Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.

Determination of thrombin-hirudin complex in plasma with an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of the thrombin-hirudin complex (TH) in plasma. The test is based on the sandwich principle and uses appropriate antibodies which selectively bind the corresponding moieties of the complex. The assay was calibrated by adding performed TH to normal human citrated plasma. The detection limit of the assay was 1.2 ng/ml. Mean coefficients of variation of 6.0% (intra-assay) and 6.4% (inter-assay) were found. The presence of TH was demonstrated in normal human plasma that was spiked with hirudin and subsequently activated in vitro by calcium-thromboplastin to generate thrombin. This complex was also found in plasma samples from pigs which had been treated with hirudin during experimentally induced septicaemia.

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

A hirudin catching ELISA for quantitating the anticoagulant in biological fluids.

A catching ELISA has been developed that permits a quantitation of the anticoagulant hirudin in buffer, urine and plasma. In plasma hirudin can be determined in concentrations ranging from 0.2 to 25 ng/ml (2.4 X 10(-3) to 0.3 AT-U/ml), in urine between 0.8 and 200 ng/ml (0.01 and 2.4 AT-U/ml). The enzyme immunoassay allows a rapid, sensitive and reproducible quantitation of hirudin, and can thus be used to assess the pharmacokinetics of the anticoagulant in patients after parenteral and/or topic administration.

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G.

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.

Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA.

A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.

Eglin-c, a polypeptide derived from the medicinal leech, prevents human neutrophil elastase-induced emphysema and bronchial secretory cell metaplasia in the hamster.

Eglin-c (Eg-c), a polypeptide with a molecular mass of 8,100 daltons, was purified from the medicinal leech Hirudo medicinalis. The Eg-c was tritiated by reductive methylation for in vitro studies. Incubation of 2.1 X 10(-10) moles of human neutrophil elastase (HNE) with 3H-elastin in the presence of 8.2 X 10(-10) moles of 3H-Eg-c inhibited 98.7% of the elastolytic activity of the enzyme. Using Sephadex G 100 chromatography and 1.7 moles of 3H-Eg-c per mole of HNE, a 34,000-dalton complex (3H-Eg-c-HNE) was observed. The stability of the complex formed between 3H-Eg-c and HNE that had been inactivated with succinyl-ala2-pro-val CH2Cl was much less than that of the 3H-Eg-c-HNE complex. In vivo studies were carried out in weight-matched groups of anesthetized golden Syrian hamsters given 100, 300, 500, or 2,000 micrograms of Eg-c in 0.5 ml saline intratracheally 1 h before 300 micrograms HNE was administered intratracheally. Control animals received saline followed by HNE or 2 doses of saline 1 h apart. Eight weeks later, lung statics and dynamics were measured in anesthetized animals, followed by histologic study of lung parenchyma and the mucosa of the large intrapulmonary airways. There were no deaths, and final mean body weights were similar in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The complete covalent structure of hirudin. Localization of the disulfide bonds.

Hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis, is a single-chain polypeptide (65 amino-acid residues) linked by three disulfide bridges. Localization of the three disulfide bonds could be assigned on the basis of the structures of cystine peptides derived by high performance liquid chromatography separations of thermolysinolytic digest of native hirudin. By characterization of the nine major fragments by amino-acid analysis, N-terminal amino-acid determination and sequence analysis, the following disulfide linkages were identified: Cys6-Cys14, Cys16-Cys28 and Cys22-Cys39. Due to the lack of any closer sequence homology and topological structural homology to other serine proteinase inhibitor proteins, hirudin seems to be unique in its primary structure and hence designates an unknown inhibitor family.

Elastase-cathepsin G inhibitors eglin b and eglin c differ by a single Tyr----His substitution. A micro-method for the identification of amino-acid substitution.

The structures of eglin b and eglin c, both potent inhibitors of human neutral granulocytic proteinase elastase and cathepsin G, were compared by micro amino-acid analysis and peptide mapping techniques. Eglin b and eglin c differ by one amino-acid substitution in the middle of the polypeptide chain. Tyrosine residue at position 35 of eglin c was substituted by histidine in eglin b. This amino-acid substitution requires one base exchange (U----C) at the DNA level and apparently does not affect the reactive site of eglins. Though without disulfide linkages, eglins are very rigid molecules and can be effectively digested by trypsin only after rigorous acid incubation.

