2-Methylthioadenosine[beta-32P]diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets.

Abstract

A B S T R A C T 2-Methylthio-ADP and its radioactive analogue [,_-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin El. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [#-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was -3.5 X 10' M`so', and the first order dissociation rate was 0.024 s-', both measured at 230C. The dissociation equilibrium constant (-15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin El-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Kj = 3.5 MM), ATP (7 AM), 2-azido-ADP (0.12 AM), inosine diphosphate (IDP, 150 AM), guanosine diphosphate (GDP, 350 MM), and AMP (800 MM). Binding of 2-methylthio-ADP was also blocked by the noncell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 320-40'C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was -15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket. INTRODUCTION ADP has two distinguishable effects on blood platelets (1): firstly, it induces aggregation of the cell by stimulating a change in their shape and exposure of fibrinogen-binding sites, and, secondly, it inhibits the adenylate cyclase of membrane preparations of platelets and prevents the accumulation of cyclic AMP by intact platelets (2). Inhibition of aggregation by pros-

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

“…As expected, DTT strongly inhibited the binding as did the chemical pCMBS which is a well-known inhibitor of ADP-induced platelet aggregation [9,[26][27][28]43]. Since DTT was present in our previous membrane preparations to preserve them from oxidation, it is not surprising that we could not observe any difference between clopidogrel treated and control rats.…”

“…One important point is to be able to quantify the number of receptors expressed on platelets in order to assess the inter-individual variability in the general population, to characterize patients with inherited deficiencies, and to monitor and study patients treated with P2Y 12 targeting drugs [4]. Various radioligands have been used to characterize and quantify the platelet P2Y receptors such as, [ 14 C]ADP [5,6], [ 3 H]ADP [6,7], [ 3 H]2-methylthio-ADP [8], [β- 32 P]2-methylthio-ADP [9,10], and [β- 33 P]2-methylthio-ADP [11], but they all share several weaknesses: (a) They are metabolically unstable and may be cleaved by a number of enzymes such as alkaline phosphatase and ectonucleotidases; (b) being agonists, they may complicate the quantification when intact, living cells are used and receptors are internalized upon activation; (c) they do not discriminate between P2Y 1 and P2Y 12 receptors. In the last decade, the only one possibility to selectively quantify P2Y 12 receptors was to use the non-selective radiolabeled ligand 2-methylthio-ADP in the presence of a P2Y 1 antagonist such as N 6 -methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) [12] or 2-iodo-N 6 -methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2500) [13].…”

Historical perspective on ADP-induced platelet activation

“…In the 1980s, by the use of 2-azido-ADP that could be photoactivated [94] and also a strong aggregating ADP analogue, 2-methylthioadenosine labelled with 32 P (2-MeS-ADP), Macfarlane and colleagues [95] obtained values ranging from 400 to 1,200 sites per platelet for the receptor that inhibits adenylyl cyclase (P2Y 12 ). More recently, using the latter analogue, Gachet et al [96] reported 600±125 specific binding sites per platelet, and Nurden and co-workers [97] found 826±126 per platelet.…”

“…In 1983, Macfarlane et al [95] had demonstrated that the thiol complexing agent p-mercuribenzene sulfonate blocks inhibition by ADP of adenylyl cyclase and blocks the binding of 2-azido-ADP and 2-MeS-ADP, but does not affect the ability of ADP to induce aggregation and the shape change. In 1993, Cristalli and Mills [98] showed that a 43-kDa protein on platelets incorporated 2-(p-azidophenyl)-ethylthio-ADP (AzPET-ADP), a photoaffinity analogue of ADP that competitively inhibits the binding of 2-MeS-ADP.…”

“…Although Macfarlane and his colleagues [95] had provided evidence in 1983 for two ADP receptors on platelets, one responsible for shape change and aggregation and the other for inhibition of adenylyl cyclase, it was not until 1996 that others began investigations that supported their findings [84]. Several groups used selective P2Y 1 antagonists and showed that they inhibited ADP-induced shape change, aggregation, and increases in calcium, but not the effect of ADP on adenylyl cyclase [84,[114][115][116][117].…”