The C-Mpl Receptor Expressed on Endothelial Cells Does Not Contribute Significantly to the Regulation of Circulating Thrombopoietin (TPO) Levels.

Abstract

Blood levels of TPO, the primary regulator of platelet production, are thought to be regulated primarily by cellular degradation following its binding to, and internalization by its cell surface receptor, c-Mpl. This physiology is supported by in vitro studies, in which TPO can be removed from solution by incubation with platelets, and accounts for the very high levels of circulating TPO in individuals with congenital amegakaryocytic thrombocytopenia (CAMT), as in this disorder there are no c-Mpl receptor-bearing cells to bind and remove the hormone. Previous reports have demonstrated that in addition to hematopoietic cells, c-Mpl is expressed on several types of endothelial cells. We hypothesized that the c-Mpl expressed on endothelial cells would contribute to the regulation of circulating TPO levels. As a model to test this hypothesis, we transplanted 10 Mpl knock-out mice and 10 wild-type (WT) controls with WT marrow cells and analyzed platelet counts and plasma TPO levels over the course of 6 months. The resulting two groups of chimeric mice both express c-Mpl on megakaryocytes and platelets, but only the WT recipients express the receptor on endothelial cells. If c-Mpl receptors normally expressed on endothelial cells take up circulating TPO and degrade it, we expected that the Mpl-null mice reconstituted with WT cells would display increased TPO levels and an increased steady state platelet count compared to the WT recipients. As expected, pre-transplant the platelet counts in the Mpl-null mice were low (average value 398x109/L) and TPO levels were high (average value 8040 pg/ml), as compared to WT controls (platelet counts average 883x109/L, TPO level 745 pg/ml). However, following transplant with WT cells, we found that TPO levels in the chimeric mice were virtually identical (954 pg/ml for the Mpl-null recipients vs. 829 pg/ml for the WT recipients) as were the steady state platelet counts up to 6 months following transplantation (891x109 /L vs. 883x109/Lx109 /L, respectively). These results indicate that the c-Mpl receptor expressed on endothelial cells does not contribute significantly to the regulation of circulating TPO levels or to steady state platelet counts. The apparent lack of TPO adsorbtion by the endothelial cells is surprising, as prior published studies indicate that Mpl receptors expressed on human umbilical vein endothelial cells bind TPO and that c-Mpl is functionally active on hepatic endothelial cells; in addition, the composite surface area of the endothelium is large. These results also imply that patients with CAMT that have successfully engrafted with normal hematopoietic stem cells should have normal (not elevated) TPO levels. Similarly, gene replacement strategies designed to restore c-Mpl in CAMT do not need to be expressed on endothelial cells to establish the normal regulation of TPO.