The pharmacokinetics and pharmacodynamics of single-dose and multiple-dose recombinant activated factor VII in patients with haemophilia A or B.

Monitoring recombinant activated factor VII (rFVIIa) treatment outcomes remains challenging. Thromboelastography (TEG) and the thrombin generation assay (TGA), measure coagulation dynamics over time and are being assessed as potential methods for evaluating and monitoring haemophilia treatment. Lack of standardized TEG/TGA methods makes it difficult to compare results and to establish a correlation with clinical outcomes. AIMS: To compare the pharmacokinetics (PK) of rFVIIa after 3×90 µg kg-1 doses vs a single dose (270 µg kg-1 ) in haemophilia patients and to evaluate TEG/TGA results postdosing to determine how these assays relate to PK findings. METHODS: Patients in this open-label, single-centre, randomized, crossover trial received one injection of 270 µg kg-1 rFVIIa crossed over with three injections of 90 µg kg-1 rFVIIa in a non-bleeding state. For TEG, kaolin and tissue factor were used as activators; TGA was performed on frozen platelet-rich and platelet-poor plasma, with and without corn trypsin inhibitor. FVIIa activity was evaluated using in vivo samples. RESULTS: TGA showed a dose-dependent effect of rFVIIa on thrombin generation; TEG revealed lower dose-dependent effects. Both showed some differences between single-/multiple-dose rFVIIa; both supported the PK findings. CONCLUSION: While TEG and TGA are not yet clinically predictive, both supported the PK results. Data suggest that, while a single dose of 270 µg kg-1 rFVIIa provides slightly higher haemostatic potential than the multiple-dose regimen of 3×90 µg kg-1 , the latter results in prolonged activity levels compared with a higher single dose.

Matrix metalloproteinase 9 is involved in Crohn's disease-associated platelet hyperactivation through the release of soluble CD40 ligand.

BACKGROUND: Patients with Crohn's disease have an increased risk for systemic thromboembolism. Their platelets are hyperactive and possess an elevated endogenous content of CD40 ligand (CD40L), a tumour necrosis factor alpha family protein member. Under basal conditions and after stimulation, these platelets express more CD40L on their surface and release higher amounts of soluble (s)CD40L than control platelets, through a mechanism that might be mediated by matrix metalloproteinases (MMPs). OBJECTIVE: The aim of this work is to study whether enhanced sCD40L release secondary to changes in the platelet content of MMPs contributes to the higher state of activation of platelets from patients with Crohn's disease. METHODS: State of activation, CD40L and metalloproteinases content of platelets isolated from patients with Crohn's disease and age- and sex-matched control individuals were analysed, respectively, by flow cytometry, western blot and gelatin zymography. RESULTS: The hyperactive state of platelets from patients with Crohn's disease might rely on their enhanced release of sCD40L, since its inhibition by a broad-range inhibitor of MMPs (GM6001) reduced fibrinogen binding induced by platelet stimulation. Analysis of the content of MMPs in platelets from patients with Crohn's disease showed an exclusive increase in MMP-9 activity. Moreover, MMP-9 inhibition diminished sCD40L release and fibrinogen binding to activated platelets. CONCLUSIONS: The results suggest that platelets from patients with Crohn's disease release more sCD40L than controls as a consequence of their higher endogenous content of CD40L and of MMP-9, which is involved in CD40L shedding. The increased levels of released sCD40L might be responsible, at least in part, for the high state of activation of platelets from patients with Crohn's disease.

Evaluation of EC50 of factor VIII as predictor of prophylaxis efficacy in patients with severe haemophilia A.

