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M V Berridge

Publications and source records attributed to M V Berridge.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.

The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.

Animals↗
PubMed1993

Cyclic adenosine monophosphate promotes cell survival and retards apoptosis in a factor-dependent bone marrow-derived cell line.

Hemopoietic growth factors promote cell survival, proliferation and differentiation, but whether these processes, which often occur in concert, are mediated through the same or different receptor signaling mechanisms is not known. Using the bone marrow-derived IL-3-dependent cell line, 32D, we show that dibutyryl cyclic adenosine monophosphate (dbcAMP) retards the rapid loss of viable cells seen in the absence of IL-3. This effect is shown to be concentration-dependent and detectable within 16 hours of culture and is not associated with cell differentiation. At earlier times (2 to 7 hours), when no significant changes in cell numbers were observed, dbcAMP stimulated the reduction of dimethylthiazoldiphenyl tetrazolium bromide (MTT), and this effect was indistinguishable from that seen with IL-3. In contrast, control cells deprived of growth factor showed a decline in MTT response over this period. The effect of dbcAMP in maintaining cell viability and MTT responsiveness was associated with a concentration-dependent inhibition of 3H-thymidine incorporation into DNA, and retardation of the intranucleosomal cleavage of DNA that is associated with apoptosis. These results suggest that in 32D cells, cAMP can act to promote cell survival and retard apoptosis, quite independently of cell proliferation, by stimulating the activity of mitochondrial enzymes involved in MTT reduction.

Adenosine Triphosphate↗
PubMed1992

The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C.

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).

Animals↗
PubMed1991

Echinococcus granulosus: development of an intermediate host mouse model for use in vaccination studies.

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.

Animals↗
PubMed1989

Subunit structure of the erythropoietin receptor.

Chemical cross-linking of the red blood cell hormone, erythropoietin (Epo), to its receptor on erythroid cells has revealed the presence of two proteins closely associated with Epo, but the relationship between these two proteins is controversial. Using the cross-linking reagents disuccinimidyl suberate and dithiobissuccinimidyl propionate, we show that 125I-Epo can be specifically conjugated in a complex of 224kDa using mouse fetal liver cells, bone marrow cells, and Friend virus-induced splenic erythroblasts as demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Under reducing conditions, the 224-kDa complex appeared as two Epo conjugates of 136 kDa and 119 kDa, and these bands were also observed to a variable extent in some nonreducing gels. Disulfide linking of the 136-kDa and 119-kDa bands was confirmed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under nonreducing followed by reducing conditions. With increasing time of 125I-Epo binding to Friend virus erythroblasts in the presence of sodium azide to inhibit receptor internalization, the 136-kDa and 119-kDa bands seen under reducing conditions increased markedly in intensity, whereas the 224-kDa band seen under nonreducing conditions declined. These results suggest that the 224-kDa Epo conjugate is inefficiently solubilized under nonreducing conditions following prolonged periods of Epo binding. A single class of saturable, high affinity receptors for Epo on each of the cell types tested is demonstrated. It is concluded that the two disulfide-linked Epo-binding proteins which can be independently cross-linked to Epo form a single ligand binding site.

Animals↗
PubMed1989

Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes.

Considerable experimental and clinical evidence suggests a relationship between erythropoiesis and thrombopoiesis. This is supported by observations that erythropoietin (Epo), the primary regulator of erythropoiesis, can affect platelet production when injected into animals. In this study we provide experimental evidence for a direct effect of Epo on thrombopoiesis by demonstrating that 125I-labeled recombinant human Epo binds to rat and mouse bone marrow megakaryocytes. Thus, autoradiographic analysis using cold competition to measure specific binding has been used to demonstrate that Epo binding to megakaryocytes increases with megakaryocyte maturation. When corrected for cell size, Epo binding sites per unit surface area increase from Stage I megakaryoblasts to Stage II megakaryocytes, and then remain approximately constant throughout further megakaryocyte maturation. Receptor density on megakaryocytes is similar to that on pronormoblasts in the rat, and in mice is 60% that on pronormoblasts. No binding of Epo to platelets or to naked megakaryocyte nuclei was detected. Equilibrium binding studies with partially purified rat megakaryocytes (20%-40% pure), where megakaryocytes are the only significant Epo binding cell population, showed a single class of saturable, high-affinity binding sites present on average at 6500 binding sites per megakaryocyte with a KD of 287 pM. Binding of [125I]Epo to rat megakaryocytes was inhibited with an antiserum against murine erythroblasts. These results suggest that the effects of Epo on thrombopoiesis may be directly mediated through specific, high-affinity binding sites for Epo on the surface of maturing megakaryocytes.

