Diadenosine tetraphosphate (Ap 4A), a constituent of platelet dense granules, and its P 1,P 4-dithio and/or P 2,P 3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y 1 and P2Y 12. The most active of those analogs, diadenosine 5′,5″″-P 1,P 4-dithio-P 2,P 3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.
To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y 1 and P2Y 12 receptor antagonism, and their metabolism in human plasma.
We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y 1-mediated changes in cytosolic Ca 2+, P2Y 12-mediated changes in VASP phosphorylation, and metabolism in human plasma.
The inhibition of ADP-induced human platelet aggregation and human platelet P2Y 12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P 1- and P 4-phosphorothioate groups, the S PS P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R PR P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S PR P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P 2,P 3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S PS P and the R PR P diastereomers.