Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

Abstract

SummaryThe equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 pM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets.The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8.A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 gM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Ql0 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism.All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

“…Prostaglandin endoperoxides have been shown to initiate platelet secretion (5,23 (24,25). In our assay system, ATP inhibited ADP-stimulated fibrinogen binding with a Ki of 3.8+1.1 ,uM.…”

Adenosine 5′‐diphosphate Antagonists and Human Platelets: No Evidence That Aggregation and Inhibition of Stimulated Adenylate Cyclase Are Mediated by Different Receptors

“…1981 (Harrison et al, 1975) andl33 pM (Lips, Sixma & Schiphorst, 1980) respectively, but in those studies washed platelets, preincubated with the inhibitors, were used. Washing platelets always results in some degree of cell damage as well as the loss of plasma cofactors, and consequently their pharmacological behaviour is different from that of platelets in their native plasma (Akkerman, Doucet-de Bruine, Gorter, de Graaf, Holme, Lips, Nijmeijer, Over, Starkenburg, Tries- The broken line was fitted by computer using least squares linear regression analysis.…”

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

“…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].…”