PubMed Health⌕ Search

Biomedical subjects

D J Kuter

Publications and source records attributed to D J Kuter.

At least 19 recordsLinked to original sources
PubMed2003

Apoptotic markers are increased in platelets stored at 37 degrees C.

BACKGROUND: PLTs for transfusion lose viability during storage in blood banking. This loss of viability is accelerated at 37 degrees C, as is the risk of bacterial contamination, and has led to the selection of 22 degrees C as the routine storage temperature. Because PLTs contain an intact apoptotic mechanism, we sought to determine whether PLTs undergo apoptosis during storage and whether storage at 37 degrees C accelerated this process. STUDY DESIGN AND METHODS: PLT-rich plasma from PLT concentrates was stored at 37 or 22 degrees C in small aliquots or whole bags, with and without cell-permeable caspase inhibitors. Number of PLTs, pH, LDH level, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium activity were analyzed over time. PLT lysates were prepared and tested for the presence and activation of apoptotic proteins by enzyme assay and Western blotting. RESULTS: PLT viability was greatly reduced after 1 to 2 days of storage at 37 degrees C; however, signs of apoptosis were evident by 3 hours after temperature shift. In temperature-stressed PLTs only, a gradual rise in caspase-3 activity was detected that correlated with the appearance of the 17- to 20-kDa cleavage products of caspase-3. Gelsolin, a caspase-3 substrate, underwent cleavage within the same time frame. Bcl-xL and caspase-2 also declined significantly; caspase-9 activity rose. Specific caspase inhibitors could prevent caspase activation but did not improve PLT cellular viability at 37 degrees C. CONCLUSIONS: PLTs contain apoptotic proteins that are activated during PLT storage at 37 degrees C and may account for the rapid decline in PLT cellular viability. Although ineffective here, inhibition of PLT apoptosis may improve PLT cellular viability.

Apoptosis↗
PubMed2001

Thrombocytopenia caused by the development of antibodies to thrombopoietin.

Thrombocytopenia developed in some individuals treated with a recombinant thrombopoietin (TPO), pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). Three of the subjects who developed severe thrombocytopenia were analyzed in detail to determine the cause of their thrombocytopenia. Except for easy bruising and heavy menses, none of these subjects had major bleeding episodes; none responded to intravenous immunoglobulin or prednisone. Bone marrow examination revealed a marked reduction in megakaryocytes. All 3 thrombocytopenic subjects had antibody to PEG-rHuMGDF that cross-reacted with endogenous TPO and neutralized its biological activity. All anti-TPO antibodies were immunoglobulin G (IgG), with increased amounts of IgG4; no IgM antibodies to TPO were detected at any time. A quantitative assay for IgG antibody to TPO was developed and showed that the antibody concentration varied inversely with the platelet count. Anti-TPO antibody recognized epitopes located in the first 163 amino acids of TPO and prevented TPO from binding to its receptor. In 2 subjects, endogenous TPO levels were elevated, but the TPO circulated as a biologically inactive immune complex with anti-TPO IgG; the endogenous TPO in these complexes had an apparent molecular weight of 95 000, slightly larger than the full-length recombinant TPO. None of the subjects had atypical HLA or platelet antigens, and the TPO cDNA was normal in both that were sequenced. Treatment of one subject with cyclosporine eliminated the antibody and normalized the platelet count. These data demonstrate a new mechanism for thrombocytopenia in which antibody develops to TPO; because endogenous TPO is produced constitutively, thrombocytopenia ensues.

Adult↗
PubMed2001

Thrombopoietin therapy increases platelet yields in healthy platelet donors.

The recombinant thrombopoietins have been shown to be effective stimulators of platelet production in cancer patients. It was therefore of interest to determine if one of these, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), could be used to increase platelet counts and consequently platelet yields from apheresis in healthy platelet donors. In a blinded, 2-cycle, crossover study, 59 platelet donors were randomized to receive a single subcutaneous injection of PEG-rHuMGDF (1 microg/kg or 3 microg/kg) or placebo and 15 days later undergo platelet apheresis. Donors treated with placebo had a median peak platelet count after PEG-rHuMGDF injection of 248 x 10(9)/L compared with 366 x 10(9)/L in donors treated with 1 microg/kg PEG-rHuMGDF and 602 x 10(9)/L in donors treated with 3 microg/kg PEG-rHuMGDF. The median maximum percentage that platelet counts increased from baseline was 10% in donors who received placebo compared with 70% in donors who received 1 microg/kg and 167% in donors who received 3 microg/kg PEG-rHuMGDF. There was a direct relationship between the platelet yield and the preapheresis platelet count: Placebo-treated donors provided 3.8 x 10(11) (range 1.3 x 10(11)-7.9 x 10(11)) platelets compared with 5.6 x 10(11) (range 2.6 x 10(11)-12.5 x 10(11)) or 11.0 x 10(11) (range 7.1 x 10(11)-18.3 x 10(11)) in donors treated with 1 microg/kg or 3 microg/kg PEG-rHuMGDF, respectively. Substandard collections (<3 x 10(11) platelets) were obtained from 26%, 4%, and 0% of the placebo, 1 microg/kg, and 3 microg/kg donors, respectively. No serious adverse events were reported; nor were there events that met the criteria for dose-limiting toxicity. Thrombopoietin therapy can increase platelet counts in healthy donors to provide a median 3-fold more apheresis platelets compared with untreated donors.

