The origin of platelet a-granule fibrinogen (Fg), whether from endogeneous synthesis or exogeneous derivation, remains unknown. Although Fg biosynthesis by megakaryocytes (MK) has been suggested, recent studies have demonstrated that certain a-granular proteins originate primarily from plasma. To study the origin of a-granule Fg, platelet-associated Fg was measured by ELISA and Western blotting, and localized by immunofluorescence and immunoelectron microscopy in a patient with symptomatic congenital afibrinogenemia before and after replacement therapy with cryoprecipitate. a-Granule Fg was detected in the majority of platelets as early as 24 h postinfusion, suggesting that direct platelet uptake was occurring. Platelet Fg reached a maximum value of 42.5% of normal values at 3 d postinfusion and was localized in the a-granules, while plasma levels followed a typical half-life profile. Significant a-granule Fg was still detectable at 13 d postinfusion, with plasma Fg virtually absent. Studies on cultured CFU-MKs from the patient also confirmed that MKs can incorporate exogeneous Fg into a-granules. These results indicate that platelet a-granule Fg can be derived from the circulating plasma pool and that Fg uptake can occur in both platelets and MKs.

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin- 6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.