Gustav Victor Rudolf Born

Effects of d- and l- enantiomers of adenosine, AMP and ADP and their 2-chloro- and 2-azido- analogues on human platelets

Aggregation of human platelets by ADP and the inhibition of this effect by adenosine are apparently mediated by different receptors. One of the criteria for receptors is that they show stereospecificity for their ligands. We have synthesized l-enantiomers of d-adenosine, AMP and ADP, together with their corresponding photolysable 2-azido analogues so that platelet receptors could be tested for stereospecificity. All of the l- enantiomers were completely inactive as aggregators or inhibitors of platelet function. None of the L-enantiomers changed levels of platelet cAMP. 2-Azido-l-adenosine, AMP and ADP are proposed as useful controls in photoaffinity experiments for non-specific labelling.

Antiplatelet drugs: Antiplatelet drugs

British Journal of Pharmacology (2006) 147, S241–S251. doi:10.1038/sj.bjp.0706401

Effect of Removing Sialic Acids from Endothelium on the Adherence of Circulating Platelets in Arteries in vivo

The contribution of the net negative charge excess due to sialic acids on endothelium in preventing adhesion of circulating platelets in vivo was investigated in anaesthetized rabbits. Platelets in the rabbit’s circulation were selectively labelled with radioactive 5-hydroxytryptamine in vivo. Segments of carotid arteries temporarily isolated from the circulation were perfused with one or other of two commercial preparations of neuraminidase; the opposite carotid artery was perfused similarly without the enzyme, as control. A neuraminidase preparation from Behringwerke free of proteolytic activity released sialic acid into the perfusate with a peak concentration after 10–15 min which decreased gradually later. A neuraminidase preparation from Sigma that contained demonstrable proteolytic activity released sialic acid similarly during the first hour and thereafter more sialic acid in a second peak. After blood flow through the carotids had been restored the adhesion of labelled platelets in the artery perfused with neuraminidase was compared with that in the artery perfused without the enzyme. The radioactivities were significantly higher in carotids that had been perfused with neuraminidase than in those that had been perfused without the enzyme. Neuraminidase perfusion had no effect on the production of prostacyclin by the carotids. Perfusion with acetylsalicylic acid before neuraminidase increased the adhesion of platelets significantly. It is concluded that diminution in electrostatic repulsion between circulating platelets and vascular endothelium from which the net negative charge excess due to sialic acids has been removed increases the adhesion of circulating platelets, irrespective of the production of prostacyclin by the arterial walls, and that inhibition of prostacyclin production augments this adhesion of platelets.

Effects of photolysable 2-azido analogues of adenosine, AMP and ADP on human platelets

The aggregating effect of ADP on human platelets and the inhibiting effect of adenosine are apparently mediated by different receptors on the cells’ outer membranes. Photolysable azido-analogues of adenosine, AMP and ADP have been prepared so that the receptors for them can be labelled. 2-Azidoadenosine inhibited platelet aggregation by ADP more than adenosine itself and increased platelet cAMP as effectively as did adenosine or 2-chloroadenosine. 2-Azido-AMP inhibited aggregation much more effectively than AMP itself or than 2-chloro-AMP. 2-Azido-ADP was about five times more potent than ADP in causing aggregation. All these 2-azido derivatives were photolysable by irradiation at 365 nm which does not affect platelet functions.

INHIBITION BY A STABLE ANALOGUE OF ADENOSINE TRIPHOSPHATE OF PLATELET AGGREGATION BY ADENOSINE DIPHOSPHATE

1 In citrated platelet‐rich plasma, freshly prepared from rabbit blood, the velocity of platelet aggregation was within limits proportional to the log of the concentration of added adenosine diphosphate (ADP). 2 Addition of either adenosine triphosphate (ATP) or its β,γ‐methylene analogue inhibited aggregation similarly except that the analogue was about half as potent as ATP. β,γ‐Methylene ATP also reversed the optical effects associated with the shape change of platelets very similarly to ATP itself. 3 As β,γ‐methylene ATP is not a substrate for nucleoside diphosphokinase, these observations do not support the proposition that inhibition of aggregation by added ATP is due to its utilization by the nucleoside diphosphokinase of platelets.