Adult↗
PubMed2001

Prophylactic platelet transfusions from healthy apheresis platelet donors undergoing treatment with thrombopoietin.

Many patients receiving dose-intensive chemotherapy acquire thrombocytopenia and need platelet transfusions. A study was conducted to determine whether platelets harvested from healthy donors treated with thrombopoietin could provide larger increases in platelet counts and thereby delay time to next platelet transfusion compared to routinely available platelets given to thrombocytopenic patients. Community platelet donors received either 1 or 3 microg/kg pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo and then donated platelets 10 to 15 days later. One hundred sixty-six of these platelet concentrates were then transfused to 120 patients with platelets counts 25 x 10(9)/L or lower. Pretransfusion platelet counts (11 x 10(9)/L) were similar for recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Early after transfusion, the median platelet count increment was higher in patients receiving PEG-rHuMGDF-derived platelets: 19 (range, -12-66) x 10(9)/L, 41 (range, 5-133) x 10(9)/L, and 82 (range, -4-188) x 10(9)/L for placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. This difference was maintained 18 to 24 hours after transfusion. Transfusion-free intervals were 1.72, 2.64, and 3.80 days for the recipients of the placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. The rate of transfusion-related adverse events was not different in recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Therefore, when transfused into patients with thrombocytopenia, platelets collected from healthy donors undergoing thrombopoietin therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.

Adolescent↗
PubMed2001

Postmarketing surveillance of new food ingredients: results from the program with the fat replacer olestra.

Market introduction of savory snacks containing olestra offered an opportunity to evaluate the safety of olestra in a free-living population and thereby compare the outcome to the previously established safety profile determined in clinical trials in which subjects were required to eat predetermined amounts at prescribed intervals. Therefore, a multifaceted postmarketing surveillance program was designed to evaluate consumer experience and safety of olestra in the marketplace. Customer comments were solicited through toll-free telephone numbers. Collected data were evaluated by both internal and external medical experts. About 10% of toll-free telephone calls reported health effects, most of which were gastrointestinal (GI) in nature. Clinical studies were designed and conducted to determine potential GI effects under the range of consumption patterns reported by toll-free calls. Health effects reported were those found commonly in the general population and analyses of the data found no biological reason to conclude that serious or meaningful health effects were the result of olestra consumption.

Adult↗
PubMed2001

The end is just the beginning: megakaryocyte apoptosis and platelet release.

Under influence of hematopoietic growth factors, particularly thrombopoietin (TPO), hematopoietic stem cells in the bone marrow go through a process of commitment, proliferation, differentiation, and maturation and become mature megakaryocytes. At this critical point, terminally differentiated megakaryocytes face a new fate: ending the old life as mature megakaryocytes by induction of apoptosis and beginning a new life as platelets by fragmentation of the large megakaryocyte cytoplasm. These events are as important as megakaryocyte commitment, proliferation, differentiation, and maturation, but the molecular mechanisms regulating these events are not well established. Although TPO drives megakaryocyte proliferation and differentiation and protects hematopoietic progenitor cells from death, it does not appear to promote platelet release from terminally differentiated megakaryocytes. Although mature megakaryocyte apoptosis is temporally associated with platelet formation, premature megakaryocyte death directly causes thrombocytopenia in cancer therapy and in diseases such as mvelodysplastic syndromes and human immunodeficiency virus infection. Also, genetic studies have shown that accumulation of megakaryocytes in bone marrow is not necessarily sufficient to produce platelets. All of these findings suggest that platelet release from megakaryocytes is an important and regulated aspect of platelet production, in which megakaryocyte apoptosis may also play a role. This review summarizes recent research progress on megakaryocyte apoptosis and platelet release.

