³¹P NMR Studies of Nucleotide Storage in the Dense Granules of Pig Platelets

Detailed ³¹P NMR measurements have been conducted on pig platelet dense granules and aqueous mixtures of ATP, ADP, MgCl₂, and 5HT. The resonance line widths of the dense granule nucleotides were temperature independent above ~30 °C; below this temperature they exhibit a strong temperature dependence, becoming undetectably broad at ~5 °C. The temperature of transition is determined by the effective solute concentrations within the granules. Spin-spin relaxation time (T₂) and line-width measurements indicate that the ³¹P resonances of dense granule nucleotides are not homogeneously broadened at 35 and 21 °C. However, the temperature-dependent changes in the intrinsic widths calculated from T₂ values parallel the changes in the measured line widths. From the T₁ and the T₂ data, a rotational correlation time of 13 ns is calculated for the dense granule nucleotides at 35 °C. Removal of 5HT and HA from the dense granules induce significant but relatively small changes in the temperature dependence of the resonance line widths. Analogous effects are seen with a gel phase separated from aqueous mixtures of ATP, ADP, and MgCl₂ in the presence or absence of 5HT. These results demonstrate that interactions involving the nucleotides and the divalent cations are predominant in determining the physicochemical state of the granule conténts; in pig platelet dense granules the nucleotides and Mg²⁺ form a relatively fluid aggregate which serves as a matrix for 5HT and possibly HA binding. Incorporation of the amines into this matrix tends to increase its fluidity.