An Enzyme in Rabbit Aorta that May Play a Role in Haemostasis and Thrombosis

Platelet interaction with blood vessel walls has been studied with the intention of elucidating regulatory mechanisms involved in haemostasis and thrombosis. Microsomal fractions (Mif) prepared from rabbit aortas have been found to inhibit ADP-induced platelet aggregation when incubated with ADP prior to addition to platelet-rich plasma. Thin layer chromatography of incubation mixtures in which [8-14C] ADP has been used has proved that the inhibition is due to degradation of ADP. The main degradation product at pH 6.0 is AMP and between the pH optimum of 7.2-8.2, it is adenosine. The rate of ADP breakdown is dependent on the duration, temperature and pH of the incubation, and on the concentrations of ADP and Mif used. Enzymic activity can be destroyed by boiling and by treatment with perchloric acid.EDTA (Na2) will remove inhibitory activity but this can be restored by addition of either calcium or magnesium to the incubate. The subcellular localization of the enzyme has been determined by isopycnic centrlfugation in a sucrose density gradient, using marker enzymes to establish the distribution of subcellular organelles in the gradient.ADP degrading activity was found to coincide with the peak of 5′ nucleotidase (5′NT) activity, the plasma membrane marker.Localization of the ADP degrading enzyme on the cell membrane means that it is in a position to destroy any ADP which might be released by platelets in their response to damage of the blood vessel wall. Its coincidental location with means that any AMP formed will be converted to adenosine which is itself an inhibitor of platelet aggregation. The ADP degrading enzyme could therefore play an important role in limiting the extent of thrombus formation.