Defining the role of Class II Phosphoinositide 3-Kinases in platelet function.

2016-11-29T02:34:36Z (GMT) by Mountford, Jessica Kate
Introduction: Arterial thrombosis causes heart attack and ischaemic stroke and is the leading cause of death in the Western world. Platelets are the key blood cell responsible for the development of arterial thrombosis and understanding signalling pathways which lead to the incorporation of platelets into these thrombi may lead to the development of new and improved anti-thrombotic therapies. The phosphoinositide 3-kinases (PI3Ks) are a family of 3 classes (I, II, and III) of intracellular enzymes which generate lipid second messengers important for many cell signalling events. The role of Class I PI3Ks in platelets has been well defined and drugs which block Class I PI3K function are in pre-clinical trials as anti-thrombotics. However, virtually nothing is known about the Class II PI3Ks in platelets. Aim: The studies of this thesis used genetic mouse models to examine the role of the Class II PI3Ks in platelets. Key Findings: Mouse platelets express two of the three Class II PI3K isoforms, PI3K-C2 alpha and PI3K-C2 beta. The function of platelets isolated from PI3K-C2 beta-/- mice was largely normal. PI3K-C2alpha-/- mice were generated but died in utero prior to haematopoietic cell development, and were affected as early as 7.5 days post-conception. However, PI3K-C2 alpha+/- mice exhibited platelet-dependent defects in haemostasis and thrombosis. Specifically, anticoagulated PI3K-C2 alpha+/- mice had a 3-fold increase in tail bleeding time over littermate wild-type mice – an impairment comparable to that observed in anticoagulated wild-type mice treated with therapeutic doses of the gold standard anti-platelet agent, aspirin. Bone marrow reconstitution studies confirmed that the impaired in vivo haemostasis of PI3K-C2 alpha+/- mice was dependent on haematopoietic cells. An ex vivo whole blood thrombosis assay revealed PI3K-C2 alpha+/- mice formed thrombi that were larger (3-fold increase) but more unstable (embolization rate of 75% versus 0%) than those of littermate wild-type mice. Detailed analyses of the function of platelets from PI3K-C2 alpha+/- mice revealed a decrease in adhesion via the collagen receptor(s), GPVI and/or alpha2beta1, and a shear-dependent increase in platelet adhesion via the integrin alphaIIbbeta3 – a phenotype which was reproduced in wild-type platelets treated with alcohols to pharmacologically increase plasma membrane fluidity. Finally, a novel mouse genetic model was developed to further reduce PI3K-C2alpha expression levels in platelets using an inducible and reversible RNAi-based approach. Platelets from these PI3K-C2 alpha-deficient mice expressed < 10% of normal PI3K-C2 alpha levels and these mice exhibited a significantly more severe in vivo haemostatic defect than that observed in PI3K-C2 alpha+/- mice. Conclusions: The studies of this thesis define a novel role for the Class II PI3Ks in platelet biology. Examination of platelet function in mice genetically deficient in the Class II PI3Ks using a combination of in vitro platelet function studies, ex vivo thrombosis assays, and in vivo models, demonstrated that PI3K-C2 alpha regulates the function of cell surface platelet adhesion receptors, possibly through changes in plasma membrane structure and/or function. These studies suggest that further investigation of the function of PI3K-C2 alpha in platelet biology is warranted to determine whether this lipid kinase represents a novel target for the development of anti-platelet agents for the prevention of arterial thrombosis.