Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell surface antigen expression during proliferation and differentiation of human erythroid progenitors was examined using a combination of sequential micromanipulations of paired daughter cells derived from erythroid burst-forming units (BFU-E) and immuno-staining with a panel of monoclonal antibodies. Single hematopoietic progenitors were identified in methylcellulose cultures containing human cord blood mononuclear cells and micromanipulated individually to secondary culture. Paired daughter cells, granddaughter cells, and subsequent generations, whose counterparts produced erythroid bursts, were stained with various cytochemical and immuno-alkaline phosphatase stainings. Most paired daughter cells of BFU-E immunostained positively with anti-platelet glycoprotein(GP) IIb, antiplatelet GPIIb/IIIa, anti-HLA-DR, and antitransferrin receptor antibodies. Acid phosphatase staining was also positive. Neither CD34 nor CD33 antigens were identified on the cells. CD36 and blood group A antigens were first identified on cells from aggregates containing 32 to 64 cells after 4 days of secondary culture and preceded the expression of glycophorin A and hemoglobin alpha. These results indicate that various cell surface antigens were sequentially expressed during the proliferation and differentiation of erythroid progenitors, and that our procedure may be useful for clarifying the morphologic and immunologic properties of hematopoietic stem cells.
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PMID:Changes in cell surface antigen expressions during proliferation and differentiation of human erythroid progenitors. 163 21

The presence of proteins antigenically related to the GPIIb/IIIa complex expressed on platelets have been investigated on tissue macrophages recovered by bronchoalveolar lavage (lung alveolar macrophages, LAM) or peritoneal lavage (peritoneal macrophages, PM) as well as on monocytes. Polyclonal antibodies (pab) directed against human platelet GPIIb/IIIa and the vitronectin receptor (VnR), and mouse monoclonal antibodies (mab) against human GPIIb, GPIIIa or the GPIIb/IIIa-complex were used. Triton X-100 extracts of bronchoalveolar cells (BAC) (containing 94% LAM) and the ultrasedimentable fraction of cell-free bronchoalveolar lavage (US) reacted with the polyclonal antibodies against the GPIIb/IIIa-complex and the VnR, but only with one (P4) of the mabs. Cell microscopy after immunogold labelling and alkaline phosphatase immunostaining, as well as immunofluorescence using the P4 mab and the polyclonal anti-GPIIb/IIIa clearly demonstrated positive membrane staining of LAM, PM and monocytes. Both BAC and US extracts gave rise to immunoprecipitates in crossed and rocket immunoelectrophoresis using anti-GPIIb/IIIa and anti-VnR. Our data indicate that monocytes and their monocyte-derived tissue counterparts constitutively express GPIIb/IIIa-like antigen(s) on their membrane. The presence of such antigen(s) on tissue macrophages makes it unlikely that platelet contamination is responsible for these findings.
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PMID:Characterization of cytoadhesion molecules on human monocytes and tissue macrophages. 170 68

In contrast to the situation in chronic idiopathic thrombocytopenic purpura (ITP) it has not been known whether acute childhood ITP has an autoimmune aetiology and to what extent autoantibodies directed to platelet autoantigens appear during the transient course of this disease. In the present study of 21 children with acute ITP an immunoblot technique was applied to detect serum antibodies to electrophoretically (SDS-PAGE) separated normal donor platelet membrane proteins. Platelet antigen binding antibodies detected by alkaline phosphatase conjugated protein A or anti-IgM antibody were observed in 13 out of 21 (62%) patients while controls were negative. In nine children antibodies were directed to antigens localized on the three major membrane glycoproteins. GPIb (four), GPIIb (one) and GPIIIa (five). Antibodies to a 250 kDa antigen were noted in one case and to smaller 25-52 kDa proteins in 12 patients. Four patients had IgG as well as IgM platelet antibodies while in nine only IgM was found. The reactions were eliminated after absorptions of sera with fresh platelets. In all of three tested patients the glycoprotein antigen specific antibodies could be detected in acid eluates prepared from the absorbing platelets. The presence of antibodies, predominantly IgM, to platelet surface antigens in a majority of the children with acute ITP, may suggest an autoimmune process, albeit transitory, similar to that in chronic ITP.
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PMID:IgG and IgM antibodies to platelet membrane glycoprotein antigens in acute childhood idiopathic thrombocytopenic purpura. 280 83

A megakaryoblastic cell line, designated MEG-01, was established from the bone marrow of a patient with blast crisis of Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia. MEG-01 cells grew in single-cell suspension with a doubling time of 36 to 48 hours. Under the usual culture conditions, approximately half of the cells adhered to the culture flask with extention of pseudopods. MEG-01 cells were positive for the periodic acid-Schiff reaction, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for myeloperoxidase, alpha-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and alkaline phosphatase. Ultrastructural platelet peroxidase was positive in MEG-01 cells. Cytoplasmic factor VIII (FVIII)-related antigen was weakly positive in larger MEG-01 cells by both an indirect immunofluorescent technique with monoclonal antibodies and a direct immunoperoxidase technique using horseradish peroxidase-conjugated conventional rabbit anti-human FVIII antibody. Platelet glycoprotein (GP) IIb/IIIa antigen was uniformly demonstrated on the surface of MEG-01 cells by both indirect immunofluorescent and immunoperoxidase techniques using antiplatelet GP IIb/IIIa monoclonal antibodies; platelet GP lb antigen was demonstrated only in the cytoplasm of larger MEG-01 cells. MEG-01 cells possessed no markers for B or T lymphocytes or for myeloid cells. Chromosome analysis of this cell line revealed a human male hyperdiploid karyotype with a modal chromosome number of 56 to 58. The Ph1 chromosome was observed in all karyotypes analyzed. This novel human megakaryoblastic cell line may provide a useful model for the study of human megakaryopoiesis and of the biosynthetic mechanisms of proteins unique to megakaryocytic lineage.
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PMID:Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. 299 11

