Tissue-plasminogen activator RNA detected in megakaryocytes by in situ hybridization and biotinylated probe

Summary Tissue-plasminogen activator (t-PA) has been identified in human megakaryocytes isolated from human rib fragments or from culture cells. The presence of t-PA antigen in the endoplasmic reticulum of mature cells was demonstrated by ultrastructural immunochemistry. These results suggest that t-PA can be synthesized by megakaryocytes as well as by vascular endothelial cells. In order to confirm this possibility, in situ hybridization using antisense RNA biotinylated probe was used in megakaryocyte culture at different steps of maturation. The presence of t-PA-RNA was clearly demonstrated at an early step of differentiation. Biotin staining was enhanced in mature cells and this was correlated with the detection of t-PA antigen by immunocytochemistry.     

Effect of retinoic acid on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cells

The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue-type plasminogen activator (t-PA) and of plasminogen activator inhibitor-1 (PAI-1) by cultured human umbilical vein endothelial cells (HUVEC). Retinoic acid produced a time- and concentration-dependent increase in the secretion of t-PA-related antigen but not of PAI-1 related antigen into the culture medium. A maximal sevenfold increase of t-PA antigen after 24 h was observed with 10 microM and a half-maximal increase with 0.1 microM retinoic acid. Retinoic acid induced a time-dependent increase of the t-PA mRNA, with a maximum at 8 h and returning to normal at 24 h. The protein kinase inhibitor H7 decreased the t-PA antigen induced by both retinoic acid and phorbol 12-myristate 13-acetate. These results suggest that treatment of HUVEC with retinoic acid increases t-PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t-PA synthesis in the absence of altered PAI-1 synthesis, which may enhance the fibrinolytic potential of the endothelium.     

Heterogeneity of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) from human melanoma cells (Bowes) was purified by immunosorbent chromatography on affinospecific polyclonal antibodies and gel filtration in the presence of KSCN. The immunosorbent eluate contained three major components of greater than 200, 85 and 65 kDa, respectively. The 65 kDa t-PA component could be separated by gel filtration on Ultrogel AcA44 in the presence of KSCN to a pure preparation yielding a unique N-terminal amino acid sequence. Immunoblot analysis, using affinospecific antibodies against t-PA, was a specific and sensitive method to identify different types of t-PA (I-IV), as well as t-PA-inhibitor complexes and degradation products in unstimulated melanoma cell culture fluids. Furthermore, the t-PA preparations, produced by phorbol ester-treated melanoma cells, were free of type IV and thus differed physiochemically from the constitutively produced t-PA preparations. The composition of t-PA from mammalian cell cultures is thus more complex than hitherto described.     

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.     

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.     

Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes

The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal alpha-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibalpha were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.     

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.     

Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells.

The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.     

t—PA昆虫细胞表达及特性分析

With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac-tPA and the linear BacPAK6 virus DNA were cotransfected with Tn-5B-1 insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by PCR identification and biological activity comparison. The t-PA activity expressed in serum-containing media reach the highest level at 72 h postinfection. The activity was 3.04 x 10(3) IU/ml, e.g. 1.8 x 10(4) IU/10(6) cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132 h postinfection). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68 kDa. Its stimulation by fibrinogen, affinity with fibrin and inactivation in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.     

Enhanced production of tissue-type plasminogen activator by estradiol in a novel type variant of human breast cancer MCF-7 cell line

ES-1 cells, which showed a higher sensitivity to the cytocidal action of estradiol were isolated from a human breast cancer MCF-7 cell line. Growth of ES-1 cells was inhibited by a dose of 17-beta estradiol that stimulated the growth of the parental MCF-7 cells. Proteins secreted from MCF-7 and ES-1 cells when cultured with 17-beta estradiol were compared by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE). Addition of estradiol to culture medium enhanced secretion of a protein of molecular mass of 52 kDa in media for both MCF-7 and ES-1 cell lines, but the secretion of a second 67 kDa protein was enhanced about 10-fold only in ES-1 cells. The analysis by SDS-PAGE of culture medium immunoprecipitated with anti-tissue-type plasminogen activator (t-PA) antibody demonstrated that the band of 67 kDa protein specifically secreted from estradiol-treated ES-1 cells contained t-PA. Zymography assays, quantitative immunoreactive assays, and Northern analysis showed about 5-fold specific increase by estradiol of t-PA with molecular mass of 65-70 kDa in ES-1 but not in its parental MCF-7 cells. Cellular level of the plasminogen activity was also specifically enhanced in ES-1 cells by estradiol, but only a slightly in MCF-7 cells. By contrast, another urokinase-type PA (u-PA) with molecular weight of 55 kDa showed very low level activity in both MCF-7 and ES-1 cell lines in the presence of estradiol. Formation of t-PA mRNA was specifically enhanced in ES-1 cells when ES-1 cells were treated for more than 12 h with 10(-8) M 17-beta estradiol. Estradiol did not elongate the lifetime of t-PA mRNA in ES-1 cells. A unique phenotype of ES-1 cells in response to estradiol is discussed in relation to activating expression of the t-PA gene.     

Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.     

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.     

A proteomic approach to the identification of molecular targets in subsequent apoptosis of HEL cells after diosgenin‐induced megakaryocytic differentiation

Diosgenin is a plant steroid which is able to induce megakaryocytic differentiation of human erythroleukemia (HEL) cells followed by apoptosis at a later stage. Apoptosis markers and phospho‐kinases involved during the subsequent apoptosis of megakaryocytes after diosgenin‐induced differentiation in these cells were detected using a proteomic approach. In mature megakaryocytes undergoing apoptosis, we observed increased expression of intrinsic apoptosis markers such as Bax/Bcl‐2 ratio and cleaved caspase‐9 as well as extrinsic apoptosis markers including cell death receptors and cleaved caspase‐8. Furthermore, we demonstrated the link between both apoptotic pathways by Bid cleavage and confirmed the executive phase of apoptosis by caspase‐3 cleavage. For the first time, we examined kinase activation and showed that kinases including Src, Tor, Akt, CREB, RSK and Chk2 may be implicated in signalling of subsequent apoptosis of mature megakaryocytes after diosgenin‐induced differentiation of HEL cells. J. Cell. Biochem. 107: 785–796, 2009. © 2009 Wiley‐Liss, Inc.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Effect of insulin on murine megakaryocytopoiesis in a liquid culture system

To examine the influence of insulin on megakaryocytopoiesis, we tested its effect on murine bone marrow cultures in a liquid culture system. In the presence of pokeweed mitogen-stimulated spleen cell conditioned medium in culture, insulin markedly enhanced megakaryocyte colony formation and increased the number and size of free megakaryocytes seen after 7 days. Many of the cells in cultures with insulin, however, were classified as immature, since they had a basophilic cytoplasm, a low cytoplasmic/nuclear ratio and low acetylcholinesterase activity. It is suggested that insulin potentiates murine marrow megakaryocytopoiesis in vitro, but that this is not accompanied by differentiation of the cells from the immature to mature state.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.