Avner Rotman

Blood platelets in psychopharmacological research.

Blood platelets are a very good model for nerve endings, for study of serotonin uptake and imipramine binding. The efficiency of various drugs, mainly antidepressants, can be evaluated using blood platelets instead of synaptosomes. A few neuronal active compounds are effective in inducing platelet secretion and shape change reaction. Platelet MAO and c-AMP levels are not yet established as useful in psychopharmacological studies.

Serotonin uptake by blood platelets of acute schizophrenic patients.

Active uptake of serotonin by blood platelets of acute schizophrenic patients has been compared to that of a control group. Preliminary results presented in this article indicate that the uptake of the schizophrenic patients was about 40% lower than that of controls. Patients were followed over a period of 5 weeks with no significant change in uptake.

The active uptake of serotonin by platelets of schizophrenic patients and their families: Possibility of a genetic marker

The active uptake of serotonin (5-HT) by blood platelets of 20 schizophrenic patients and their families was studied. The uptake was studied over a wide range of 5-HT concentrations (0.1–20μM), and Km and Vmax of the uptake process were calculated. Of 20 patients, 18 exhibited a lower rate of uptake than the family average at 5-HT concentrations lower than the Km value. At a 5-HT concentration of 0.1 μM, the average 5-HT uptake of patients was 2.15 pmol/108 platelets/min, while that of families was 2.99 pmol/108 platelets/min (33% difference). At a high 5-HT concentration, only the drug-treated patients had lower Vmax than the family average, and this might be attributed to the effect of the drugs. Km of patients and families were very similar. In eight families, one or more of the family members showed 5-HT uptake patterns very similar to that of the patient. We termed these healthy family members “schizophrenic risks”. Our findings indicate the involvement of some genetic factors in this disease.

Platelet uptake of serotonin in psychotic children.

Platelet serotonin (5-HT) uptake was assayed in ten long-term hospitalized children subdivided into two diagnostic groups ‘autistic’ and ‘other psychotic’. Weekly blood samples were taken from these children over a period of 6–7 weeks and serotonin uptake was determined. Constant and significant uptake values (13.28±2.0 pmol serotonin/108 platelets/5 min for autistics and 9.59±1.58 pmol serotonin/108 platelets/5 min for other psychotics) were obtained. There was no correlation with drug treatment or with behavioral fluctuations in individual children. Three weekly uptake assays performed on six parents suggest the possible importance of genetic factors in these diseases. Biochemical-kinetic methodological problems of platelet 5-HT assessment are discussed.

Photoaffinity labelling of the serotinin carrier protein in platelets and brain synaptosomes

Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focusing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [3H]azidoimipramine.

Platelet dopamine uptake in autistic and other psychotic children. Inhibition by imipramine

Abstract 1. 1. The dopamine uptake by blood platelets of Autistic and other Psychotic children was measured during 3–4-week period. 2. 2. A pilot genetic study was carried out in 3 families over a 3-week period of time. 3. 3. Results indicate that the two groups have a different rate of dopamine uptake. 4. 4. However, the value of platelets as a model for dopamine uptake is questioned because of low specificity and efficiencies of the uptake mechanism.

Platelet activation and wheat germ agglutinin-binding

Platelet membrane contains a few glycoproteins, which are important to the aggregation phenomena [ 1,2]. Platelets interact with wheat germ agglutinin (WGA), possibly through glycoprotein I [3-51, and are agglutinated by this lectin. A glycoprotein extract, isolated from human platelet plasma membrane, exhibits haemagglutination and, in addition, causes agglutination of platelets [6]. We have postulated two possible mechanisms to explain this activity: (1) A lectin is buried in the membrane which, upon activation, becomes exposed. (2) Both lectin and the receptor may be present at the surface of the cell, but are self-neutralized in the freely-circulating platelet. Once the cell is activated, the pair-complex dissociates and intercellular interaction occurs outside the plane of the surface membrane. To further examine the availability of WGA receptors on the platelet membrane, and their possible exposure due to activation, we studied the binding of WGA to platelets before and during aggregation. We demonstrate here that there is no change in the binding of 1251-labeled WGA to washed human platelets, as a result of thrombin or ADP-induced aggregation. On the contrary, a marked decrease in the lectin binding was observed when the aggregation was performed in platelet-rich plasma (PRP).

Agglutination activity associated with a glycoprotein extract of human platelet plasma membranes Possible involvement in platelet aggregation

Platelets are extremely active cells of the blood circulation system. Much attention has been focused on the components of the carbohydrate-rich glycocalyx surrounding these cells in an attempt to elucidate the role of the surface glycoproteins in the haemostatic function of platelets [ 11. The recent demonstration that agglutination activity is associated with the major glycoprotein of erythocyte membranes and the likelihood that both a lectin and receptor are present as a self-neutralized pair within the membrane [2], led us to investigate the possibility of the presence of an analogous lectin and receptor in platelets. We demonstrate here that a glycoprotein extract isolated from human platelet plasma membranes exhibits haemagglutination and in addition causes agglutination of platelets.

Receptor and non receptor-mediated activation of blood platelets. Effect on membrane-cytoskeleton interaction

Platelets were activated by receptor and non receptor-mediated reagents. The effect of these reagents on aggregation, secretion, cytoskeleton formation, interaction of alpha-actinin and other membrane proteins with the cytoskeleton was studied. Results show that receptor-mediated activation (e.g. ADP or thrombin activation) leads to a high extent of association of alpha-actinin with the cytoskeleton while non receptor-mediated activation (e.g. ionophore A-23187, arachidonic acid) leads to a low association between the two species. The degrees of aggregation, secretion and total amount of protein in the two modes of activation were the same.

Azidofluorescein diacetate — A novel intracellular photolabelling reagent

Many colloidal properties of the cell, such as those essential to sol-gel transformation, intracellular motion (cyclosis), ameboid movement, spindle formation and cell cleavage, depend for the most part, on the cytoplasmic matrix. Furthermore, the cytoplasmic matrix is the site of many fibrillar differentiations such as keratin fibers, myofibrils, microtubules and filaments. The viscosity of the cytoplasm is probably dependent on environment and internal factors such as the state of the microfilaments and microtubules. Therefore, it is of utmost importance and interest to label the cell cytoplasm and monitor molecular processes through the microviscosity changes. The viscosity of the membrane lipids [l-3] and the rotational relaxation time of lectins bound to the cellular surface membrane [4] have been measured using fluorescent probes. They were used also to study the lateral movement of membrane proteins [5,6] and the vertical displacement of membrane proteins [7]. In [8] the lipid soluble fluorescent probe 1,6diphenyl-1,3,5-hexatriene (DPH) was used to study membrane microviscosity change during platelet activation. Changes of the intracellular microviscosity of malignant and normal cells were studied by the introduction of fluorescein into the cell [9,10]. These studies suffer from the disadvantage that the free fluorescein leaks out of the cell, especially ai 37°C. We report here the introduction of an intracellular photoreactive labelling probe. This probe, azidofluorescein diacetate, is trapped inside the cell by rapid hydrolysis of the ester groups then is activated by irradiation. Using this method the fluorescein deriva-