Partial purification of thrombopoietin using lectin chromatography

Thrombopoietin (TPO) was partially purified from the plasma of thrombocytopenic rabbits by ammonium sulfate fractionation and lectin chromatography. Thrombopoietic activity was measured with an in vivo assay that measures platelet production by the incorporation of 75Se-selenomethionine (75SeM) into newly forming platelets in mice. Thrombopoietin was precipitated from rabbit plasma by an ammonium sulfate saturation of 60%-80%. The ammonium sulfate precipitate was further fractionated by application to a column of wheat germ agglutinin (WGA) bound to agarose. The biological activity was eluted from the WGA column with 0.2 M N-acetylglucosamine. The minimum dose that stimulated thrombopoiesis was decreased 1000-fold to 1.3 microgram/g body weight by this first lectin step. The biological activity eluted from the WGA column was then applied to a column of concanavalin A (ConA) bound to agarose and eluted with 0.1 M alpha-methylmannose. The ConA step produced an additional two-fold increase in specific activity and decreased the minimum dose to 0.65 microgram/g body weight. Stimulation of thrombopoiesis was not associated with an increased peripheral platelet count. Although the purification scheme presented provided a 7000-fold increase in specific activity compared to plasma from thrombocytopenic rabbits, analysis of the WGA and ConA fractions by gel-permeation high-performance liquid chromatography (GP-HPLC) and SDS-PAGE indicated that many proteins were still present at this stage of the purification procedure.     

Thrombocytosis-induced suppression of small acetylcholinesterase- positive cells in bone marrow of rats

Transfusion of platelet concentrates was used to establish a thrombocytosis of approximately three times normal platelet levels in male rats. This thrombocytosis resulted in a rebound thrombocytopenia to 60% of normal counts. Examination of the small acetylcholinesterase (ACh-E) positive cells of the marrow at this time showed a reduction to 50% of normal levels without significant changes in control animals. A second group of experiments indicated that this suppression developed as early as the third day posttransfusion, persisted until day 7, and returned to baseline levels by day 9. Incorporation of 75SeM indicated that the reduction in platelet count was due to decreased platelet production. Little or no changes were observed in the hematocrit or WBC. This evidence supports the hypothesis that these cells are early cells in the megakaryocytic series. They are the earliest cells of the series seen to be affected by thrombocytosis. Feedback control by platelets or platelet extracts of this cell population may represent one level of regulation of megakaryopoiesis.     

Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits.

Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate-phosphate buffer) and carboxymethyl cellulose (0/003 M citrate-phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta-globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis-stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.     

Relationships between thrombopoiesis and erythropoiesis: with studies of the effects of preparations of thrombopoietin and erythropoietin

The effects of administration of partially purified human urinary erythropoietin and rabbit thrombopoietin, and of endogenously produced erythropoietin and thrombopoietin on both red cell and platelet production were examined in mice. Partially purified thrombopoietin was prepared from rabbit plasma by sequential fractionation with ammonium sulfate precipitation, and DEAE and Sephadex G-100 chromatography. Preparations of thrombopoietin and partially purified human urinary erythropoietin (NIH No. H-11-TaLSL) were administered subcutaneously to normal mice, and the rate of incorporation of selenomethionine-75 Se into platelets was measured as an index of thrombopoietic activity of the infused material. Erythropoietin and thrombopoietin were assayed for erythropoietic activity by measuring the rate of appearance of 59Fe in the red cells of posthypoxic polycythemic mice. Preparations containing thrombopoietin had barely measurable erythropoietic activity, and 7 units of partially purified erythropoietin had little thrombopoietic activity. When endogenous levels of erythropoietin were increased by hypoxia, platelet production was not enhanced. Similarly, increased levels of thrombopoietin, induced in response to thrombocytopenia produced by platelet antiserum, did not alter red cell production. These data suggest that physiologically increased levels of thrombopoietin do not stimulate erythropoiesis, and that physiologically increased levels of erythropoietn do not stimulate thrombopoiesis. However, currently available, partially purified preparations of erythropoietin and thrombopoietin may be capable of stimulating both platelet and red cell production if used in sufficient quantities.     

