Relationships between chemical structure and inhibition of ADP-stimulated human thrombocyte release of serotonin and platelet factor 4

The inhibitory potencies of carbamoylpiperidinoalkane and N-alkylnipecotoylpiperazine derivatives on ADP-stimulated human blood platelet aggregation, serotonin (5-HT) release and platelet factor 4 (PF-4) release were evaluated. The procedure was designed to allow concurrent determination of all three sets of values. Most compounds were more than twice as potent in blocking PF-4 (X = 91 +/- 1 (S.E., n = 7)%) compared to their inhibition of 5-HT (X = 38 +/- 1(S.E., n = 6)%) release; the one compound which failed to meet these criteria was still decidedly more powerful in impeding PF-4 than 5-HT release. Since the compounds' platelet aggregation-inhibitory values were within the same range as their 5-HT release-blocking potencies, but had a strikingly greater impact in arresting PF-4 release, it is suggested that the platelet plasma membrane and the lining enveloping the dense bodies may share certain commonalities, while the sheathing encasing the alpha-granules may differ from both in a tangible manner.     

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.