The immunophenotype of peripheral blood blast cells from 14 patients in the chronic phase of chronic myeloid leukemia (CML) was studied using a panel of monoclonal antibodies (McAb) directed against megakaryocytic, granulomonocytic, erythroid and lymphoid antigenic determinants. The blast cells were enriched by a simple bovine serum albumin (BSA) density-cut separation and cooled in liquid nitrogen. The study was done using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique on the thawed blast cells. A consistent pattern of reactivity with McAb was found in all patients, showing that blast cells were heterogeneous. A minor component of the blast cells react with platelet antibodies, most of them being labelled with anti-GPIIb-IIIa McAb. Anti-GPIb and Von Willebrand factor McAb detected 4 times fewer megakaryocytic blast cells, suggesting that these cells are located very early in the differentiation scheme. Two major blast cell compartments were labelled with early myelomonocytic (anti-CD13: MY7) and early erythroid (anti-CD36: FA6-152) McAb. The CD34 (My10) and DR antigens which are expressed by immature blast cells and myeloid progenitors of human bone marrow (BM) were present on more than 50% of the CML blast cells. Thus, the blast cells of chronic phase CML patients, showed the same cellular diversity as the increased progenitor cell compartment observed in this disease, and their differentiation stages seemed to be very closely related.
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PMID:Immunophenotype of blast cells in chronic myeloid leukemia. 319 45

Human megakaryocyte colony formation was observed in serum-free agar cultures with a medium containing deionized bovine serum albumin, iron-saturated transferrin, 2-mercaptoethanol, and recombinant human interleukin 3 (IL-3). Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet glycoprotein IIb/IIIa. Colony numbers reached a peak at day 14, with a mean of 47 megakaryocyte colonies (range 23-104) per 2 x 10(5) bone marrow mononuclear cells. Recombinant human IL-3 stimulated the growth of megakaryocyte progenitors. Similar results were obtained using nonadherent, T-cell-depleted mononuclear cells. These findings suggest that IL-3 stimulates the growth of megakaryocyte progenitors directly without activation of accessory cells, or the requirement of factors present in the serum or plasma. This serum-free culture system may be useful for studying the effects of purified natural and recombinant biological regulators on human megakaryocyte progenitors.
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PMID:Clonal growth of human megakaryocyte progenitors in serum-free cultures: effect of recombinant human interleukin 3. 326 26

A method is detailed for the solubilization of human platelets using a dialyzable detergent, decanoyl-N-methylglucamide (Mega-10). At a detergent/protein ratio of 1:12, the efficiency of solubilization was 27%. This platelet lysate (PLy) was then bound to nitrocellulose (NC) discs to assay retention of the native immunological functions of the platelet membrane antigens. Using alkaline phosphatase-coupled anti-IgG, the major platelet membrane glycoproteins GPIb, GPIIb, and GPIIb/IIIa were detectable with as little as 20 ng of monoclonal antibody. Antisera to the class I histocompatibility antigens HLA-A1, B7, B8, the PlA1 allodeterminant, and serum from multiply transfused, alloimmunized patients were reactive even after 100 days storage of the discs at 4 degrees C, and with as little as 1.0 micrograms of NC-bound PLy. The binding of the same antisera to intact, immobilized platelets as well as specific complement-mediated lymphocytotoxicity was also inhibited by PLy. PLy from HLA-A3- or B44-positive donors, however, did not inhibit cytotoxicity of lymphocytes expressing either antigen using several different antisera. Our results indicate that Mega-10 is an excellent solubilizing agent for the immunological study of platelet membranes. The fact that clinically relevant platelet membrane antigens are preserved, immunologically reactive, and stable over long periods of storage, makes this assay amenable to a routine crossmatching procedure for platelet transfusions.
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PMID:An enzyme-linked immunosorbent assay for the detection of platelet antibodies using detergent-solubilized platelets immobilized on nitrocellulose discs. 368 Sep 64

A synthetic gene encoding ornatin E (OrnE), a 50-amino acid glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist and platelet aggregation inhibitor isolated from the leech Placobdella ornata, was designed, constructed, and expressed in Escherichia coli. The OrnE gene was fused to the heat stable enterotoxin stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. This construction directed secretion of recombinant ornatin E (rOrnE) into the extracellular medium at levels of 7-19 mg/liter. The protein was purified to apparent homogeneity in 18-38% yields by ammonium sulfate precipitation, Q-Sepharose and S-Sepharose ion exchange chromatography, and reverse-phase HPLC. Purified rOrnE was found to be indistinguishable from leech-derived OrnE as judged by amino acid composition, N-terminal sequencing, mass spectroscopic analysis, and HPLC coelution. In addition, rOrnE exhibits similar activity in fibrinogen/GP IIb-IIIa ELISA and platelet aggregation assays. Purified rOrnE possesses three disulfide bonds, the reduction and carboxymethylation of which results in a ca. 60-fold reduction in biological activity. A misfolded variant of rOrnE was characterized and shown to have a ca. 6-fold reduction in activity. These data demonstrate that the native disulfide bonds are required for the optimal GP IIb-IIIa antagonist activity of the ornatins.
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PMID:Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist. 837 97

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and > 200-fold, respectively. The conservative D51E mutant was still > 100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa.
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PMID:Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. 845 99

In the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of microvesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.
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PMID:Microvesicles bind soluble fibrinogen, adhere to immobilized fibrinogen and coaggregate with platelets. 949 96


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