Trough factor (F) VIII level is a not reliable bleeding risk indicator to predict prophylaxis efficacy in severe haemophilia A (SHA), therefore, accurate biomarkers are much needed. Thrombelastography (TEG) monitors both thrombin and clot formation addressing the global haemostatic status but its usefulness to tailor prophylaxis in haemophilia has been poorly evaluated. In this study, correspondence between individual pharmacodynamic/pharmacokinetic profile of FVIII and joint condition, physical activity and bleeding phenotype of SHA patients under prophylactic treatment was assessed. Nineteen SHA patients < 18 years old on long-term prophylaxis treatment with FVIII were studied in an observational cross-sectional study. Whole blood was withdrawn before FVIII administration and at five time-points after infusion for a TEG-based pharmacodynamic- and pharmacokinetic-study. Type of prophylaxis and joint condition at inclusion and physical activity as well as onset of treated spontaneous bleeding events in the previous two years were retrospectively assessed. Six patients had suffered at least one treated spontaneous bleeding event and were named as "bleeders". The rest were named as "non-bleeders". Only the half maximal effective concentration of FVIII (FVIII-EC50) for TEG parameters R-time, K-time and α-angle correlated with the bleeding phenotype being significantly higher in bleeders suggestive of a poorer response to FVIII. Poorer joint condition, trough FVIII levels or type of prophylaxis were not definitive predicting variables of bleeding phenotype. In conclusion, this study reveals FVIII-EC50 for the first time as a valuable biomarker to anticipate individual efficacy of prophylaxis in SHA.

Characterization of the alpha 1-adrenoceptor-mediated responses in perfused rat liver.

The present work aimed to further characterise the hepatic alpha 1-adrenergic actions by studying the influence of nutritional status and/or extracellular medium composition in the alpha 1-adrenoceptor-induced responses. The experiments were performed in a non-recirculating liver-perfusion system featuring continuous monitoring of vascular resistance, as well as the effluent perfusate changes in pO2, pCa2+, pK+ and pH. The alpha 1-adrenoceptor activation produced biphasic responses to most parameters studied. The acute phase lasted for about 3 min and it was followed by a phase of sustained stimulation that lasted as long as the receptor activation was maintained. Our data indicate that there is not a single pattern of alpha 1-adrenergic responses but variable patterns depending on the nutritional status and the experimental conditions. Gluconeogenic substrates alone produced reciprocal changes in the outflow perfusate pH and Ca2+ activity. The magnitude of these changes indicates that the diversity of alpha 1-adrenoceptor responses are the result of the superposed effects of different rates of substrates and/or metabolites transport. The sustained alpha 1-adrenoceptor stimulation produced extracellular acidification and increases in respiration, vascular resistance and Ca2+ release. These responses required physiological extracellular [Ca2+]. At low extracellular [Ca2+], the alpha 1-adrenoceptor activation failed to acidify the extracellular medium, suggesting that receptor-induced H+ efflux demands normal rates of Ca2+ influx. The correlation between alpha 1-adrenergic-induced increase in O2 uptake and Ca2+ release indicates that the increased energy production can be accounted for by the energy cost of Ca2+ release. The alpha 1-agonist concentration-response studies have shown significant differences in the [alpha 1-agonist]0.5 for each type of response, suggesting the existence of multiple alpha 1-adrenoceptor-coupled signal-transduction pathways.

Impaired protein kinase C activation is associated with decreased hepatic alpha 1-adrenoreceptor responsiveness in adrenalectomized rats.

The present work aimed to study the influence of corticosteroids on the alpha 1-adrenoreceptor-induced activation of hepatic metabolic functions. The experiments were performed in a nonrecirculating liver perfusion system featuring continuous monitoring of pO2, pCa2+, Ca2+, pH, and portal pressure. The alpha 1-adrenergic-induced stimulation of respiration, H+ and Ca2+ release, glycogen breakdown, and gluconeogenesis, were diminished in livers from adrenalectomized animals. The normal liver responsiveness was restored on administration of exogenous corticosteroids but not mineralocorticoids. The following observations support the conclusion that corticosteroids control a hepatocyte-specific early postreceptor step in the alpha 1-adrenergic signaling pathway: 1) the alpha 1-adrenergic stimulation of vascular smooth muscle contraction was not impaired by corticosteroid deficiency; 2) the alpha 1-adrenoreceptor ligand-binding affinity does not seem to be altered by adrenalectomy; 3) the alpha 1-adrenergic-induced intracellular alkalosis, protein kinase C activation, and Ca2+ mobilization were diminished in hepatocytes from adrenalectomized rats, indicating that both Ca(2+)-dependent and -independent processes were altered; and 4) non-receptor-mediated homeostatic mechanisms of metabolic or intracellular pH control were not impaired by adrenalectomy.

