Dual structural requirements for multilineage hematopoietic-suppressive activity of chemokine-derived peptides.

Many chemokines have direct suppressive activity in vitro and in vivo on primitive hematopoietic cells. However, few chemokine-derived peptides have shown a significant activity in inhibiting hematopoiesis. Interestingly, a peptide derived from the 34-58 sequence of the CXC chemokine platelet factor 4 (PF4) produced a 30-40% inhibition of proliferation of murine hematopoietic progenitors (CFU-MK, CFU-GM, and BFU-E) in vitro, at concentrations of 30-60-fold lower than PF4. The aim of the present work was to define the structural parameters and motifs involved in conferring biological activity to the peptide PF4(34-58). Both structural predictions and determinations revealed a new helical motif that was further localized between residues 38 and 46. This helix was necessary for binding of the peptide and for permitting the functional DLQ motif at position 54-56 to activate the putative receptor site. Peptides lacking either the helical or the DLQ motif were devoid of inhibitory activity on the hematopoietic progenitors in vitro. However, among inactive peptides, only those having the helical motif counteracted the inhibition induced by the active peptide PF4(34-58). This suggested that the helix might be required for peptide interactions with a putative receptor site, whereas the DLQ motif would be implicated in the activation of this receptor. These results identify for the first time the dual requirements for the design of chemokine-derived peptides with high suppressive activity on hematopoiesis, as well as for the design of molecules with antagonistic action.

New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides.

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34(+) marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.

Quantitation of megakaryocytopoiesis by computerized automatic in culture image analysis.

A computer-assisted automatic image procedure was karyocytopoiesis in culture. This analysis system was based on acetylcholinesterase staining, a specific staining for murine bone marrow megakaryocytes, and an image capturing instrument with a computer program. Two kinds of routine megakaryocyte culture methods were used, the plasma clot and the serum-free agar systems. A comparison between manual counting and the instrument was made. The image analysis software was able to distinguish between megakaryocytes (MK) at different stages of maturation. The results show that this analysis system can simultaneously detect not only the number of megakaryocytes and their colonies in each dish, but also the surface area of individual megakaryocytes. In addition, this analysis system functions automatically 24 hours a day and the results obtained are reproducible. Using this system, we have confirmed previous observations that thrombopoietin (TPO) and heparin stimulate both proliferation and maturation of megakaryocytes. In addition, we found that platelet factor 4 (PF-4) significantly reduced the number of megakaryocytes but not their cell surface area, whereas TGFbeta1 decreased both number and surface area of megakaryocytes, suggesting that PF4 and TGFbeta1 negatively regulate megakaryocytopoiesis by different mechanisms. We noticed that megakaryocytes grown under agar culture conditions regularly had an increased size in comparison with those grown in a plasma clot system, which may be an indication that the plasma clot culture media contains an inhibitor(s) of megakaryocyte maturation. Our data indicate that this image analysis system, in addition to its automatic and reproducible features, is more efficient and allows detection of more parameters than routine manual microscopic detection.