Animals↗
PubMed2001

The effect of unfractionated vs. low molecular weight heparin on tissue factor pathway inhibitor levels in hospital inpatients.

Although heparin is widely used as an antithrombotic agent, its multiple mechanisms of action are not fully defined. Recent work has suggested that tissue factor pathway inhibitor (TFPI) may contribute to the antithrombotic activity of heparin by inhibiting the extrinsic pathway of coagulation. We have investigated the effect of heparin on TFPI and have found that when unfractionated heparin is given by continuous intravenous infusion to hospitalized inpatients, TFPI levels increase 2.3-fold and remain high as long as heparin is continued, but return to baseline levels soon after the infusion is stopped. In contrast, therapeutic doses of the low molecular weight heparin, dalteparin, resulted in significantly less TFPI induction. Given the increasing number of studies establishing the clinical efficacy of low molecular weight heparins as antithrombotic agents, these results suggest that TFPI may not be a major contributor to the antithrombotic effect of heparin.

Anticoagulants↗
PubMed2000

Cloning and functional characterization of a novel c-mpl variant expressed in human CD34 cells and platelets.

The thrombopoietin receptor, c-mpl, is a crucial element not only in thrombopoietin (TPO)-initiated signaling pathways but also in the regulation of the circulating amount of TPO. We have identified a new c-mpl isoform, called c-mpl-del, that lacks 72 bp (24 amino acids) in the extracellular region of c-mpl and arises as a consequence of alternative RNA splicing between exons 8 and 9. c-mpl-del is expressed along with c-mpl-wt in blood mononuclear cells, CD34(+)cells, megakaryocytes, and platelets prepared from either normal donors or ET patients, although its relative expression appears to increase with megakaryocyte differentiation. The c-mpl-del-transfected cells expressed greater amounts of c-mpl-del RNA and protein than the comparable c-mpl-wt-transfected cells, however flow cytometry analysis could not detect any c-mpl receptor on the surface of the c-mpl-del-transfected cells. Further evidence for the absence of surface c-mpl-del was that in contrast to cells transfected with c-mpl-wt, those transfected with c-mpl-del did not grow in response to TPO, failed to undergo tyrosine phosphorylation of TPO-specific signal molecules, and did not bind(125)I-rHuTPO. Taken together, these results demonstrate that c-mpl-del, a naturally occurring variant of c-mpl, fails to be incorporated into the cell membrane but might serve as a mechanism to decrease the overall expression of functional c-mpl late in megakaryocyte differentiation.

Alternative Splicing↗
PubMed2000

Future directions with platelet growth factors.

Since the purification of thrombopoietin 6 years ago, c-Mpl ligands such as recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) have undergone extensive clinical investigation. Both recombinant forms have been shown to reduce the thrombocytopenia associated with nonmyeloablative chemotherapy. Several areas of research have been identified for further clinical development of c-Mpl ligands. One future direction is to continue to explore the intravenous route of administration of rhTPO and PEG-rHuMGDF, as well as fusion proteins of interleukin-3-thrombopoietin and thrombopoietin peptide mimetics, which may be as potent as thrombopoietin, but may lack antigenicity. Another focus would be on the use of these molecules in treating non-chemotherapy-induced thrombocytopenia associated with myelodysplastic syndrome (MDS), idiopathic thrombocytopenic purpura (ITP), human immunodeficiency virus (HIV)-related ITP, and liver disease. Additionally, c-Mpl ligands may have a role in improving apheresis yields when administered to normal platelet donors. Considerable data demonstrate the effectiveness of PEG-rHuMGDF in raising the platelet yields in apheresis donors. In the past few years, investigation into the use of thrombopoietin for ex vivo expansion of pluripotent stem cells has been extensive. Last, thrombopoietin may serve as a radioprotectant by preventing radiation-induced apoptosis of pluripotent stem cells. In the coming years, the clinical role of rhTPO, PEG-rHuMGDF, and related molecules such as the thrombopoietin peptide mimetics will probably be established for both chemotherapeutic and nonchemotherapeutic indications.

Animals↗
PubMed2000

Thrombopoietin and the TPO receptorduring platelet storage.