Role of protein kinase-C in the alpha 1-adrenoceptor-mediated responses of perfused rat liver.

The present work aimed to determine the role played by protein kinase-C (PKC) in the alpha 1-adrenoceptor-induced activation of hepatic metabolism. The following observations indicate that activation of PKC is a condition necessary for alpha 1-adrenoceptor activation of hepatic functions, but not sufficient to mimic the receptor-mediated effects in the absence of external physiological stimuli. 1) alpha 1-Adrenoceptor activation promoted the translocation of PKC from the cytosol to its active form in the plasma membrane. 2) Activation of PKC by the phorbol ester 12-myristate 13-acetate or exogenous diacylglycerols or by elevation of endogenous levels of diacylglycerols by inhibiting diacylglycerol kinase mimicked the alpha 1-adrenoceptor-mediated actions. However, the time course and magnitude of the nonreceptor responses differ from those mediated by alpha 1-adrenoceptor activation. In addition, nonreceptor-mediated activation of PKC decreased the alpha 1-adrenoceptor responsiveness. 3) Inhibition of PKC by either H-7 [1-(5-isoquinolinilsulfonyl)2-methylpiperazine] or staurosporine inhibited all of the alpha 1-adrenoceptor-induced responses, except gluconeogenesis. The vasopressin effects were not inhibited by H-7, indicating that PKC activation is a distinct feature of the hepatic alpha 1-adrenoceptor activation that is not shared by all the Ca(2+)-mobilizing agonists. The diacylglycerol-PKC branch of the alpha 1-adrenoceptor signaling pathway seems to control the sustained phase of stimulation of hepatic functions. In these studies we have also observed that phorbol 12-myristate 13-acetate produces a concentration-dependent inhibition of hepatic respiration. However, decreased energy availability does not seem to be the cause of its action to decrease alpha 1-adrenoceptor responsiveness.

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (

Compound heterozygosity of the GPIbalpha gene associated with Bernard-Soulier syndrome.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

Effects of L-glutamate on the responses to nerve stimulation in rat isolated atria.

In rat atria isolated with their sympathetic fibres the chronotropic responses to nerve stimulation with pulses of 2 ms duration were reduced in a concentration-dependent manner by 10 microM to 1 mM L-glutamate (Glu) and by 0.01 to 1.00 microM (R,S)-3-hydroxy-5-methoxyloxasole-4-propionic acid (AMPA), whereas they were unaffected by other agonists of Glu receptors such as 1 microM to 1 mM N-methyl-D-aspartic acid (NMDA), 10 microM to 1 mM kainate and 1 to 100 microM (+/-)-2-amino-4-phosphonobutyric acid (AP4). The reductions in the atrial responses to nerve stimulation caused by Glu were not accompanied by alterations in either the basal efflux of [3H]noradrenaline or its overflow in response to the stimulation. The sensitivity of the atria to exogenous noradrenaline was not modified by either Glu or AMPA. The decreases in the chronotropic responses caused by Glu and by AMPA were prevented by both the non-selective Glu receptor antagonist, 100 microM kynurenic acid, and the selective AMPA receptor antagonist, 10 to 50 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX). In addition, the adenosine receptor antagonist, 8-phenyltheophylline (10 microM), as well as the muscarinic acetylcholine receptor antagonist, atropine (3 microM), prevented the inhibitory effects of both Glu and AMPA on the chronotropic responses of rat isolated atria. Since both adenosine and acetylcholine are known to exert negative inotropic and chronotropic effects in cardiac tissues, it is proposed that Glu could contribute, through the interaction with receptors of the AMPA type, to facilitate the release of adenosine and acetylcholine from the atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of phenylarsine oxide on hepatic alpha 1-adrenoreceptor responsiveness. Dissociation between ionotropic and metabolic responses.

