75Se-seleno-methionine-actin as a probe for determination of platelet production rate.

Synthesis of actin is a reasonably correct representation of the platelet production rate. Measurement of actin production obviates some of the drawbacks encountered in currently available methods. Platelet actin was characterized on polyacrylamide gel electrophoreses using methylated radioactive rabbit actin as a reference. Platelet actin was unambiguously identified by showing that it forms a complex with DNAase-I similar to the complex obtained with pure rabbit actin. Six days after intravenous injection of 75Se-seleno-methionine actin was one of the most highly labelled platelet components. The platelet pellet (one rat per sample) was solubilized with 1% Triton X-100, 0.75 M guanidine-HCl and dialyzed in a medium containing 1% SDS (to eliminate Triton X-100 and guanidine-HCl). A carefully measured aliquot was deposited on a polyacrylamide gel. After electrophoresis in the presence of SDS the radioactivity of the actin band was measured and the ratio of the total actin radioactivity to the injected radioactivity was taken as a measure of platelet production. The validity of the actin probe was tested with populations of normal animals. The specific radioactivity of actin was proportional to the injected dose of 75Se-seleno-methionine up to 1 mCi/kg animal. A semi-log plot of actin specific radioactivity vs time exhibited a pseudo-first order decrease. Another constituent with a high specific radioactivity (XM) was excluded as a suitable probe because it was shown to be an adsorbed plasma protein.

In vivo platelet kinetics in 31 diabetic patients. Correlation with the degree of vascular impairment.

In vivo platelet survival was estimated, in 31 diabetic patients, using 51Cr-labelled autologous platelets. A mathematical analysis attempted to measure the "potential" life span (senescence process) and the degree of a superimposed aleatory destruction (consumption process). The potential platelet life span in diabetics did not differ from normal values. However, excessive consumption was observed in one third of the studied cases, without correlation with the age of the patients, the clinical duration of diabetes, and the degree of vascular impairment. Thus, platelet kinetic studies did not provide presently useful indications, in a particular patient, regarding the prognosis of the vascular disease, and the justification of anti-aggregant therapy.

New evaluations of circulating granulocyte and macrophage stem cells in healthy adults using conditioned media and recombinant human growth factors.

Peripheral human blood contains granulo-monocyte (CFU-GM) and eosinophil (CFU-Eo) progenitors. In vitro, the number of colony forming units is thought to range from 0.1-14 per 2 x 10(5) plated cells. We show that the number of CFU-GM, Eo depends on culture methods. By modifying the usual assay method (using human umbilical cord plasma and the association of 2 stimulating conditioned media: activated lymphocyte conditioned medium and bone marrow fibroblast conditioned medium), we found different circulating CFU-GM, Eo numbers. The mean number of circulating CFU-GM, Eo in 107 healthy adults was 22.4 per 2 x 10(5) plated cells (range: 1-84). There was a slight difference between males (mean number: 23.6) and females (mean number: 20.4). The mean number of CFU-GM, Eo harvested on Percoll gradient was 123/ml of peripheral blood (range: 7-513). These results are far over those commonly reported in literature. This suggests that these latter results were probably underestimated. The use of recombinant human interleukin 3 and recombinant human GM (granulocyte-monocyte) colony-stimulating factors shows that CFU-GM, Eo numbers are found to be comparatively increased compared to that obtained with our modified method (using rhIL-3 alone), or that the size of those colonies is notably increased (using rhIL-3 + rhGM-CSF).

[Thrombopoietin and the control of platelet production (author's transl)]. / La thrombopoïétine et le contrôle de la production plaquettaire.

Under normal conditions the circulating platelet mass is remarkably constant, which suggested early on that a negative feedback mechanism was involved in the control of thrombocytopoiesis. Furthermore, plasma or serum from thrombocytopenic animals or humans has been reported to stimulate platelet production when injected into laboratory animals, thus suggesting that it contained a thrombopoietin(s). During the past decade, the biochemistry and physiology of thrombopoietin have been studied. However, it is not now well established that thrombopoietin is a glycoprotein produced, at least in part, by the kidney. Moreover, particular attention may be directed to its machanism of action.