Animals↗
PubMed1989

Monoclonal antibodies that bind to hemopoietic stem cells: characterization and immunohistochemical localization of cells expressing gp50-65.

The cell surface of hemopoietic stem cells has been shown to express several antigens in common with more mature hemopoietic cells. One set of stem cell antigens is defined by a group of three monoclonal antibodies (13C6, 1C10, and 1A9), selected on the basis of binding to subpopulations of spleen colony-forming stem cells (CFU-S). These antibodies are shown to recognize cell surface glycoproteins of 50-65 kd (gp50-65) that occur widely on hemopoietic cells. Each cell type investigated shows a distinctive pattern of expression of these glycoproteins. To further investigate the presence of gp50-65 on stem cells, low-density bone marrow cells were labeled with 1C10 and sorted according to fluorescence intensity. Most (73%) of the stem cells (CFU-S10) were recovered in the two most highly fluorescent fractions containing 2.6% of starting marrow cells. Immunohistochemistry of frozen sections of normal spleen and spleens during repopulation after lethal irradiation and bone marrow transplantation showed that the most strongly 1C10-labeled cells occurred under the splenic capsule and along trabeculae. Although many of these cells were also alpha-naphthylacetate esterase positive and Mac-1 positive, indicating cells of the monocyte-macrophage lineage, a distinctive population of singular cells were stained with 1C10 alone. These cells were negative for surface Ig and closely corresponded with a small population of cells in a similar location that were doubly labeled with 1C10 and anti-Thy-1. These results show that stem cells express high levels of gp50-65 and suggest that stem cells can be identified by immunohistochemical methods using dual labeling procedures.

Animals↗
PubMed1988

Expression and modulation of specific, high affinity binding sites for erythropoietin on the human erythroleukemic cell line K562.

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor-potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.

Culture Media↗
PubMed1988

Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL.

The principal growth factor involved in the regulation of erythropoiesis, erythropoietin (Epo), is currently under clinical trial for the treatment of anemia. Despite the advanced state of these trials, little is known about the nature and distribution of the receptor for Epo on human hemopoietic cells or about the cellular mechanisms of signal transduction. In the present study 125I-labeled recombinant human Epo has been used to demonstrate expression of saturable, high affinity binding sites for Epo on density-fractionated human bone marrow cells and on the human erythroleukemic cell line, HEL. Binding was reversible and proportional to cell number, and HEL cells were shown to express on average 34 receptors per cell (range 30-35) with an affinity of 293 pM (range 275-300 pM) at 37 degrees C in the presence of sodium azide to block receptor internalization. Autoradiographic analysis of Epo binding to human bone marrow cells showed that specific binding, measured as the difference in grain counts between total binding and binding in the presence of excess unlabeled Epo, was greatest to pronormoblasts and declined during erythroid cell maturation to undetectable levels on nucleated red cells. Autoradiography also revealed significant Epo binding to marrow megakaryocytes, which comprise less than 1% of nonerythroid cells. In contrast to erythroid cells, Epo binding to megakaryocytes increased with cell maturation, with stage IV megakaryocytes exhibiting the highest specific binding. Grain density per surface area however, remained constant during megakaryocyte maturation and was approximately 25% that on pronormoblasts.

Autoradiography↗
PubMed1988

Down-modulation of high-affinity receptors for erythropoietin on murine erythroblasts by interleukin 3.

Erythropoiesis is regulated by the glycoprotein hormone erythropoietin (Epo) and by several other factors including interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor. The possibility that IL-3 and GM-CSF may act by modulating Epo receptor expression was investigated using erythroblasts purified from the spleens of phenylhydrazine-treated mice. AT 37 degrees C, in the presence of sodium azide to inhibit receptor internalization. 125I-labeled human recombinant Epo bound to a single class of high-affinity receptors on splenic erythroblasts (450 sites/cell, Kd = 700 pM). Autoradiographic studies indicated that 94% of specifically bound Epo was associated with erythroblasts, decreased Epo binding being observed with increasing erythroid cell maturation. Whereas recombinant mouse IL-3 and GM-CSF did not compete with 125I-Epo for binding to the Epo receptor, preincubation of cells with IL-3 resulted in a concentration-dependent loss of 125I-Epo binding without altering the affinity of residual receptors for Epo. Complete loss of Epo receptors was effected within 2 h at IL-3 concentrations above 2500 U/ml. Preincubation with recombinant mouse GM-CSF had no effect on binding, even at 100,000 U/ml. In comparison, preincubation of cells with Epo (50 U/ml) caused complete loss of 125I-Epo binding within 30-60 min, an effect not explained by receptor saturation with unlabeled Epo. Thus, in addition to trans-down-modulating growth factor receptors of the granulocyte-macrophage series, IL-3 also trans-down-modulates a growth factor receptor of the erythroid lineage.