The present studies analyze the effect of the tervalent arsenical compound phenylarsine oxide (PAO) on hepatic response to alpha 1-adrenoreceptor stimulation. PAO, while not significantly altering the rate of glycogen breakdown, was found to inhibit many characteristic alpha 1-adrenoreceptor mediated responses including H+ and Ca2+ release, increased energy production, and vascular smooth muscle contraction. PAO inhibited basal gluconeogenesis but failed to inhibit the alpha 1-agonist induced stimulation of glucose production. These data suggest that alpha 1-adrenoreceptor mediated stimulation of metabolism and rates of ion flux across the plasma membrane are separate processes and that exchange in ion homeostasis is not essential to elicit the receptor-mediated metabolic responses. The selective effect of PAO offers an interesting tool for studying the alpha 1-adrenoreceptor signaling mechanisms.

Serotonin uptake inhibitors and the prejunctional effects of serotonin on peripheral sympathetic nerves.

The experiments were designed to study whether the inhibitors of the uptake of serotonin (5-HT) potentiated the prejunctional effects of 5-HT on peripheral sympathetic nerves. The effect of two selective 5-HT uptake inhibitors, citalopram and fluoxetine, were studied on the presynaptic actions of 5-HT in the cat isolated nictitating membrane and in the guinea-pig isolated atria. Frequency-effect curves to nerve stimulation and concentration-response curves to noradrenaline (NA) were performed in both preparations. The facilitation that 0.1 microM 5-HT causes on the contractile responses to nerve stimulation of the nictitating membrane of the cat was not potentiated but entirely prevented by both 0.01 microM citalopram and 1.0 microM fluoxetine. On the other hand the diminution that 1.0 microM 5-HT evokes on the chronotropic responses to nerve stimulation of guinea-pig isolated atria was not modified at all by 0.1 and 1.0 microM fluoxetine and only partially prevented by 10.0 microM fluoxetine and by 0.001 microM, 0.01 microM and 0.1 microM citalopram. This latter effect of citalopram was unrelated to the concentration employed. The 5-HT uptake inhibitors did not modify per se either the responses to nerve stimulation or the sensitivity to exogenous NA in both tissues studied. In addition, the 5-HT uptake inhibitors did not interfere with the contractile responses caused by 5-HT in the cat isolated nictitating membrane. Taken together, these observations might indicate a pharmacological rather than a physiological role for the effects of 5-HT in guinea-pig isolated atria and cat nictitating membranes. It is concluded that the 5-HT uptake inhibitors do not potentiate but even antagonize the presynaptic effects of 5-HT. Our results also show that 5-HT uptake inhibitors are more effective to interfere with the facilitation rather than with the inhibition that 5-HT causes on sympathetic responses.

[Annual distribution of serotypes of Salmonella, Shigella, and infantile enteropathogenic Escherichia coli in the Republic of Argentina, 1979-1981]. / Distribución anual de serotipos de Salmonella, Shigella y Escherichia coli enteropatógeno infantil (EPI) en la República Argentina durante el período 1979-1981.

We report data of isolation of 3,665 strains of Salmonella, 1,855 of Shigella and 697 of E. coli infantile enteropathogenic (EPI) from different sources: human, animal, food and water, in Argentina during the triennium 1979-1981, considering their importance in the chain of transmission of enterobacteria. It appears that S. typhimurium is the most common among all the isolated serotypes of Salmonella, following in order of importance, S. oranienburg, S. derby, S. panama, S. agona, S. anatum, S. newport, S. bredeney and S. montevideo. It is important to emphasize the appearance of new Shigella serotypes in Argentina, particularly Sh. dysenteriae 2. We found that E. coli EPI 0111:B4 was the most frequent serotype and in 1981 the serotype 0112:B13 was also found.