The use of 75Se-methionine as a tracer of thrombocytopoiesis. I. In vivo incorporation of the tracer into platelet proteins: a biochemical study.

This study was undertaken to define to which platelet components 75Se-methionine is bound after its injection to normal rats, and to study the curves of specific radioactivity of each labelled fraction. It has been shown that, for a part, platelet labelling is due to adsorption of plasma proteins (albumin and fibrinogen) after their synthesis. For another part, methionine is directly incorporated in the bone marrow precursors into platelet actomyosin, a protein constituent of the cells; the variations of the specific radioactivity of this component indicate that 75Se-methionine can be used as a cohort label of platelets.

[Double radio-active labelling for the simultaneous study of autologous and homologous human platelets' life span (author's transl)]. / Double marquage des plaquettes par 51Cr et 111In. Application à l'étude simultanée de la durée de vie des plaquettes autologues et homologues.

In cases of thrombopenia of obscure origin, labelling human platelets in plasma medium with both radio-active indium and chromium makes it possible to study simultaneously the life span of infused autologous (111In) and homologous (51Cr) platelets. Examples are given where this method revealed a corpuscular abnormality of platelet kinetics.

An analysis of the effect of random and ageing mechanisms on the survival of platelets.

Human platelets survival curves make it possible to estimate two factors which could be held responsible for the disappearance of cells: a) random destruction b) ageing of the platelets. This study was devoted to non-thrombocytopenic patients with chronic vascular disease. In 40% of the cases, the platelet destruction was found to be due to normal ageing of the cells combined with a random destruction process. The estimation of both of these parameters is of interest for the clinical survey of these patients. The statistical analysis of the remaining 60% of the cases presented here has demonstrated a pure linear disappearance of the labelled platelets. This obsveration favours ageing as the cause of the destruction of the platelets.

[Study of proteins binding radioactive indium in vivo]. / Etude des protéines fixant in vivo l'indium radio-actif

After in vivo infusion, radio-active indium is fixed by liver and bone marrow, but more slowly and less completely than iron. It does not incorporate to haem. It is found as labelled ferritin in the liver, after the fifth day. In the bone marrow, indium is almost exclusively found as labelled transferrin; several arguments suggest the intra-cellular site of the labelled protein. Using indium may enable a better knowledge of the intermediary iron pools.

[Platelet kinetics during arterial diseases. Macro-angiopathy, cardiac valve prosthesis and prosthesis of the aortic bifurcation: 73 cases (author's transl)]. / Cinétique plaquettaire au cours des maladies artérielles. Artérites, prothèses valvulaire et vasculaire: 73 observations.

From 73 platelet kinetic studies in vascular diseases, some conclusions can be drawn: excessive platelet consumption is not as usual as previously published; abnormal platelet recovery is an usual feature in patients with vascular or valvular prosthesis; correlation between platelet kinetic abnormalities and thrombo-embolic complications needs reevaluation; constant abnormal platelet kinetics in the cases of aorto-femoral graft made these cases a valuable model for the clinical and biological evaluation of the drugs committed to a change of the platelet functions.

Partial purification of a thrombocytopoiesis stimulating factor present in the serum of thrombocytopenic rats.

Successive chromatographic procedures made it possible to isolate thrombocytopoietin from the serum of thrombocytopenic rats. The following steps were taken: DEAE-cellulose phosphate chromatography, Sephadex chromatography, exclusion chromatography on DEAE-Sephadex A-50 gel. The apparent molecular weight of thrombocytopoietin was about 48,000 daltons. Like erythropoietin, thrombocytopoietin is a glycoprotein, its molecular weight is similar. A single form is obtained only when the different purification steps last less than 10 days. Beyond that time, partial degradation may occur.

Chromatographic techniques for the separation of a thrombocytopoiesis-stimulating factor from aplastic rats.