Animals↗
PubMed1988

Effects of recombinant human erythropoietin on megakaryocytes and on platelet production in the rat.

The contention that erythropoietin (Epo) affects platelet production was investigated in the rat with recombinant human Epo (rHuEpo). In normal rats, Epo caused a dose-dependent increase in both reticulocyte and platelet numbers, the reticulocyte response preceding that of platelets. Withdrawal of Epo resulted in reticulocytes and platelets returning to control levels. [75Se]-selenomethionine incorporation into platelets was also enhanced in response to Epo. Chronic daily administration of rHuEpo resulted in steady state erythrocyte levels after 12 to 14 days, which were elevated 20% above controls. Attainment of this steady state was associated with both reticulocytes and platelets returning to control levels despite continued administration of Epo, an effect not associated with a change in the half-life of circulating Epo. In polycythemic rats a platelet response was observed before an effect on reticulocytes. Erythropoietin caused a 2.4-fold increase in the frequency of small acetylcholinesterase-positive cells within 24 hours, and increased the mean megakaryocyte diameter within 48 hours. Furthermore, the [3H]-thymidine labeling index of megakaryocytes from rats treated for 24 hours with rHuEpo was increased for all stages of megakaryocyte maturation. These results support the proposal of an effect of Epo on rat megakaryocytes causing increased platelet production.

Animals↗
PubMed1987

Induction of B-lymphocyte antigens on the chronic myeloid leukemic cell line K562 using sodium butyrate.

Chronic myeloid leukemia (CML) is a disorder arising from a defect in the hemopoietic stem cell. Consequently, the malignant clone can involve all cells within the stem cell's capacity for differentiation, including erythrocytes, granulocytes, monocytes, megakaryocytes, and lymphocytes. Similarly, the K562 cell line, which was derived from a patient with CML, has been shown to be capable of differentiation towards erythrocytes, granulocytes, monocytes, and megakaryocytes, and in this respect may represent a model of the hemopoietic stem cell. However, although K562 shows properties of a myeloid stem cell, no lymphocyte-specific features or differentiation have yet been described. In the present study, K562 cells have been induced to differentiate by culture in the presence of sodium butyrate. The direction and extent of induced differentiation over 12 days were determined with a panel of monoclonal antibodies and with cytochemical stains. This treatment consistently induced expression of pre-B-cell markers, including B-lymphocyte-specific B4 and B1, and of the common acute lymphoblastic leukemia antigen (CALLA), recognized by J5. In addition to the increased expression of B-lymphocyte markers, butyrate induction of K562 resulted in a decrease in granulocyte markers, increases in certain monocyte and platelet markers, and an increase in beta 2 microglobulin expression. Butyrate-induced expression of B-lymphocyte markers was not observed with the myelomonocytic cell line U937. The expression of B-lymphocyte-specific antigens on butyrate-induced K562 may result from the relaxed control of gene expression, but alternatively these observations may indicate the lymphoid-myeloid stem cell nature of K562.

Antibodies, Monoclonal↗
PubMed1987

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement.

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet-dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.

Animals↗
PubMed1986

Expression of antigens of the platelet glycoprotein IIb/IIIa complex on human hematopoietic stem cells.

