Loss of PIKFyve In Murine Platelets Leads to Aberrant Platelet Granule Biogenesis and a Pleomorphic Phenotype with Multiorgan Failure

Abstract 159

Platelets contain numerous biologically active substances that are packaged into three different types of lysosome-related secretory granules that are released upon activation. Once released from the platelets, the contents of these granules contribute not only to hemostasis and thrombosis, but also to a wide range of pathologic processes, such as tumor metastasis formation, osteoporosis, and hepatitis, among others. However, the signaling events involved in platelet granule formation and exocytosis are not well understood. The phosphoinositide, phosphatidylinostol 3,5 bisphosphate (PtdIns(3,5)P₂) is critical for membrane trafficking at the vacuole (the lysosome equivalent) in yeast. However, very little is known about the function of PtdIns(3,5)P₂ production in higher eukaryotes. In mammals, PtdIns(3,5)P₂ is the product of PtdIns(3)P phosphorylation by the lipid kinase, PIKFyve. Platelets contain PIKFyve, and synthesize PtdIns(3,5)P₂ following thrombin stimulation. Given the function of PtdIns(3,5)P₂ in yeast membrane trafficking, we hypothesized that PtdIns(3,5)P₂ production by PIKFyve regulates the biogenesis and/or secretion of platelet granules. To test this hypothesis, we genetically engineered mice to lack PIKFyve activity specifically in their megakaryocytes and platelets. Homologous recombination lox P sites were introduced to flank exons 42 and 43, which encode the activation loop of the PIKfyve kinase domain. Resultant PIKFyve ^fl/fl mice were crossed with PF4 Cre+ mice that express Cre from the Platelet factor 4 promoter. This breeding strategy results in PIKFyve ^fl/fl PF4 Cre+ offspring that are predicted to have lost PIKFyve expression in only their megakaryocytes and platelets. A total of 40 PIKFyve ^fl/fl PF4 Cre+ mice were examined and compared with their control littermates. RT-PCR analysis demonstrated a complete loss of normal PIKFyve mRNA in platelets from the PIKFyve ^fl/fl PF4 Cre+ mice. Surprisingly, by 3–4 weeks of age, all 40 PIKFyve ^fl/fl PF4 Cre+ mice, but not PIKFyve ^fl/fl PF4 Cre- or PIKFyve ^f/+ PF4 Cre+ mice, developed diffuse dorsal alopecia and progressive weight gain associated with general body swelling. This was more pronounced in the abdomen, perineum, and limbs, and became more prominent as the mice aged. By 5–6 months of age, PIKFyve ^fl/fl PF4 Cre+ mice displayed severe body swelling with abdominal distention, disabling them from normal ambulation and feeding. A DEXA scan for total body fat distribution and bone mineral density revealed that PIKFyve^fl/fl PF4 Cre+ mice had approximately 50% lower body fat content and 20% lower bone mineral density as compared to their control littermates. Necropsy of 5-month-old PIKFyve^fl/fl PF4 Cre+ mice demonstrated that large vacuolated cells extensively infiltrated multiple organs, including bone marrow, kidney, pancreas, liver, heart, thymus, lung, small and large intestines, genital organs, and skin. These cells resembled brown fat or foamy histiocytes in that the cytoplasm was entirely replaced by giant vacuoles, but their identity is currently unknown. We hypothesize that these invasive cells are of hematopoietic or vascular origin, since they initially infiltrate the organs in a perivascular distribution before they ultimately invade and replace the majority of the organs' cellular architecture. Despite this lethal abnormality, PIKFyve^fl/fl PF4 Cre+ mice have normal platelet counts. Furthermore, in response to thrombin or collagen, platelets from these mice have essentially normal P-selectin exposure and ATP release, reflecting normal release of contents from alpha granules and dense granules, and their aggregation profile is similar to that of control platelets. Nevertheless, flow cytometric analysis indicates that mepacrine uptake and storage in dense granules and lysosomes is markedly impaired. This suggests that PIKFyve and its product, PtdIns(3,5)P₂ play a role in the incorporation of specific components into either dense granules or lysosomes. In conclusion, we observed that loss of PIKFyve activity in murine platelets is associated with defective incorporation of selected contents within granules, and a pleomorphic phenotype that is due to multiple organ infiltration with largely vacuolated cells of undetermined origin. These findings provide further evidence that platelets contribute to a wide range of biologic processes that extend beyond hemostasis.