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

Inositol 1,3,4,5-tetrakisphosphate (IP₄) is formed from inositol 1,4,5-trisphosphate (IP₃) by IP₃ 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca²⁺ entry, IP₃ regulation, and phosphoinositide 3-kinase antagonism.

To better elucidate a function for IP₄, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP₄ directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP₃) binding to pleckstrin-homology (PH) domain-containing proteins in platelets.

Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca²⁺ studies, or resuspended in high K⁺ and low Na⁺ buffers for permeabilization experiments. Phosphorylation of AKT-Ser⁴⁷³ and Rap1-GTP formation were measured by Western blotting and PIP₃ binding using PIP₃ beads.

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 Ca²⁺ concentration, elevated basal levels of IP₃, and enhanced the peak height of Ca²⁺ elevated by agonists. In permeabilized platelets, IP₄ inhibited GTPγS induced formation of AKT-Ser⁴⁷³ phosphorylation and platelet aggregation. IP₄ reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP₃ beads.

ITPK and IP₄ are negative regulators of platelet function. IP₄ regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.