Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation.

Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.

[Platelet aggregation: a tool for clinical investigation and pharmacological study. Methodology]. / L'agrégation plaquettaire: outil d'investigation clinique et d'étude pharmacologique. Méthodologie.

Platelet aggregation can be measured quantitatively by continuous recording of the transmission of a beam of light across a suspension of platelets in constant agitation in an aggregometer. In routine clinical investigation, the study of platelet aggregation is performed on platelet-rich citrated plasma (PRPc). The blood sample has to be excellent to eliminate all traces of thrombin. The blood is collected in 3.8% sodium dihydrate citrate (1 volume for 9 volumes of blood). It is centrifuged at 190 g for 15 minutes at ambiant temperature. The PRPc obtained can be diluted with platelet-poor plasma (PPP) to adjust the concentration of platelets to 3 X 10(5)/microliters. The PRPc is stored at ambiant temperature in stoppered tubes under CO2 to avoid variations in pH. The maximal delay between the collection of the blood and the end of the study of aggregation is 3 hours. In certain cases, in order to define the platelet lesion and to eliminate the influence of plasma proteins, clotting factors and anti-coagulants, a suspension of washed platelets is used. The blood is collected on acid-citrate-dextrose (ACD) and centrifuged at + 37 degrees C to obtain the PRPc. The platelet deposit obtained by centrifugation of the PRPc is washed twice, according to Mustard's method, in Tyrode-albumin buffer at a concentration of 0.35% at + 37 degrees C. It is re-suspended in the same buffer solution at + 37 degrees C in the presence of apyrase and the platelet concentration is adjusted to 3 X 10(5)/microliters. The aggregation or agglutination of the platelets is induced by several agents: ADP, adrenalin, collagen, thrombin, arachidonic acid, ionophor A 23187, PAF-acether and ristocetin. The quantitative study of the aggregation curves of human platelets allows us to study the physiological and biochemical mechanisms control platelet aggregation, to recognize and classify the hereditary or acquired platelet abnormalities which lead to clinical haemorrhagic or thrombotic manifestations and to study the effect of drugs which inhibit platelet aggregation and to understand their mechanism of action.