Negative Paracrine Effect of Platelet Factor 4 on Megakaryopoiesis Occurs through Lipoprotein Related Protein Receptor-1 on Megakaryocytes.

Abstract

Platelet Factor 4 (PF4) is a paracrine inhibitor of megakaryopoiesis in vivo, with platelet counts inversely related to platelet PF4 levels in mice. Duration of thrombocytopenia after chemotherapy-induced thrombocytopenia (CIT) was directly related to platelet PF4 levels. Defining the molecular basis by which PF4 has this effect may have clinical implications in patients with CIT. Based on a recent observation that PF4 could activate endothelial cells through the Lipoprotein Related Protein Receptor-1 (LRP-1), we investigated whether a similar receptor might be involved in the negative paracrine effect of PF4 on megakaryopoiesis. We found that the addition of receptor-associated protein (RAP), a general inhibitor of members of the LRP family of receptors by binding to the alpha subunit, could block the inhibitory affect on in vitro megakaryocyte (MEG) colony number per plated marrow cells in a specific and dose-dependent fashion. Thus, at 0.2 μM RAP could prevent ∼50% of the inhibitory affect of the addition of 20 μg/mL of recombinant PF4 and at 0.4 μM RAP, the inhibitory effect of the added PF4 was completely abolished (p = 0.01 of no RAP vs. 0.4 μM RAP). Since a number of LRP receptors are inhibited by RAP, we repeated these studies using a specific anti-LRP-1 monoclonal antibody that blocks LRP-1 activation by binding to its alpha subunit (American Diagnostic). We have previously shown that transgenic mice overexpressing human PF4 by 6-fold have a 50% decrease in MEG-containing colonies in the above colony assay relative to WT littermates due to increase release of PF4 from developing megakaryocytes. We now found that the number of colonies derived from these high PF4 marrows increased to that seen in WT littermates when 25 μg/mL of the anti-LRP-1 antibody was included (p = 0.01). Since there are no prior studies showing that LRP-1 is expressed on human and murine bone marrow and murine fetal liver-derived MEGs, we performed RT-PCR on the appropriate RNAs as well as flow cytometry for the presence of LRP-1. RT-PCR of MEG RNA shows that LRP-1 message is present in MEGS. By flow cytometry, LRP-1 is clearly present on both human and murine MEGs. These studies support that PF4 inhibits megakaryopoiesis through LRP-1 on developing megakaryocytes. Studies were also done on the megakaryocyte-erythroid progenitor cell line G1ME. These cells do not express LRP-1, but when stimulated to differentiate, LRP-1 is detectable by flow cytometry on maturing megakaryocytes, suggesting that PF4 exerts its negative paracrine effect on developing MEGS and not on earlier progenitor cells. Consistent with this finding, we observed that in mice with different levels of platelet PF4, MEG content in the marrow is inversely related to platelet PF4 levels, but the size and ploidy are not correlated to PF4 content. Thus, we believe that we have defined the receptor system involved as well as the target cell of the observed inhibitory effect of PF4 on megakaryopoiesis. Such studies on the mechanism by which PF4 exerts this negative paracrine effect should lead to a more complete understanding of the regulation of megakaryopoiesis and may offer novel strategies to intervene in clinical thrombocytopenia, especially CIT.