The long-term objective is to identify and characterize kinetic mechanisms by which coronary arteries can control the temporal and spatial extent of their response to events that trigger thrombus formation. We propose that the reaction sequence ATP yield ADP yield AMP yield Adenosine, occurring extracellularly, provides one such mechanism. Transient high concentrations of extracellular adenine nucleotides occur in the region of an incipient thrombus. Platelets respond oppositely to ADP and adenosine, and ATP, ADP, and adenosine all affect vascular smooth muscle tension. We suggest that the kinetic properties of the hydrolysis pathway, especially those of the last enzyme, 5 feet-nucleotidase, provide quite subtle means of regulating the balance of ADP and adenosine in the extracellular milieu, and thus of modifying the time course of platelet aggregation. Further, by determining the rate of appearance of adenosine especially, the properties of the hydrolysis pathway may play a major role in determining the time course of vasoconstriction and vasodilation in the neighborhood of a forming thrombus.
The specific aims for this project period are: 1) to determine the time course of hydrolysis of extracellular ATP and ADP as a function of initial substrate concentration, using cultured endothelial cells and smooth muscle cells from swine coronary arteries, and swine aorta; 2) to correlate cellular production of prostaglandins, and synthesis and release of cAMP with the concentrations of adenine nucleotides and adenosine occurring during hydrolysis; and 3) to determine whether individual differences in the activity of 5 feet-nucleotidase significantly affect the time course of nucleotide hydrolysis and cellular response. Nucleotides will be separated and measured using high performance liquid chromatography. Prostaglandins and cAMP will be measured by radioimmunoassay.
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