Key Points• Inositol hexakisphosphate kinase 1 (IP6K1) knockout mice display lower inorganic polyphosphate levels in platelets.• Low platelet polyphosphate leads to lengthened clotting time, altered clot architecture, and protection against pulmonary thromboembolism.Polyphosphate (polyP), a polymer of orthophosphate moieties released from the dense granules of activated platelets, is a procoagulant agent. Inositol pyrophosphates, another group of phosphate-rich molecules, consist of mono-and diphosphates substituted on an inositol ring. Diphosphoinositol pentakisphosphate (IP 7 ), the most abundant inositol pyrophosphate, is synthesized on phosphorylation of inositol hexakisphosphate (IP 6 ) by IP 6 kinases, of which there are 3 mammalian isoforms (IP6K1/2/3) and a single yeast isoform. Yeast lacking IP 6 kinase are devoid of polyP, suggesting a role for IP 6 kinase in maintaining polyP levels. We theorized that the molecular link between IP 6 kinase and polyP is conserved in mammals and investigated whether polyP-dependent platelet function is altered in IP6K1 knockout (Ip6k1) mice. We observe a significant reduction in platelet polyP levels in Ip6k1 2/2 mice, along with slower platelet aggregation and lengthened plasma clotting time. Incorporation of polyP into fibrin clots was reduced in Ip6k1 2/2 mice, thereby altering clot ultrastructure, which was rescued on the addition of exogenous polyP. In vivo assays revealed longer tail bleeding time and resistance to thromboembolism in Ip6k1 2/2 mice. Taken together, our data suggest a novel role for IP6K1 in regulation of mammalian hemostasis via its control of platelet polyP levels.

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Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P < 0.05) when cells subjected to serum starvation, contact inhibition, and 3 mM sodium butyrate (NaBu) treatment when compared with cycling cells. However, 3 mM NaBu treatment caused alterations in cell morphology and increase in dead cells. Thus, interspecies nuclear transfer was carried out using fibroblast cells subjected to contact inhibition for 72 h, serum starvation for 48 h, and cells treated with 1.0 mM NaBu for 48 h. The fusion rates, the proportion of fused couplets that cleaved to two-cell and developed to blastocyst, were highest in all three species when the donor cells were treated with 1.0 mM NaBu for 48 h. But, the blastocyst percentage of interspecies nuclear embryos (5-6%) was significantly lower when compared with rabbit-rabbit nuclear transfer embryos (22.9%). In conclusion, fibroblast cells of leopard, lion, and tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.

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