Loss of PIKFyve Kinase Function Driven by Platelet Factor 4 Promoter Results in Platelet Lysosomal Storage Defects and Infiltration of Multiple Organs with Vacuolated Macrophages

Abstract 697

Phosphatidylinositol kinases are crucial in the generation of diverse membrane phosphoinositides that regulate the intracellular signaling for essential cellular processes including membrane trafficking. PIKFyve is a kinase that generates phosphatidylinostol 3,5 bisphosphate (PtdIns(3,5)P₂) from PtdIns(3)P. Yeast mutants lacking the PIKFyve homologue were unable to synthesize PtdIns(3,5)P₂ and exhibited enlarged vacuoles, defective membrane recycling and abnormal protein sorting to multivesicular bodies. In human platelets, PtdIns(3,5)P₂ synthesis increased following thrombin stimulation, and our data indicated that murine platelets express PIKFyve mRNA. We hypothesized that PIKFyve and its phospholipid product, PtdIns(3,5)P₂ would be critical in the membrane trafficking during the biogenesis of platelet granules. To test this hypothesis, we engineered mice to lack PIKFyve specifically within their platelets and megakaryocytes. The resulting PIKFyve^fl/fl mice contained a conditional targeting mutation within two exons encoding for a critical component of its kinase domain. The conditionally targeted mice were crossed with transgenic mice expressing Cre recombinase under the control of platelet factor 4 (PF4) promoter. As expected, RT-PCR analysis confirmed a complete loss of normal PIKFyve mRNA in platelets derived from PIKFyve^fl/fl PF4 Cre+ mice. Surprisingly, by 3 weeks of age, PIKFyve^fl/fl PF4 Cre+ mice started to display a robust pleomorphic phenotype characterized by dorsal hair loss and progressive weight gain due to generalized body swelling. By 5–6 months of age, the body weight of the PIKFyve^fl/fl PF4 Cre+ mice was about 30% higher than that of the control littermate mice (PIKFyve^fl/fl PF4 Cre- or PIKFyve^fl/+ PF4 Cre+) and appeared acutely ill with decreased ambulation and feeding. A DEXA scan revealed that PIKFyve^fl/fl PF4 Cre+ mice had approximately 50% lower body fat content compared to their control littermates. Necropsy of PIKFyve^fl/fl PF4 Cre+ mice at about 6 months of age demonstrated that their weight gain was due to massive infiltration of largely vacuolated F4/80 expressing macrophages in multiple organs including skin, muscle, lung, heart, liver, pancreas, small and large intestines, kidney, genital organs, thymus and bone marrow. The vacuoles within these macrophages occupied the entire cytoplasm and were reminiscent of PIKFyve null yeast mutant cells. Unexpectedly, PCR analysis of genomic DNA from tissues infiltrated by these vacuolated macrophages revealed that PIKFyve deletion had occurred within these cells. This indicated that the PF4 Cre was expressed in these macrophages and led to targeting of PIKFyve and ultimately to the development of vacuolated macrophages that infiltrated multiple organs. Despite the pleomorphic phenotype, the platelet counts of the PIKFyve^fl/fl PF4 Cre+ mice were normal. Furthermore, platelets lacking PIKFyve normally exposed P-selectin from alpha granules and released ATP from dense granules in response to agonist stimulation. Nevertheless, the PIKFyve^fl/fl PF4 Cre+ platelets exhibited a three-fold increase of basal b-hexosaminidase, a lysosomal hydrolase. This data suggests a role for PIKFyve in the biogenesis of platelet lysosomes. Furthermore, in vivo platelet thrombus formation analysis using the ferric chloride induced carotid artery injury model revealed that PIKFyve^fl/fl PF4 Cre+ mice made an occluding thrombus significantly faster than the control littermate mice demonstrating that they are prothrombotic. Histological analysis of the carotid artery specimen at the site of thrombus demonstrated that the intravascular side did not contain vacuolated macrophages. This suggests that the enhanced thrombosis formation was due to the increased lysosomal content within the platelets, and not due to infiltration by abnormal macrophages. We conclude that PF4 promoter driven selective deletion of PIKFyve kinase is associated with defective biogenesis of platelet lysosomes and a prothrombotic effect in vivo. Furthermore, PIKFyve^fl/fl PF4 Cre+ mice display a pleomorphic phenotype associated with vacuolated macrophage accumulation in multiple organs and suggest that PF4 promoter driven Cre expression is not restricted to megakaryocytes and platelets. This study also highlights a potential problem in previous and current work using the PF4 Cre transgenic model.