[Serotypes of Salmonella in the Republic of Argentina isolated from human and non-human sources, 1979-1981]. / Serotipos de Salmonella en la República Argentina aislados de fuentes humanas y no humanas durante el período 1979-1981.

A total of 49 serotypes were isolated in Argentina during the triennium 1979-1981, from 3,665 Salmonella strains isolated from different sources: human, animal, food and water. The degree of correlation among every one of them was analyzed in order to establish their influence in the cycles of transmission and human infection. S. typhimurium prevailed in human materials, being also isolated from water and animal, although in a smaller degree. Something similar occurred with S. oranienburg. It was observed that S. typhi retained its usual feature of endemic disease. Other serotypes of Salmonella were also found, particularly: S. paratyphi, S. panama, S. derby, S. agona, S. bredeney, S. newport, S. anatum and S. montevideo. From 3,665 Salmonella strains studied, 73.73% were isolated from human sources and the other 26.27% from non human sources, 5.40% corresponding to animal isolations, 15.39% from water and 5.48% from food.

Effects of thrombopoietin receptor agonists on procoagulant state in patients with immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of immune thrombocytopenia (ITP), an anti-platelet-antibodies autoimmune disease. The observation of a low frequency of bleeding episodes despite their thrombocytopenia suggests the existence of a compensatory mechanism. This study aimed to evaluate the effect of TPO-RA treatment on platelet function and on the procoagulant state in ITP patients before (ITP-bR) and after responding (ITP-aR) to treatment. Plasma- and microparticle (MP)-associated procoagulant capacity from ITP patients was similar before and after responding to the TPO-RA regimen but higher than the healthy control values. High MP-associated procoagulant activity did not seem to be due to increased platelet activation, since platelet stimulation by agonists was reduced in ITP-bR and ITP-aR patients. It could be related to increased platelet apoptosis, evaluated in terms of surface phosphatidylserine (PS), observed in both ITP groups. In summary, TPO-RA treatment increased platelet count but did not ameliorate their function and did not change plasma- and MP-associated procoagulant state of ITP patient responders to this therapy.

Platelet apoptosis and agonist-mediated activation in myelodysplastic syndromes.

Patients with myelodysplastic syndromes (MDS) have a defect in the differentiation of bone marrow multipotent progenitor cells. Thrombocytopenia in MDS patients may be due to premature megakaryocyte death, but platelet apoptotic mechanisms may also occur. This study aimed to study function and apoptotic state of platelets from MDS patients with different platelet count. Reticulated platelets, platelet activation, activated caspases and annexin-V binding were evaluated by flow cytometry. Pro-apoptotic Bax and Bak proteins were determined by western blots and plasma thrombopoietin by ELISA. Microparticle-associated procoagulant activity and thrombin generation capacity of plasma were determined by an activity kit and calibrated automated thrombography, respectively. High plasma thrombopoietin levels and low immature circulating platelet count showed a pattern of hypoplastic thrombocytopenia in MDS patients. Platelets from MDS patients showed reduced activation capacity and more apoptosis signs than controls. Patients with the lowest platelet count showed less platelet activation and the highest extent of platelet apoptosis. On this basis, patients with thrombocytopenia should suffer more haemorrhagic episodes than is actually observed. Consequently, we tested whether there were some compensatory mechanisms to counteract their expected bleeding tendency. Microparticle-associated procoagulant activity was enhanced in MDS patients with thrombocytopenia, whereas their plasma thrombin generation capacity was similar to control group. This research shows a hypoplastic thrombocytopenia that platelets from MDS patients possess an impaired ability to be stimulated and more apoptosis markers than those from healthy controls, indicating that MDS is a stem cell disorder, and then, both number and function of progeny cells, might be affected.

Podocalyxin enhances the adherence of cells to platelets.

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

Production and characterization of murine monoclonal antibodies against human podocalyxin.

Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.