The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood.

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitrarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37 degrees C with a saturating concentration of exogenous thymidine. In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 +/- 11 units (mean +/- S.D., n = 20, range 45-89). The apparent Michaelis constant for thymidine was of the order of 10(-4) M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin. Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity +/- S.D. was 96 +/- 27 units; range 58-140, n = 8). Thymine formation from thymidine was temperature- and pH-dependent in whole blood. 2'-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3'-azido-3'-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously. In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.

The role of blood platelets in nucleoside metabolism: regulation of platelet thymidine phosphorylase.

Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an unusually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiological and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylase thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism. In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.