Reduction of hematotoxicity and augmentation of antitumor efficacy of cyclophosphamide by dopamine

Modulatory effects of dopamine (DA) on hematotoxicity and antitumor efficacy of cyclophosphamide (CY) were studied in Swiss mice bearing transplantable Ehrlich ascites carcinoma (EAC). DA was administered i.p. at a dose of 50 mg/kg/day for 5 consecutive days beginning day 3 after tumor transplantation. CY (200 mg/kg i.p.) was injected 24 hour after completion of DA treatment. DA pretreatment reduced the suppressive effects of CY on hemoglobin, RBC, total WBC, neutrophil, platelet, and bone marrow nucleated cell counts. Likewise, DA partially prevented the CY-induced fall in pluripotent (CFU-S12) and lineage-specific stem cells for granulocytes (CFU-C) in bone marrow. Moreover, mice receiving a combination of DA and CY illustrated greater reduction in tumor volume, viable tumor cell count and mitotic index along with upregulation of tumor cell apoptosis than CY-only group. As a result, the former group demonstrated prolonged hosts survival. Thus, DA protected to a great extent the hematopoietic cells of tumor bearing hosts from the suppressive action of CY and concomitantly augmented its antitumor efficacy resulting in improved hosts survival.     

Stress-induced cholinergic signaling promotes inflammation-associated thrombopoiesis

To study the role of the stress-induced "readthrough" acetylcholinesterase splice variant, AChE-R, in thrombopoiesis, we used transgenic mice overexpressing human AChE-R (TgR). Increased AChE hydrolytic activity in the peripheral blood of TgR mice was associated with increased thrombopoietin levels and platelet counts. Bone marrow (BM) progenitor cells from TgR mice presented an elevated capacity to produce mixed (GEMM) and megakaryocyte (Mk) colonies, which showed intensified labeling of AChE-R and its interacting proteins RACK1 and PKC. When injected with bacterial lipopolysaccharide (LPS), parent strain FVB/N mice, but not TgR mice, showed reduced platelet counts. Therefore, we primed human CD34+ cells with the synthetic ARP26 peptide, derived from the cleavable C-terminus of AChE-R prior to transplantation, into sublethally irradiated NOD/SCID mice. Engraftment of human cells (both CD45+ and CD41+ Mk) was significantly increased in mice that received ARP26-primed CD34+ human cells versus mice that received fresh nonprimed CD34+ human cells. Moreover, ARP26 induced polyploidization and proplatelet shedding in human MEG-01 promegakaryotic cells, and human platelet engraftment increased following ex vivo expansion of ARP26-treated CD34+ cells as compared to cells expanded with thrombopoietin and stem cell factor. Our findings implicate AChE-R in thrombopoietic recovery, suggesting new therapeutic modalities for supporting platelet production.     

Platelet Production in Hypoxic and RBC-Transfused Mice

Platelet production rates were studied in hypoxic, red blood cell (RBC) transfused, and normal mice. In addition, platelet depletion was induced in some of the mice by injection of rabbit anti-mouse platelet serum (RAMPS) to stimulate platelet production. Hypoxia alone caused an increase in haematocrit and platelet count at 1–3 d, followed by a decrease in platelet counts to below normal values at 6–7 d. On the other hand, RBC transfusion caused increased haematocrit and decreased platelet count of mice at 1–4 d, with a return of platelet counts to normal by 5–6 d. Normal mice and mice transfused with RBC responded to platelet depletion with rebound-thrombocytosis with maximum platelet production 3–5 d later and elevated platelet counts on days 5–6. However, platelet production in platelet-depleted mice exposed to hypoxia was less marked, and platelet counts did not reach normal levels. The data are consistent with the hypothesis that hypoxia causes thrombocytopenia by stem cell competition between erythroid and megakaryocytic cell lines and/or inhibition of thrombopoietin production.     

