Megakaryocytes Exchange Significant Levels of Their Alpha-Granular PF4 with Their Environment

Platelet factor 4 (PF4, CXCL4) is a major chemokine in megakaryocytes (megs). It is synthesized almost exclusively by megs during their development and may have important roles in regulating both hematopoietic stem cell and megakaryocyte proliferation. We now show that megs both release significant amounts of PF4 into their environment as well as take up PF4 into alpha granules. This PF4 is then available for release by thrombin activation. We examined PF4 recycling during megakaryopoiesis based on the observation that in vitro-cultured human meg hematopoietic precursors release significant amounts of PF4 into the media beginning after approximately 7 days of culture, when definitive megs begin to emerge. Using immunohistochemistry, we find that in vivo in murine bone marrow, human PF4 (hPF4) is released by hPF4 transgenic (hPF4+) megs during the steady-state, and this release is markedly accentuated 48 hours after sub-lethal 660 cGy whole body irradiation from an X-ray source to induce bone marrow injury. By comparison, animals without endogenous PF4 expression (Pf4-/-) showed only background staining. After irradiation, the levels of PF4 staining within the hPF4+ megs decreased with a concomitant increase in background staining suggesting that the stored PF4 was released into the bone marrow milieu. The increase in the PF4 staining in the intramedullary space was not due to released PF4 from entrapped platelets as similar changes were seen in untreated hPF4+ mice and in mice made thrombocytopenic by injection of antiCD41 antibody. We then asked whether the released PF4 could be taken back up by the megs and whether internalized PF4 could reach significant levels compared to endogenously synthesized PF4. We show that murine megs can take up significant levels of hPF4 so that peak hPF4 uptake at 24 hours (19±2 ng/10e6 cells) is equivalent to the amount of mouse (m) PF4 (30±1 ng/10⁶ cells) natively present within the megs. Blocking antibodies to either PF4 itself or to lipoprotein receptor related protein 1 (LRP1) prevented PF4 uptake (53±17 IU/10e6 cells and 32±9 IU/10e6, respectively, vs 95±9 IU/10e6 cells, p <0.01, for either vs. no treatment), consistent with our previous report that LRP1 was necessary for PF4’s negative paracrine effect on megakaryopoiesis. The PF4 that was taken up by megs localizes at least in part to alpha granules, as evidenced by co-localization with P-selectin by immunofluorescence microscopy. Quantification showed a higher degree of colocalization between endogenous mPF4 and internalized hPF4 than between other alpha-granule markers, including vWF, P-selectin and internalized fibrinogen. Moreover like endogenous mPF4, the internalized PF4 can be re-released upon thrombin-induced meg activation. Finally, we asked whether the PF4 uptake was unusual and began by studying uptake of the related chemokine, platelet basic protein (PBP, CXCL7), another protein synthesized by megs and stored in alpha-granules. Unlike PF4, PBP was not internalized by megs as judged by immunohistochemistry or ELISA, indicating that the ability to be internalized and re-released is a relatively unique property of PF4. In summary, we demonstrate that PF4 - an important regulator of megakaryopoiesis and hematopoiesis - is released by megs in the intramedullary space at steady-state and even more so when stressed. Moreover, the released PF4 can be taken up into alpha-granules and stored for potential rerelease. Whether this complex cycle of PF4 in megs is unique to PF4 or applies to other alpha-granular proteins and whether it is necessary for the PF4 effect on hematopoiesis/ megakaryopoiesis needs further investigation