SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3⁺) and suppressor (Leu 2⁺) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×10⁵/ml) were co-cultured with Leu 3⁺, or Leu 2⁺ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:

These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.