TXAS-deleted mice exhibit normal thrombopoiesis, defective hemostasis, and resistance to arachidonate-induced death

Besides its well-recognized role in hemostasis and thrombosis, thromboxane A(2) synthase (TXAS) is proposed to be involved in thrombopoiesis and lymphocyte differentiation. To evaluate its various physiologic roles, we generated TXAS-deleted mice by gene targeting. TXAS(-/-) mice had normal bone marrow megakaryocytes, normal blood platelet counts, and normal CD4 and CD8 lymphocyte counts in thymus and spleen. Platelets from TXAS(-/-) mice failed to aggregate or generate thromboxane B(2) in response to arachidonic acid (AA) but produced increased prostaglandin-E(2) (PGE(2)), PGD(2), and PGF(2 alpha). AA infusion caused a progressive drop of mean arterial pressure (MAP), cardiac arrest, and death in wild-type (WT) mice but did not induce shock in TXAS(-/-) mice or in WT and TXAS(-/-) mice treated with antagonist to the thromboxane-prostanoid (TP) receptor. The TXAS(-/-) mice were able to maintain normal MAP upon AA insult when TP was present but were unable to do so when TP was blocked by an antagonist, suggesting a role of endoperoxide accumulation in influencing MAP. We conclude that TXAS is not essential for thrombopoiesis and lymphocyte differentiation. Its deficiency causes a mild hemostatic defect and protects mice against arachidonate-induced shock and death. The TXAS-deleted mice will be valuable for investigating the roles of arachidonate metabolic shunt in various pathophysiologic processes.     

Thrombokinetics in idiopathic thrombocytopenic purpura

Summary Measurements of platelet production in 16 patients with idiopathic thrombocytopenic purpura (ITP) demonstrate that megakaryocytopoiesis (total thrombopoiesis) and platelet turnover (effective thrombopoiesis) are increased in parallel to as much as 8 times normal. The marrow megakaryocytes show changes characteristic of stimulated thrombopoiesis.     

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

Filamin A (FlnA) is a large cytoplasmic protein that crosslinks actin filaments and anchors membrane receptors and signaling intermediates. FlnA(loxP) PF4-Cre mice that lack FlnA in the megakaryocyte (MK) lineage have a severe macrothrombocytopenia because of accelerated platelet clearance. Macrophage ablation by injection of clodronate-encapsulated liposomes increases blood platelet counts in FlnA(loxP) PF4-Cre mice and reveals the desintegration of FlnA-null platelets into microvesicles, a process that occurs spontaneously during storage. FlnA(loxP) PF4-Cre bone marrows and spleens have a 2.5- to 5-fold increase in MK numbers, indicating increased thrombopoiesis in vivo. Analysis of platelet production in vitro reveals that FlnA-null MKs prematurely convert their cytoplasm into large CD61(+) platelet-sized particles, reminiscent of the large platelets observed in vivo. FlnA stabilizes the platelet von Willebrand factor receptor, as surface expression of von Willebrand factor receptor components is normal on FlnA-null MKs but decreased on FlnA-null platelets. Further, FlnA-null platelets contain multiple GPIbα degradation products and have increased expression of the ADAM17 and MMP9 metalloproteinases. Together, the findings indicate that FlnA-null MKs prematurely release large and fragile platelets that are removed rapidly from the circulation by macrophages.     

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.     

Compensatory mechanisms in platelet production: the response of Sl/Sld mice to 5-fluorouracil