BACKGROUND: For most cells, the addition of a specific growth factor has improved cellular viability by preventing programmed cell death (apoptosis). To determine whether the platelet-specific hematopoietic growth factor thrombopoietin (TPO) might improve platelet viability, endogenous TPO and the platelet TPO receptor were analyzed during storage, and the effect of recombinant TPO on platelet viability was assessed. STUDY DESIGN AND METHODS: During platelet storage, TPO stability was assessed by SDS-PAGE, TPO receptor function was measured, and the platelet TPO receptor was characterized by a (125)I-rHuTPO competitive-binding assay. A recombinant TPO, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), was added to platelet concentrates during storage, and its effect on pH, LDH, and metabolic activity was determined. RESULTS: During storage, the molecular weight and concentration of endogenous TPO (125 +/- 19 pg/mL) and exogenous TPO (5720 +/- 140 pg/mL) were constant for 12 days; the number (33 +/- 4), binding affinity (149 +/- 33 pM), and function of the platelet TPO receptors were constant for 7 days. Metabolic activity measured with the MTT and MTS assays closely correlated with changes in the pH and LDH. The addition of PEG-rHuMGDF did not alter the pH, LDH, or metabolic activity of platelets during storage, but it did increase by 65 percent the uptake of (35)S-methionine into platelets. Finally, platelet concentrates obtained from donors treated with PEG-rHuMGDF retained normal metabolic activity for 12 days, as compared with 5 to 6 days for normal platelet concentrates. CONCLUSIONS: TPO and its platelet receptor are present in normal amounts and have normal function during platelet storage. The addition of recombinant TPO increased platelet methionine transport but did not alter platelet viability during storage. Other means to prevent apoptosis during platelet storage should be considered, and the measurement of platelet metabolic activity by MTT and MTS assays may assist this effort.

Blood Platelets↗
PubMed2000

The mechanism of apoptosis in human platelets during storage.

BACKGROUND: Although it is usually involved only in nucleated cells (NCs), artificially enucleated cells also lose viability by a programmed process of cell death called apoptosis. Because platelets undergo loss of viability during storage, an attempt was made to determine whether platelets contained the apoptotic mechanisms and whether it was activated during platelet storage. STUDY DESIGN AND METHODS: Platelet viability was measured by reduction of a tetrazolium dye (MTS) and annexin V binding. Members of the death receptor, caspase, and Bcl-2 families were detected by RNase protection assay and Western blotting. Caspase 3 activation was measured by enzyme and Western blot assays and by cleavage of gelsolin. RESULTS: After 5 days of storage under standard blood banking conditions, platelets display biochemical signs of apoptosis by losing MTS activity and increasing the amount of phosphatidylserine on their surface. The mRNA and the proenzyme for several members of the caspase, death receptor, and Bcl-2 families are expressed at high levels in platelets. An increase in caspase 3 activity and the amount of the biologically active p17 subunit of active caspase 3 were observed to coincide with the appearance of apoptotic markers during storage. These effects were not due to platelet activation. The caspase 3 substrate, gelsolin, began to undergo proteolysis after 3 to 4 days of storage, and the addition of the caspase inhibitor z-VAD-fmt substantially inhibited this process. CONCLUSION: Platelets contain many of the components of the apoptotic mechanism and show activation of caspase 3 and consequent cleavage of gelsolin during storage, independent of platelet activation. Evaluation of the mechanism of apoptosis in platelets may provide a basis for developing novel strategies to enhance platelet viability during storage.

Apoptosis↗
PubMed2000

The platelet thrombopoietin receptor number and function are markedly decreased in patients with essential thrombocythaemia.

Essential thrombocythaemia (ET) is a relatively common myeloproliferative disorder characterized by an elevated platelet count. As thrombopoietin (TPO) and the TPO receptor (c-mpl) regulate platelet production in normal physiology, their role in ET was investigated. A well-characterized cohort of 23 ET patients was evaluated and followed for 3 years. The TPO levels in these ET patients (189 +/- 131 pg/ml) were the same as in normal subjects (179 +/- 112 pg/ml) and TPO was not produced by ET platelets. There were 5.6 +/- 5.5 TPO binding sites/ET platelet vs. 56 +/- 17 TPO binding sites/normal platelet and this was associated in ET patients with normal-sized platelet c-mpl protein and mRNA, but a 10-fold reduction in platelet c-mpl mRNA. The K(d) for the TPO receptor on ET platelets was 66 +/- 30 pmol/l vs. 163 +/- 31 pmol/l on normal platelets, but the c-mpl cDNA had a normal nucleic acid sequence. The decreased number of ET platelet TPO receptors resulted in a fourfold decrease in the platelet-dependent TPO clearance (0.30 +/- 0.14 ml/h/10(9) ET platelets vs. 1.24 +/- 0.38 ml/h/10(9) normal platelets) at a time when the platelet count in ET patients was 2.7-fold above normal. The fourfold decrease in the TPO clearance, elevated platelet mass and resulting normal total TPO clearance explain the normal TPO levels. These results also suggest that the thrombocytosis in ET may be attributed to an alteration of the normal feedback interaction between TPO and its receptor and not as a result of any defect in the structure of TPO or c-mpl.