Thrombocytopoietin seems to be only partially responsible for the regulation of platelet production. We determined precisely the differences between a second thrombocytopoiesis-stimulating factor (TSF2), and thrombocytopoietin (TSF1) and erythropoietin (Epo). Fractionation was carried out, first on a DEAE-cellulose phosphate column and then on a Sephadex G-75 column. Thrombocytopoietic activity in the various fractions was assessed using 75Se-methionine platelet incorporation into normal recipients. Epo concentrations were determined using a radioimmunoassay. We showed that the apparent molecular weight of TSF2 is 14,000 daltons. It differs from TSF1 (48,000 daltons) and from Epo (39,000 daltons). For doses of 8-12 mU Epo/rat, found in whole serum injected, no effect on thrombocytopoiesis was found. On the contrary, a significant effect (p less than 0.01) was found when the same quantities of Epo present in the Sephadex G-75 F4' fraction were injected (2-10 mU/rat). TSF2 can be separated from TSF1 and Epo, using biochemical techniques.

[Isolation and function of platelets. I. Platelet rich plasma. Comparison between 2 methods: gel filtration and albumin density gradient centrifugation. II. A new method using total blood: metrizamide gradient centrifugation]. / Isolement et fonctions des plaquettes I. A partir de plasma riche en plaquettes. Comparaison de deux méthodes: filtration sur gel et centrifugation sur gradient d'albumine. II. Nouvelle méthode à partir de sang total: centrifugation sur gradients de métrizamide

Human platelets separated from platelet rich plasma (PRP) by two different methods: gel filtraton and centrifugation on albumin gradient have been compared for yield, cellular and protein contamination, ultra-structure, platelet populations, shape change, aggregability and functional preservation. A new method for the separation of platelets from total blood on metrizamide gradients has been established. This technique is rapid and easy; it avoids the initial centrifugation of the PRP where about 30% of the platelets are lost and needs of blood 5 to 10 ml of blood.

Balance between human peripheral blood CFU-GEMM and CFU-Meg in sustained and acute thrombocytopenia.

Quantitative variations of blood cell progenitors were studied in thrombocytopenic post-chemotherapy patients and in healthy plateletpheresis donors with the aim of better understanding the reasons for their varying presence in peripheral blood. In 6 post-chemotherapy patients with severe thrombocytopenia on days 21-27 after initiation of chemotherapy when white blood cell counts were approximately 2.1-6.0 x 10(9)/l, a balance was observed between the most and least immature progenitors: levels of CFU-Meg were significantly lower than control values whereas levels of CFU-GEMM were 2-fold higher than in controls. Results suggest that an activator distinct from the known poietins may stimulate very immature progenitors. In 15 plateletpheresis donors, numbers of CFU-GEMM and CFU-Meg were greatly increased, respectively 5.5-fold and 10-fold, following plateletpheresis. Data once again indicate a major role of CFU-GEMM in the production of mature blood cells. As the most immature progenitors and thrombocytopoiesis stimulating factor(s) are both present in peripheral blood, such factors may be responsible for the initial engagement of these very early progenitors into a specific cell line.

Peripheral blood stem cells collected before and after leukapheresis in the very early remission phase of hematopoietic malignancies.

Human peripheral blood obtained after chemotherapy-induced remission in hemopoietic malignancies has been suggested to be a potential substitute for autologous bone marrow as regards autologous hematopoietic reconstitution. The schedule and consequences of early leukapheresis are, however, still imprecise. We report a study performed in two series of, respectively, 10 and 14 patients where sequential leukapheresis (total number = 84) was evaluated with regard to colony-forming unit (CFU) potency. Our data demonstrate that adequate numbers of progenitor cells can be collected by leukapheresis and that, even when this is performed at an early stage after remission, subsequent hematopoietic reconstitution is not impaired.

Immunological evaluation of harvested stem cells obtained by leukapheresis after chemotherapy.

Some patients suffering from malignancies may benefit of myeloablative chemotherapy followed by hematological reconstitution with autologous peripheral blood reinfusion. A quick evaluation of the number of hematopoietic progenitors present in leukapheresis blood samples is necessary to ensure the collection of a sufficient number of these cells. A study was performed on a series of 25 leukapheresis following initial chemotherapy. The number of granulomonocytic colony-forming unit (CFU-GM) and the number of CD34+ cells were evaluated simultaneously, in each sample. Results have shown a relatively strong linear correlation between both methods of evaluation of hematopoietic progenitors, suggesting that immunophenotyping could be a useful method to estimate the number of progenitors.