Sl/Sld mice are a unique animal model for studying platelet production in that they sustain normal platelet mass despite reduced marrow activity. The aim of this study was to determine if the compensatory mechanisms operating in these mice could be augmented by further reducing bone marrow activity with the drug 5-fluorouracil (5-FU), known to induce a strong stimulatory effect on platelet production. The platelet recovery in Sl/Sld mice after 5-FU administration contrasted that found in their normal littermates. Sl/Sld mice did not display the sustained thrombocytosis that was observed in +/+ mice between days 10 and 14. Platelet number was elevated in Sl/Sld mice at day 20, when the marrow megakaryocyte compartment had normalized. A significant increase in marrow megakaryocyte number and size was observed at days 8 and 11 in both +/+ and Sl/Sld mice after 5-FU administration. The data suggest that the increase in megakaryocyte size and number following 5-FU treatment was not able to significantly contribute to a sustained rebound thrombocytosis at the time of increased marrow megakaryocytopoiesis. It is concluded that the already compromised marrow of Sl/Sld mice is able to respond to the damage invoked by 5-FU to produce larger than normal megakaryocytes. In contrast to normal mice (+/+ littermates), the increase in marrow megakaryocytopoiesis observed does not lead to a thrombocytosis, indicating that platelet production and release in Sl/Sld mice cannot be further amplified by a strong marrow stimulation.     

Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis

Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7^flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.     

A Comparison of Platelet Size, Platelet Count, and Platelet 35S Incorporation as Assays for Thrombopoietin

SUMMARY. Average platelet size, platelet count, and ³⁵S-incorporation into platelets were compared as methods for the measurement of thrombopoietin-stimulated thrombopoiesis. In mice injected with rabbit anti-mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, ³⁵S-measurements on platelets of these mice showed significant increases in cpm/average platelet 2–4 days after RAMPS treatment. Significant increases in ³⁵S-incorporation into the total circulating mass of platelets were found on days 3–4. In normal mice or mice in rebound-thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet count and ³⁵S-incorporation into platelets were shown to be significantly increased. Moreover, a dose-response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The %³⁵S-incorporation into platelets showed a significant linear dose-response, i.e. as the dose of thrombopoietin was increased, an increase in %³⁵S-incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the %³⁵S-incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.     

Dopamine induces platelet production from megakaryocytes via oxidative stress-mediated signaling pathways

Dopamine (DA), a catecholamine neurotransmitter, is known to for its diverse roles on hematopoiesis, yet its function in thrombopoiesis remains poorly understood. This study shows that DA stimulation can directly induce platelet production from megakaryocytes (MKs) in the final stages of thrombopoiesis via a reactive oxygen species (ROS)-dependent pathway. The mechanism was suggested by the results that DA treatment could significantly elevate the ROS levels in MKs, and time-dependently activate oxidative stress-mediated signaling, including p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and caspase-3 signaling pathways, while the antioxidants N-acetylcysteine and L-glutathione could effectively inhibit the activation of these signaling pathways, as well as the ROS increase and platelet production triggered by DA. Therefore, our data revealed that the direct role and mechanism of DA in thrombopoiesis, which provides new insights into the function recognition of DA in hematopoiesis.     

Thrombocytopoietic response to immunothrombocytopenia in nude mice

Thrombocytopoiesis was evaluated in T cell-deficient nu/nu mice and in T cell-replete nu/+ controls to determine if abnormalities would be associated with the deficiency of T cells. Mice were studied in the unperturbed steady state and after acute immunothrombocytopenia was induced by an injection of guinea pig antimouse platelet serum (APS). The state of thrombocytopoiesis was determined from platelet counts, megakaryocyte size, megakaryocyte number, and numbers of Meg-CFC. Splenic lymphocytes were evaluated by response to the mitogens bacterial lipopolysaccharide (LPS), phytohemagglutinin (PHA), and concanavalin A (Con A). Hematocrits, reticulocyte counts, leukocyte counts, marrow cellularity, GM-CFC, and BFU-E also were measured. Steady state thrombocytopoiesis was identical in nu/nu and nu/+ mice. In response to an injection of APS, acute thrombocytopenia was followed by macromegakaryocytosis and rebound thrombocytosis in mice of both genotypes. Splenic Meg-CFC increased in nude mice after APS or an injection of normal guinea pig serum (NGpS), and splenic GM-CFC increased after APS. Neither Meg-CFC nor GM-CFC increased in the spleens of nu/+ mice, but they showed early transient increases in bone marrow that did not occur in nu/nu mice. Sporadic, but weak, mitogenic responses to PHA or Con A were occasionally observed with nu/nu spleen cells, but these did not correlate with the state of thrombocytopoiesis. The results demonstrated that platelet production was normal in nu/nu mice and that megakaryocytopoiesis and platelet production responded to the stimulus imposed by acute immunothrombocytopenia. Increases in megakaryocyte size and platelet production occurred independently of changes in numbers of Meg-CFC, GM- CFC, or BFU-E. A normal complement of T cells appears to be unnecessary for normal platelet production and its augmentation in response to the stimulus of acute immunothrombocytopenia in vivo.     

