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Biomedical subjects

J Lawler

Publications and source records attributed to J Lawler.

At least 145 records · Page 8Linked to original sources
PubMed1986

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Amino Acid Sequence↗
PubMed1986

Thrombospondin in essential thrombocythemia.

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000-dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.

Antibodies, Monoclonal↗
PubMed1985

Hemin-mediated dissociation of erythrocyte membrane skeletal proteins.

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

The structure of human platelet thrombospondin.

Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1985

Sp alpha I/65: a new variant of the alpha subunit of spectrin in hereditary elliptocytosis.

Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two-dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Adult↗
PubMed1985

Primary human marrow cultures for erythroid bursts in a serum-substituted system.

To determine environmental requirements for erythroid burst formation in primary culture, we added human bone marrow cells to serum-depleted methylcellulose, agar, or fibrin clot cultures. Optimal BFU-E proliferation was present in cultures prepared with Iscove's modified Dulbecco medium, 248 micrograms/ml transferrin, 1.63 micrograms/ml ferric chloride, 117 micrograms/ml bovine serum albumin, and each of seven preparations of erythropoietin. Burst number was comparable to that in serum containing culture. Reducing sodium dodecyl sulfate electrophoresis of commercial albumin preparations showed them to contain abundant lipoproteins. Results of experiments with human plasma albumin found to be greater than 98% pure by one- and two-dimensional gel electrophoresis and delipidated albumin indicate that an albumin source is needed for burst formation to occur. Together with albumin, exogenous phosphatidylcholine but not phosphatidylserine augmented burst number. Bursts routinely appeared in serum-depleted culture without added burst-promoting activity (BPA). However, leukocyte-conditioned medium (LCM) and its high-speed supernatant and pellet fractions enhanced burst formation. Antimembrane IgG capable of neutralizing BPA reduced burst number to a level below that achieved in LCM-depleted culture, suggesting that endogenous BPA was inactivated. We conclude that human marrow BFU-E proliferation requires iron-saturated transferrin, albumin, and erythropoietin. Exogenous BPA and phospholipids enhance but are not essential for burst formation to proceed in primary culture.

Blood↗
PubMed1984

Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding.

Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix.

Binding, Competitive↗
PubMed1984

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Cooperative binding of calcium to thrombospondin. The effect of calcium on the circular dichroism and limited tryptic digestion of thrombospondin.

Removal of calcium from thrombospondin with EDTA results in a decrease in the intensity of the negative CD peak between 200 and 250 nm. Quantitative analysis of the CD spectrum of thrombospondin indicates that thrombospondin contains approximately 11% alpha-helix, 43% beta-sheet, and 46% random coil in the presence of calcium and that a small change in secondary structure may occur upon removal of calcium with EDTA. When the change in the CD at 220 nm is measured as a function of calcium concentration, a sigmoidal curve with a transition midpoint of 120 microM is obtained, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed a Hill coefficient of 12.3. Calcium was also found to affect the peptide pattern produced by limited tryptic digestion of thrombospondin, with some portions of the molecule being resistant to trypsin in the presence of calcium. When the change in quantity of a 65,000-dalton tryptic fragment was measured as a function of calcium concentration, a sigmoidal curve was again obtained. The midpoint of this transition is achieved at a free calcium concentration of 45 microM at 0 degrees C and 103 microM at 25 degrees C. These data indicate that thrombospondin contains at least 12 binding sites for calcium and that cooperative interactions between sites are associated with a conformational change in the thrombospondin molecule.

Blood Platelets↗
PubMed1983

Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro.

To investigate serum requirements for optimal erythropoiesis in vitro, we studied the response of erythroid progenitor cell proliferation in culture to platelet-derived growth factor (PDGF). Human bone marrow cells cultured with platelet-poor plasma-derived serum (PDS) form fewer erythroid colonies than do cells cultured with human whole blood serum or fetal calf serum (P less than 0.05). Treatment of washed platelets with thrombin releases a low molecular weight (less than 100,000) factor that enhances colony growth. This secreted factor appears to be PDGF, based upon the ability of partially purified and electrophoretically pure PDGF to restore colony-forming capacity of PDS-containing cultures to 70-96% of the level found in control cultures with whole blood serum or fetal calf serum. Enhancement of colony growth by PDGF was noted only in marrow cultures supplemented with erythropoietin and PDS. Presence of bioactive erythropoietin in PDGF preparations was excluded by assay in hypertransfused, polycythemic mice, and in fasted rats. Although PDGF stimulates erythroid burst formation in marrow cultures containing optimal concentrations of burst-promoting activity (BPA), it does not influence proliferation of circulating erythroid bursts, regardless of BPA concentration added to culture. We conclude that PDGF is a serum determinant of optimal erythroid progenitor cell proliferation in marrow culture. The activity of PDGF is distinct from that of the apparent erythroid specific growth factors erythropoietin and BPA.

Animals↗
PubMed1983

Molecular heterogeneity of hereditary pyropoikilocytosis: identification of a second variant of the spectrin alpha-subunit.

In hereditary pyropoikilocytosis (HPP), the red cell membrane skeletons exhibit a mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To determine the underlying molecular defect, we have subjected HPP spectrin to limited tryptic digestion, followed by one- and two-dimensional separations of the peptides. Two of the HPP kindreds exhibited a marked decrease in 80,000-dalton peptide (previously identified as the spectrin dimer-dimer contact domain of the alpha-subunit) and a concomitant increase of the 74,000-dalton polypeptide (presumably derived from the 80,000-dalton domain) and a decrease in a 22,000-dalton polypeptide. We now report tryptic digests of two other HPP kindred that are characterized by a decrease or complete absence of the 80,000-dalton tryptic fragment, with a concomitant increase in fragments at 46,000 and 17,000 daltons. The 46,000-dalton fragment separated into multiple spots on isoelectric focusing, ranging in isoelectric point from 5.25 to 5.35, and the 17,000-dalton fragment focused to a single spot at 5.4. Minor fragments at 56,000 and 22,000 daltons were also decreased, while a 38,000-dalton fragment increased. Limited tryptic digestion of the separated alpha- and beta-subunits revealed that the 74,000-dalton fragment in the first group of patients and the 46,000-dalton fragment in the second group of patients were derived from the alpha-subunit. Both subtypes exhibited a similar defect of spectrin self-association, with 30%-38% of spectrin dimers in O degrees C extracts. The results indicate that at least two distinct forms of structurally defective spectrin may give rise to the clinical presentation of HPP.

Adolescent↗
PubMed1982

Molecular defect of spectrin in hereditary pyropoikilocytosis. Alterations in the trypsin-resistant domain involved in spectrin self-association.

In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0 degrees C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.

Electrophoresis, Polyacrylamide Gel↗