Phosphoinositide metabolism in resting and thrombin-stimulated human platelets: Evidence for metabolic homogeneity

On the basis of differences in specific radioactivity (SA), separate pools of phosphoinositides have recently been proposed in platelets. Human platelets were labelled for 60 min with [³²p]p- and subsequently transferred to a phosphate- and Ca²⁺free Tyrode’s solution by gel-filtration. Thereafter, the platelets were either incubated at 37°C for 120 min, a condition which induces increase in specific labelling of the diester phosphate of phosphatitylinositol (PI), or stimulated with 0.5 U/ml of thrombin. The changes in SA of both diester and monoester phosphates of the phosphoinositides were detrmined. Immediately after the gel filtration, the SA of the diester phosphate of phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP₂) were both similar to that of PI and amounted to 4% or the SA of the monoester groups of PIP and PIP₂- Whereas the SA of the monoester phosphate essentially remained constant and the same for PIP and PIP² during the entire incubation, the SA of their diester phosphates increased gradually in parallel to that of PI, and reached 20% of the monoster groups after 120 min. The effect of thrombin was studied at 15, 60 and 180 sec after the addition. The absolute radioactivity of both diester and monoester phosphates of all phosphoinositides increased conciderably after an initial decrease. However, for the monoester groups, the changes in radioactivity were parallelled by the changes in mass for both PIP and PIP₂. Thrombin therefore induced no changes in SA of the monoester phosphates. In contrast, the SA of the diester phosphates increased 5-fold and remained similar for all three phosphoinositides during the 180 sec of stimulation.

In conclusion, our results demonstrate close metabolic equilibrium between all three phosphoinositides.

Thrombin-induced changes in SA of PIP and PIP₂ are purely secondary to changes in specific labelling of the diester phosphate.