PB0356 - The emerging role of Kupffer cells in thrombopoietin generation

Background: Thrombopoietin (TPO) is the physiological regulator of the hematopoietic stem cell niche (HSC), megakaryopoiesis and platelet production. We reported that platelet GPIbα is required for platelet-mediated TPO generation (Blood, 2018). However, the mechanism of the platelet-hepatocyte interaction is unknown. Platelets in the vessel are separated from hepatocytes by a fenestrated endothelium. Potential intermediaries for TPO generation include hepatocyte luminal protrusions, and Kupffer cells, which are essential for desialylated platelet (dPLT) clearance.

Aims: Determine whether Kupffer cells contribute to TPO generation independently or in concert with hepatocellular protrusions.

Methods: Murine Kupffer cells were depleted with Clodronate Liposomes. Murine liver endothelial fenestrations were reduced with treatment of 250ppb arsenite in their drinking water. Kupffer cell depleted and arsenite-treated mice were transfused 3x108 dPLTs. Sera and platelet counts were recorded, and TPO quantified by ELISA. Primary murine Kupffer cells were isolated and cultured alone or with dPLTs, and media supernatant was added to hepatocytes for TPO qPCR analysis.

Results: Kupffer cell depleted mice showed a TPO decrease of 43.6%(±16) 2 days post depletion, which could not be rescued with dPLT transfusion. Arsenite-treated mice had a nadir of 61.3%(±4) baseline TPO levels, and were also non-responsive to dPLT transfusions. These findings demonstrates that Kupffer cells facilitate platelet-hepatocyte luminal protrusion contact for TPO generation. Surprisingly, Kupffer cell depletion in GPIbα-deficient mice, which lack platelet-mediated TPO generation, had a TPO decrease of 22.5%(±5) and in vitro Kupffer cell supernatant increased hepatocellular TPO expression by 2.43 fold. Interestingly, these data suggest that Kupffer cells promote baseline hepatocellular TPO production via secretory factor release.

Conclusion(s): Our data demonstrates that Kupffer cells are essential for platelet-mediated TPO generation and promote constitutive hepatocellular TPO production. These findings have broad impacts for the HSC niche, megakaryopoiesis and platelet production, as well as therapeutic discovery for thrombocytopenia patients.