Amiram Eldor

The effect of streptozotocin diabetes on platelet function in rats.

Abstract Streptozotocin diabetes in rats induced alterations in platelet aggregation. These could be ascribed to both intrinsic and extrinsic factors. A pronounced increase in thrombin induced platelet aggregation and serotonin release, observed in citrated plasma as well as in platelet suspension indicated an intrinsic anomaly of platelet function. A diminution of ADP induced platelet aggregation in diabetic plasma which could be reversed by washing and a similar influence of diabetic plasma on normal platelets was clearly indicative of the influence of a plasma factor. These anomalies were observed to occur rapidly after the onset of diabetes, but before the development of retinal vascular changes. In view of the considerable speculation about the relationship between abnormal platelet function and the development of diabetic vascular disease, this model would appear to be uniquely suitable for further study of this problem.

Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina.

Cultured bovine endothelial cells produce an extensive underlying extracellular matrix (ECM) which closely resemble the vascular subendothelial basal lamina in its organization and chemical composition. This naturally produced ECM was used to study the interaction of platelets with the subendothelium when exposed or covered with vascular endothelial cells. Incubation of platelet rich plasma with the ECM induced a rapid and massive platelet adherence, aggregation, thromboxane formation and release reaction. These were demonstrated using phase and scanning electron microscopy, Indium-111 or (14C)-serotonin labelled platelets, and a radioimmunoassay for thromboxane B2. In contrast to the ECM no platelet activation was induced either by uncoated plastic dishes or ECM covered with a confluent endothelial cell monolayer. Aspirinized platelets failed to undergo aggregation and degranulation, when incubated with the ECM. Culture dishes coated with characteristic constituents of the basal lamina such as collagen type IV and type V or fibronectin induced a much lower platelet reactivity as compared with ECM coated dishes. Digestion of ECM components (collagen, fibronectin, hyaluronic acid, and chondroitin sulphate) by specific enzymes was not associated with a substantial decrease in its platelet reactivity. Furthermore, exposure of ECM to sodium dodecyl sulphate or sodium periodate, or freezing and thawing did not decrease its biological activity. In contrast, platelet activation was completely abolished following heat denaturation or glutaraldehyde fixation of the ECM. The availability of a naturally produced ECM provides an appropriate model to study the interaction of platelets with the subendothelium in a controlled system which is isolated from other components of the vessel wall.

A low molecular weight brain substance interacts, similarly to clonidine, with α2-adrenoceptors of human platelets

In the present study, we explored the effects of a clonidine-displacing substance (CDS) which was isolated and partially purified from bovine brain. The low molecular weight brain substance competes with clonidine and rauwolscine in rat brain membranes, and mimics clonidines inhibitory action in rat vas deferens. We find that CDS competes with [3H]rauwolscine-labeled alpha 2-adrenoceptors in human platelets. Further characterization of CDS in human platelets reveals that, like clonidine, it inhibits the epinephrine-induced aggregation, potentiates the ADP- and the collagen-induced aggregation however, by itself, CDS is unable to induce aggregation. Unlike clonidine, CDS does not affect the prostacyclin (PGI2)-stimulated cAMP accumulation in intact platelets. The presence of CDS in human plasma, as we have recently shown, implies a possible role of CDS in the regulation of platelet action.

ENTERAL ADMINISTRATION OF INSULIN IN THE RAT

1 The effect of the surfactant, Cetomacrogol 1000, on the absorption of insulin across the rectal mucosa has been studied. 2 Rectal administration of microenemata containing Cetomacrogal 1000 and insulin causes a rise in the plasma concentration of insulin and a consequent fall in the blood glucose concentration in diabetic and non‐diabetic rats. 3 The hypoglycaemic response is dependent on both the concentration of surfactant and the dose of insulin administered. 4 The results suggest that the transport of insulin across the rectal mucosa is facilitated by Cetomacrogal 1000.

DISTRIBUTION OF MYOSIN, ACTIN AND ACTIN-BINDING PROTEIN IN MEMBRANE AND SOLUBLE FRACTION OF HUMAN BLOOD PLATELETS

Contractile proteins play an important role in a number of platelet functions such as aggregation, clot contraction and serotonin release [l] . The understanding of the molecular mechanism of these basic platelet activities requires the elucidation of the structure, biological activity and localization of the contractile proteins. Both actin [2] and myosin [3] were found in the plasma membrane as well as in the soluble fraction of platelets. Actin-binding protein, another contractile protein with apparent subunit molecular weight of 260 000 was described recently in platelets [4] , however its localization was not reported. Considering the role of contractile proteins in platelet function, it seemed of interest to quantitatively determine the distribution of contractile proteins between the membrane and cytosol fractions.

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.

The distribution of platelet contractile proteins in normal and streptozotocin diabetic rats

Abstract Streptozotocin diabetes in rats produces changes in platelet aggregation. As contractile proteins and glycoproteins are associated with this process, their quantity and distribution was studied in this model on unfractionated platelets and on platelet fractions. K + (EDTA) activated myosin ATPase activity was significantly higher in the KCl extract of diabetic rat platelets. No significant differences were observed in the amount of myosin, actin, actin-binding protein and glycoproteins in unfractionated platelets and platelet fractions. The contractile protein content of normal rat platelets were compared to those of healthy humans and differences were observed in the amount of these proteins and in the specific activity of myosin ATPase.

Distribution of Myosin, Actin and Actin-Binding Protein in Thrombasthenic Platelets

The quantity of myosin, actin, and actin-binding protein (ABP) in platelets and platelet fractions of 5 patients with Glanzmanns thrombasthenia were compared to those of normal individuals. No significant differences were observed between the average amounts of these contractile proteins in unfractionated platelets or platelet membrane obtained after osmotic shock. However, the amount of myosin was significantly decreased in the KCl extract of thrombasthenic platelets ruptured by freezing and thawing,while its specific ATPase activity was increased.