Several cell surface and cytoplasmic markers specific for the megakaryocyte-platelet lineage have been described. However, as yet, none of these has been shown to be expressed on cells earlier than the committed megakaryocyte progenitor, CFU-Meg. The present study was aimed at determining whether platelet lineage antigens could be detected on human pluripotential stem cells. Rabbit antiserum against human platelets (APS) was extensively absorbed with erythrocytes and either platelets, neutrophils, monocytes, or cells of the monocytic cell line U937. The anti-stem cell antibodies in each absorbed antiserum were determined using a complement-dependent cytotoxic assay for the pluripotential stem cell CFU-mix. Platelets alone removed anti-stem cell antibodies from APS. Absorption of APS with platelets from a patient with Glanzmann's thrombasthenia failed to remove the anti-stem cell activity, providing evidence for involvement of the platelet glycoprotein IIb/IIIa complex. Antiserum against purified glycoprotein IIb and against glycoprotein IIIa also recognized stem cells, resulting in reduced formation of mixed colonies. Absorption of these antisera with normal platelets removed the anti-stem cell activity, indicating that both IIb and IIIa are represented on stem cells. Hence, cell surface antigens specific for the stem cell-megakaryocyte-platelet pathway are expressed on the platelet glycoprotein IIb/IIIa complex.

Antibody Specificity↗
PubMed1986

Expression of the leukocyte functional molecule (LFA-1) on mouse platelets.

Platelet involvement in adhesion, hemostasis, and immune adherence is mediated by functionally associated cell surface molecules. Platelets are also involved in cytolytic reactions, but little is known about the mechanisms or biologic significance of these processes. To further investigate cell surface molecules concerned with platelet function, antisera against mouse platelets, thymocytes, and macrophages and monoclonal antibodies against Mac-1 (complement receptor type 3) and leukocyte function-associated glycoprotein type 1 (LFA-1) were used to demonstrate LFA-1--like molecules on mouse platelets. The alpha subunits of platelet and thymocyte LFA-1 showed identical electrophoretic mobility but differed significantly from the alpha subunit of macrophage Mac-1. Peptide mapping demonstrated the identity of the beta subunits of these three molecules but showed that the alpha subunit of Mac-1 was distinct from the alpha subunits of platelet and thymocyte LFA-1. Platelet LFA-1, as demonstrated by surface iodination with lactoperoxidase and by labeling sialic acid residues with sodium borohydride, was not a major component of the platelet membrane. The functional significance of LFA-1 on mouse platelets has yet to be demonstrated, monoclonal antibodies against LFA-1 having little effect on adenosine diphosphate-induced platelet aggregation and immune adherence. In contrast, although Mac-1 could not be demonstrated on mouse platelets in immunoprecipitation studies, its presence was clearly demonstrated by low levels of antibody binding in enzyme-linked immunosorbent assays and by the ability of M1/70 monoclonal antibody to inhibit platelet immune adherence. Human platelets, which are inactive in immune adherence assays, are shown to lack LFA-1 and Mac-1.

Animals↗
PubMed1985

Cell-lineage antigens of the stem cell-megakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex.

The stem cell-platelet lineage is uniquely defined by platelet cell-lineage antigens. These antigens are present on all stem cells measured by the spleen colony assay and become restricted to the platelet cell lineage as differentiation proceeds. In this study, anti-platelet serum (APS) has been used to identify cells in the bone marrow that express platelet cell-lineage antigens and to identify platelet cell surface molecules expressing these antigens. Anti-platelet IgG extensively absorbed with brain, thymus, and peritoneal cells bound selectively to stem cells, megakaryocyte progenitor cells (Mk-CFC), and megakaryocytes in CBA mouse bone marrow and to blood platelets. No other hemopoietic cell type, tissue, cell line, or tumor cell bound significant amounts of antibody against platelet cell-lineage antigens as determined by ability to absorb the anti-stem cell activity in APS. Studies with lactoperoxidase-labeled platelets showed that two major iodinated proteins of Mr = 114,000 and 138,000 were immunoprecipitated with APS and with antiserum that had been extensively absorbed. These proteins correspond to the platelet IIb-IIIa glycoprotein complex, which is known to express receptors for collagen and fibrinogen, molecules known to influence hemopoietic cell proliferation and tumor cell growth. A panel of six monoclonal antibodies against human IIb-IIIa inhibited spleen colony formation by 17% to 100%, J15 and A5.15 also being cytotoxic for granulocyte-macrophage progenitor cells and Mk-CFC. Other platelet monoclonal antibodies did not inhibit spleen colony formation. Although APS inhibited fibrinogen binding to platelets and platelet aggregation, these activities were greatly reduced with absorbed antiserum. Furthermore, fibrinogen treatment of bone marrow did not block the anti-stem cell activity in APS. Thus the evidence is consistent with expression of platelet cell-lineage antigens on the platelet IIb-IIIa glycoprotein complex at a site removed from the fibrinogen binding site.

Animals↗