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A role for phosphatidylinositol 3-kinase in platelet aggregation Stevens, Christiaan Simeon Michael

Abstract

The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin GPIIb-IIIa and subsequent binding of fibrinogen. Previous studies have demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) is activated both prior to, and following, activation of GPIIb-IIIa in human platelets, but the role of PI 3-kinase in platelets has not been elucidated. The objective of the work contained in this thesis was to better understand the function of PI 3-kinase in platelets. I showed that the p85 subunit of PI 3-kinase associates with a tyrosine-phosphorylated 105 kDa protein. This association was dependent on GPIIb-IIIa activation and was not inhibited by PI 3-kinase inhibitors. Furthermore, the protein was not immunoreactive with several antibodies to known proteins in this weight range. PI 3-kinase inhibitors were also used to probe the dependence of thrombin-induced aggregation on PI 3-kinase. It was found that when platelets were stimulated with high concentrations of thrombin in the presence of LY294002, a competitive inhibitor of PI 3- kinase, the inhibitors had no affect on aggregation nor GPIIb-IIIa activation. However, with lower concentrations of thrombin, aggregation and GPIIb-IIIa activation were almost completely inhibited by LY294002. These results indicate that aggregation and GPIIb-IIIa activation is less dependent on PI 3-kinase activity when high concentrations of agonist are used, indicating that there are PI 3-kinase-independent pathways that can function under these conditions. Finally, the role of the SH2 domain containing inositol 5-phosphatase (SHIP) in regulating the products of PI 3-kinase in platelets was investigated using mice with a targeted gene disruption. It was found that the level of phosphatidylinositol 3,4,5- trisphosphate [PI(3,4,5)P3] was increased in SHIP"7" platelets in response to Collagen Related Peptide (CRP) whereas formation of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was reduced demonstrating a role for the 5-phosphatase in the metabolism of PI(3,4,5)P3 . I also demonstrated that the elevation of PI(3,4,5)P3 was insufficient to activate phospholipase C but it enhanced the elevation of the protein tyrosine kinase, Btk, and calcium influx.

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