Adult↗
PubMed1999

Interaction of thrombopoietin with the platelet c-mpl receptor in plasma: binding, internalization, stability and pharmacokinetics.

Thrombopoietin (TPO) is the primary regulator of platelet production and acts through binding its receptor, c-mpl, found on megakaryocyte progenitor cells, megakaryocytes and platelets. Circulating levels of TPO are regulated primarily by the clearance of TPO after it binds to c-mpl receptors on circulating platelets. In this study the interaction of TPO with the platelet c-mpl receptor has been analysed under physiological conditions using radiochemical and pharmacokinetic approaches. 125I-rHuTPO was prepared using a novel method of gentle iodination that preserved its biological activity and used to demonstrate that platelets, but not endothelial cells, have a single class of binding sites (56 +/- 17 binding sites/platelet) with high affinity (Kd = 163 +/- 31 pM). Cross-linking experiments confirmed that TPO, but not erythropoietin (EPO), specifically associated with the 95 kD platelet c-mpl receptor. Upon addition of TPO to platelets, 80% of the TPO binding sites were internalized within an hour and were not recycled. TPO that was not bound by platelets was stable for up to 6 d in both platelet-poor and platelet-rich plasma. Using unlabelled recombinant human TPO (rHuTPO), standard pharmacokinetic analysis demonstrated that platelets have an average TPO clearance of 1.24 +/- 0.38 ml/h/109 platelets and that TPO clearance was reduced by low temperature but not by a number of drugs or metabolic inhibitors. The maximal amount of TPO removed by platelets in vitro was identical to that predicted by the total number of TPO binding sites. These results provide a biochemical and pharmacokinetic basis for the clinical use of TPO and for understanding possible disorders of platelet production.

Blood Platelets↗
PubMed1999

The physiological response of thrombopoietin (c-Mpl ligand) to thrombocytopenia in the rat.

It has been suggested that circulating levels of thrombopoietin (TPO) are determined primarily by platelet and megakaryocyte clearance of TPO and not by changes in hepatic TPO production. The experimental evidence accumulated so far to support this hypothesis is incomplete. We have therefore developed a new model of non-immune thrombocytopenia in the rat and used it to assess the relationship of TPO (c-mpl ligand) to the platelet mass. 14 d following the administration of busulphan, the platelet count reached a nadir of <2% of its initial value and remained at this level for up to 6 d. Circulating TPO was measured by two different bioassays which were sensitive enough to measure normal levels of TPO and levels rose from 106 +/- 29 pg/ml in animals with a normal platelet count to 2015 +/- 544 pg/ml in those with thrombocytopenia. These elevated levels of TPO were solely a response to the low platelet count since transfusion of a normal mass of platelets into the thrombocytopenic animals returned the TPO levels exactly to normal. The increase in TPO levels in thrombocytopenic animals was not due to increased TPO production since the thrombocytopenic animals did not show any increase in TPO mRNA in total or polysome-associated hepatic RNA. Rather, rat platelets were able to bind and stoichiometrically remove TPO from thrombocytopenic plasma via high-affinity receptors (Kd = 38 +/- 10 pm; 233 +/- 32 receptors/platelet). These results serve as a proof that the circulating level of TPO is determined not by alterations in TPO transcription or translation but by the ability of the platelet mass to bind and remove TPO from the circulation.

Animals↗
PubMed1999

Cancer, coagulation, and anticoagulation.

Thromboembolic disease affects about 15% of cancer patients and presents a challenge to the oncologist for both prophylaxis and treatment. Although long known to be associated with malignancy, the underlying biochemical mechanisms are poorly understood. Both low-dose warfarin and low molecular weight heparin are effective strategies for prophylaxis of venous thromboembolism, including those involving venous access devices. Current treatment options for venous thromboembolism include heparin (unfractionated and low molecular weight), warfarin, and internal vena cava filters. The appropriate use of these therapeutic options in cancer patients is reviewed herein. There is suggestive evidence that heparin may be superior to warfarin in the long-term treatment of venous thromboembolism. Whether anticoagulants might also improve cancer survival rates independent of their effect on thromboembolism deserves further investigation.

Anticoagulants↗