Evidence that inositol 1,4,5-trisphosphate 3-kinase and inositol 1,3,4,5-tetrakisphosphate are negative regulators of platelet function.

Background: Inositol 1,3,4,5-tetrakisphosphate (IP) is formed from inositol 1,4,5-trisphosphate (IP) by IP 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca entry, IP regulation, and phosphoinositide 3-kinase antagonism.

Objectives: To better elucidate a function for IP, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP) binding to pleckstrin-homology (PH) domain-containing proteins in platelets.

Methods: Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca studies, or resuspended in high K and low Na buffers for permeabilization experiments. Phosphorylation of AKT-Ser and Rap1-GTP formation were measured by Western blotting and PIP binding using PIP beads.

Results: GNF362-enhanced platelet aggregation stimulated by low concentrations of ADP, collagen, thrombin, U46619, and thrombus formation in collagen-coated capillaries. GNF362 induced a transient elevation of Ca concentration, elevated basal levels of IP, and enhanced the peak height of Ca elevated by agonists. In permeabilized platelets, IP inhibited GTPγS induced formation of AKT-Ser phosphorylation and platelet aggregation. IP reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP beads.

Conclusion: ITPK and IP are negative regulators of platelet function. IP regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.