Influence of Dietary Lipids on the Effect of Chlorpromazine on Membrane Properties of Rabbit Red Cells

The effect of diets containing different types of common natural oils on physical properties of red cells was investigated by using rabbits. The rabbits were fed for 18 months on a standard diet in which 8% of its energy content was provided by safflower oil and 32% energy by either more safflower oil or fish oil, linseed oil, olive oil or palm oil. Erythrocyte deformability was significantly decreased by the fish oil diet compared with each of the other diets. Osmotic fragility was significantly less (66 mM) for red cells from rabbits fed on the linseed oil diet, and significantly greater (71 mM) for red cells from rabbits on the fish oil diet, than for red cells from rabbits on the other three diets which did not differ significantly from each other (68 mM). With rabbits on the standard diet, the resistance of their erythrocytes to osmotic haemolysis was increased by chlorpromazine at concentrations below and decreased by concentrations above 30 μM. The dietary oils caused significant changes in the effects of chlorpromazine on osmotic fragility. The concentration at which the effect of chlorpromazine reversed from antihaemolytic to prohaemolytic was decreased by the safflower and linseed oil diets and increased by the fish oil diet, compared with the olive and palm oil diets. Analysis of the fatty acid compositions of the dietary oils on the one hand and of the red cell phospholipids on the other established, specifically, that in the presence of 30 μM chlorpromazine the percentage haemolysis was directly proportional to the linoleate content of the red cell phospholipids.

Inhibition of Adenosine Deaminase and of Platelet Aggregation by 2-azidoadenosine, a Photolysable Analogue of Adenosine

Aggregation of human blood platelets induced by ADP was strongly inhibited by 2-azidoadenosine, a photolysable analogue of adenosine. The deamination of adenosine by adenosine deaminase (E. C. 3.5.4.4) was competitively inhibited by 2-azidoadenosine. In the presence of 2-azidoadenosine the enzyme was inactivated by u. v. radiation. The inactivation was diminished in the presence of a protein scavanger.

Inhibition by N-acetyl neuraminic acid of platelet thrombogenesis induced by laser injury to rat and hamster venules.

1 In rats and hamsters under barbiturate anaesthesia, laser radiation to venules about 50 μm in diameter in mesoappendix and cheek pouch respectively caused the formation of platelet thrombi which occluded the vessels in about 9 min. 2 This occlusion time was significantly prolonged by the intravenous injection of N‐acetyl neuraminic acid (NANA) but not by D‐glucuronic acid or β‐methoxyneuraminic acid, in doses which had no effect on blood pH or on the condition of the animals. 3 The results confirm the antithrombotic effect of NANA previously demonstrated with another technique.

Increases in aggregation by and uptake of 5-hydroxytryptamine with platelets from rabbits treated with chlorpromazine.

1 Citrated platelet‐rich plasma was prepared from New Zealand white rabbits before, during and after administration of chlorpromazine (2 mg/kg) intramuscularly once daily for 3 to 4 weeks. 2 In these plasmas, the velocity of platelet aggregation by 5‐hydroxytryptamine (5‐HT) added at 1, 3 and 10 μm increased greatly, beginning 3 to 4 days after the start of chlorpromazine injections and lasting for a similar period after they were terminated The increase had two maxima, the first after 6 to 10 days and the second after 17 to 24 days. Chlorpromazine treatment did not affect aggregation by adenosine diphosphate (ADP). 3 The uptake of 5‐HT by rabbit platelets was very fast and linear for less than 10 s. In platelets from untreated rabbits the uptake had a Km of 0.35 ± 0.08 μm and a Vmax of 39.8 ± 6.1 pmol 10−8 platelets 10−1 (n = 5). 4 In platelets from rabbits injected with chlorpromazine (see (2) above) both kinetic constants increased significantly, the Km to 0.88 ± 0.08 μm and the Vmax to 67.8 ± 5.5 pmol. 10−8 platelets. 10−1 s (n = 9).

Comparison of the Numbers of Plasmalemmal Vesicles in Arterial and Venous Endothelia of Rats

The numbers of plasmalemmal vesicles in endothelial cells of rat blood vessels were determined on electron microscopic sections. In all vessels examined which included aorta and carotid and femoral arteries, vena cava and femoral vein, and lung and brain capillaries, the numbers were of the same order of magnitude. For arteries the numbers were about double those for the corresponding veins. About one-third of all vesicles could be stained with ruthenium red after its infusion into the vessels. The results make it improbable that differences in numbers of ‘transport’ vesicles in different types of blood vessel contribute significantly to the selective accumulation of atherogenic plasma proteins in arteries.