L-asparaginase: acute effects on protein synthesis in rabbits with normal and increased fibrinogen production

The acute effects of a single intravenous dose of L-asparaginase on protein synthesis were studied in normal rabbits and in animals that had received turpentine to stimulate fibrinogen production. Male New Zealand rabbits received L-asparaginase (500 U/kg) 16 hr before the injection of the radiolabeled amino acid [75Se]selenomethionine (75SeM). Incorporation of 75SeM into fibrinogen and serum proteins in the L-asparaginase-treated rabbits was the same as for saline-treated controls, with fibrinogen representing approximately 5% of the labeled plasma proteins. In turpentine-treated rabbits, the maximal incorporation of 75SeM into serum proteins remained unchanged, whereas 75SeM-fibrinogen increased sixfold and accounted for 25% of the labeled proteins. Animals that received L-asparaginase at the same time as turpentine or 14 hr later showed significant decreases in synthesis of both serum proteins and fibrinogen. 75SeM-fibrinogen that was purified from L-asparaginase-treated rabbits underwent normal catabolism when injected into normal recipient rabbits. These data indicate that L- asparaginase can acutely cause partial inhibition of both serum protein and fibrinogen synthesis when administered to rabbits shortly before or during a period of increased fibrinogen production. Fibrinogen that is synthesized in the presence of L-asparaginase does not have an abnormal rate of catabolism.     

A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice

During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1-deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1-deficient MKs in response to a gradient of stromal cell-derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1(-/-) MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be explained by an increase in integrin activation. The defect of migration and polarization was confirmed in vivo with demonstration of altered spatial localization of MKs within the bone marrow in PECAM-1-deficient mice, following immune-induced thrombocytopenia. This study identifies a novel role for PECAM-1 in regulating MK migration and thrombopoiesis.     

Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders.

Thiazole orange (TO), a fluorescent dye originally synthesized for reticulocyte analysis, is characterized by a large fluorescence enhancement and high quantum yield on binding to nucleic acids, particularly RNA. In addition, the dye readily permeates live cell membranes. We applied TO staining, followed by fluorescence-activated flow cytometric analysis, to platelets in whole blood samples from hematologically normal subjects and patients with various quantitative platelet disorders. The percentage of TO-positive platelets in 50 control subjects was 8.6 +/- 2.8% (mean +/- SD) ranging from 2.8% to 15.8%. In 21 thrombocytopenic patients whose bone marrow contained normal to increased numbers of megakaryocytes, the percentage of fluorescently labeled platelets was significantly elevated (P less than .0001) to 26.9 +/- 10.9% (range, 13.3% to 57.1%). In contrast, the proportion of positively stained platelets in 23 patients with thrombocytopenia due to impaired platelet production (various conditions with reduced marrow megakaryocytes) did not significantly differ from the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (P less than .0001). Differences in the distributions of the percentage values as well as of the absolute counts for TO-positive platelets between the two patient groups were again highly significant (P less than .0001). Both the sensitivity and the specificity of this method in distinguishing between these categories of thrombocytopenia were greater than or equal to 95%. We conclude that flow cytometric analysis of platelets after staining with TO is a sensitive and specific test that rapidly provides information on the thrombopoietic activity in thrombocytopenic disorders. Our data further suggest that increased amounts of residual RNA characterize platelets released under conditions of "stress thrombopoiesis."