No abstract

Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non-hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (Kd approximately 5 nM). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites. The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein-coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of D-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.

The aim of the present study was to determine whether dietary intake of monounsaturated or long chain n-3 fatty acids could be effective in lowering platelet responsiveness through modulation of platelet phospholipid composition. Rats were fed diets containing 20% fat with equal cholesterol and 13a-tocopherol contents. These diets were supplemented with saturated, oleic or n-3 fatty acids, n-3 polyunsaturated fatty acids being added either pure, as eicosapentaenoic and docosahexaenoic ethyl esters, or as MaxEPA oil. Dietary n-3 fatty acids did not affect the oxidation status of plasma lipids. Oleic acid- and saturated fatty acid-rich diets led to similar enrichment of platelet phospholipids in arachidonic acid and to comparable thromboxane A(2) generation on stimulation with collagen or thrombin. Platelets of n-3-fed groups were differently enriched in eicosapentaenoic and docosahexaenoic acids at the expense of arachidonic acid. These groups displayed similar thromboxane A(2) production, although levels were lower than those for groups fed with oleic- or saturated fatty acid-rich diets. Only the MaxEPA diet led to a reduction in platelet reactivity, measurable as a small decrease in the aggregation induced by collagen. This diet was also responsible for a high cholesteroUphospholipid ratio and low a-tocopherol content in platelets. Overall results indicated that (i) only MaxEPA reduced platelet reactivity and (ii) this effect was moderate and apparently unrelated to platelet arachidonic acid content, membrane cholesterol to phospholipid ratio or thromboxane A(2) production.