiments in mice demonstrated that the three conjugates and underivatized BSA are equally stable in the circulation. These prototypic 5-HT drug-protein conjugates may be useful for probing 5-HT2 receptor-ligand interactions in human platelets.     

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).     

Effects of reserpine and emipramine on vesicular serotonin uptake and storage in intact human platelets

The effects of reserpine and imipramine on intact human platelets have been investigated, utilizing brief thrombin treatment to evaluate serotonin (5HT) uptake into and loss from the vesicular (thrombin-releasable) compartment. Less than five seconds after its addition, reserpine (10^?6M) almost completely inhibited the uptake of 5HT into storage vesicles; but induced an outward flux of stored 5HT from vesicles only after more than two minutes following its addition. Imipramine (10^?6M), acting over a 30-minute period, caused no loss of vesicular 5HT, but acted within five minutes to inhibit markedly the movement of cytoplasmic 5HT into storage vesicles. It thus seems likely that in human platelets, inhibition of vesicular 5HT uptake does not necessarily lead to the loss of vesicular 5HT.     

In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

Normal serotonin uptake by blood platelets and brain synaptosomes but selective impairment of platelet serotonin storage in mice with Chediack-Higashi syndrome

The Chediack-Higashi syndrome (CHS) is an autosomal recessive disorder reported in man and in several animal species including the "beige mice" (bg/bg). Among several manifestations of this genetic trait, deficiency of secretable substances - including serotonin - normally stored in platelet dense granules is a characteristic feature. The animal model of Chediak-Higashi syndrome used in the present study provides a unique opportunity to compare the kinetics of serotonin (5-hydroxytryptamine, 5-HT) uptake in platelets and brain synaptosomes in conditions of selective reduction of 5HT concentration in the platelets. The kinetics of 5HT uptake, as measured in the present study, was normal in synaptosomes and platelets from the same animals. The lower intraplatelet 5HT levels in bg/bg animals as compared to normal synaptosomes levels in the presence of normal uptake offer an indirect proof that the 5HT defect described in the CHS is due to an impaired 5HT storage mechanism. This is supported by the observation that spontaneous release of 5HT was markedly increased in platelets from CH5 mice but was normal in synaptosomes from the same animals. Thus platelets are a reliable model to study 5HT uptake, but not 5HT storage and release in brain synaptosomes.     

Effect of pharmacologically selective antidepressants on serotonin uptake in rat platelets

We tested a hypothesis that a long-term administration of antidepressants acting through different primary biochemical mechanisms is associated with changes in the platelet serotonin (5-hydroxytryptamine, 5-HT) transport. Laboratory rats were administered norepinephrine reuptake inhibitors (desipramine, maprotiline), selective 5-HT reuptake inhibitor (citalopram), reversible monoamine oxidase inhibitor (moclobemide), and lithium (inositol monophosphatase inhibitor among others) during a 4-week period. Apparent kinetic parameters of platelet 5-HT transport were analyzed. Significant decrease in apparent Michaelis constant (K(M)) was found after the administration of all tested antidepressants except for desipramine. There was certain increase in maximal velocity (V(max)) values following the administration of desipramine, maprotiline, and citalopram; however, the all V(max) changes were not significant. V(max)/K(M) ratio representing limiting permeability at low extracellular concentrations of 5-HT was systematically increased in all the tested drugs, but significant changes were occurred only in maprotiline- and citalopram-treated rats. Adaptive changes in platelet 5-HT transport induced by citalopram were opposite to the acute inhibitory effect of this drug on 5-HT transporter activity. An increase in limiting membrane permeability for 5-HT could be included in the common adaptive effect of the long-term administration of antidepressants that differ in pharmacologic selectivity.     

ADP stimulates IP3 formation in human platelets

Aspirinated human platelets labeled with 32PO4 showed a 1.7-fold increase in [32P]IP3 when stimulated with ADP. ADP-stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.     

GABA uptake by human blood platelets: effects of lithium and rubidium chlorides

The methodologic and kinetic characteristics of GABA uptake by platelets were determined in blood platelets of volunteer donors. Extrapolation of a reciprocal plot indicate two uptake systems: a high affinity and a passive diffusion mechanism. When LiCl and RbCl were added in vitro, they had no effect on platelet GABA uptake. Our data suggest that platelet GABA and 5-HT uptake are carried out by different systems.     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

Uptake of gamma-aminobutyric acid by human blood platelets: comparison with CNS uptake

Human blood platelets show a sodium and temperature dependent uptake of gamma-aminobutyric acid (GABA) and other neuroactive amino acids. The most potent inhibitors tested of platelet GABA uptake were taurine and beta-alanine, while nipecotic acid and cis-3-aminocyclohexanecarboxylic acid were relatively weak inhibitors. These results suggest GABA is transported by a beta-amino acid uptake process in human platelets. Thus, platelet GABA uptake may more closely resemble glial rather than neuronal uptake.     

Effects of antimycin a plus 2-deoxyglucose on uptake and vesicular storage of serotonin in human platelets

The effects of the metabolic poisons antimycin A (4.1 μg/ml) and 2-deoxyglucose (32.2 mM) on the uptake and vesicular storage of serotonin in washed human platelets have been examined. Within 15 seconds after the addition of the metabolic poisons, H³-5HT begins to move from vesicles into the cytoplasm; by 30 minutes after poison addition, essentially all the platelet 5HT appears to be cytoplasmic. The metabolic poisons also act rapidly to decrease plasma-membrane uptake of H³-5HT from the extracellular medium by approximately 20% within 1 minute after their addition. This may represent a direct effect rather than one resulting from altered cytoplasmic or vesicular 5HT, since platelets with <10% of the normal number of vesicular storage sites exhibit a similar reduction after addition of the metabolic poisons.