Eglin c, a pharmacologically active elastase inhibitor.

Eglin c is an elastase/cathepsin G inhibitor from leech Hirudo medicinalis. The gene for this 70 aminoacid peptide was synthesized chemically, cloned and expressed by E. coli. Here we report biochemical and pharmacological studies. The rate of complex formation between Eglin c and human leukocyte elastase (HLE) or human cathepsin G (H. Cat. G) was determined and compared to those of a number of other proteinase/proteinase-inhibitor interactions (alpha 1 PI and alpha 2M). The association rate constants of Eglin c with the leukocyte enzymes are of the same order of magnitude as those with the naturally occurring inhibitors alpha 1 PI and alpha 2M. The association rate constant of Eglin c (extracted from leech) and Eglin c (biotechnology product) with HLE was found to be identical. The equilibrium constants Ki of the Eglin c/HLE and the Eglin c/H. Cat. G interactions are in the order of 10(-10) M. In an experiment with the hamster emphysema model, 0.5 mg or 2 mg of Eglin c applied intratracheally one hour before an HLE-insult completely protected the animals against emphysema and no signs of toxicity due to Eglin c were observed.

Inhibition of cells in culture by polyamines does not depend on the presence of ruminant serum.

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the inhibitory dose-response relationships of spermine and spermidine in the presence of selected sera have been examined. In contrast to previous studies which used [3H]thymidine uptake as an index of proliferation, in vitro inhibition by polyamines was shown to require neither foetal calf serum (FCS) nor the addition of any exogenous polyamine oxidase. Cells grown in the absence of FCS were between 5-50% as sensitive to polyamines as in its presence. By using specific inhibitors of polyamine oxidase, it was shown that polyamine-elicited mitotic inhibition in the absence of FCS was still dependent on a polyamine oxidase, and evidence is presented to show that the source of the enzyme is the cells themselves.

Elucidation of the different effects of polyamines and other naturally-occurring inhibitors of cell proliferation (chalones) on T-lymphocyte and granulocyte colony growth in vitro.

Using T-lymphocyte and granulocyte colony assays with truly proliferating cells the effects of the polyamine spermine and of other naturally-occurring inhibitors of cell proliferation have been differentiated. It has been confirmed that spermine, in the presence of fetal calf serum, is a potent inhibitor of cell proliferation. This inhibition could be reversed by the addition of either 3-hydroxybenzyl-oxyamine or 4-bromo-3-hydroxybenzyl-oxyamine, both of which are inhibitors of the polyamine oxidase. In comparison, fractions isolated from calf thymus were shown to inhibit lymphocyte, but not granulocyte colony growth, indicating their tissue specificity and lymphocyte chalone activity. Further this inhibition was not reversed by polyamine oxidase inhibitors demonstrating that polyamines were not the inhibitory principles in this preparation.

Lymphocyte chalone from calf thymus: problems of large scale extraction and assay.

Large scale extraction and assay of the lymphocyte chalone (LC), the natural lymphocyte proliferation inhibitor, require optimized, reproducible and standardizable methods. To this end we compared the effects on LC yield of storing thymus at different temperatures after collection from calves: Both quick freezing on solid CO2 and slow freezing at -50 degrees C led to a 30% loss of LC yield relative to that from unfrozen tissue kept at O degrees C. Moreover we found that the apparent decrease or total loss of LC activity upon storage of a purified LC fraction may result from an occasional lack of LC-responsiveness of human blood lymphocytes depending on the donor's physiological state, in spite of normal PHA reactivity. This suggested the use of LC-responsive, cryopreserved lymphocytes, the advantages of which are documented and discussed in detail.

A micromethod to culture granulocyte colonies from human bone marrow in agar-containing glass capillaries.

A micromethod was developed to grow granulocytic colonies from human bone marrow in agar-containing glass capillary tubes. Treatment of the bone marrow samples and the culture conditions (type and quantity of serum, CSF, cell seeding density) were optimized. Up to 60 colonies were obtained from 3.5 x 10(4) nucleated cells seeded into 50 microliter of total incubation medium/capillary with horse serum (13%) and leukocyte and partially purified bovine lung conditioned medium as CSF (17 and 3%, respectively). The micromethod requires less culture materials (about 1/20), cells, CSF and less time for colony counting, but higher cell densities for seeding, resulting in an increased sensitivity for drug or factor testing. Colony morphology can be easily examined. The micromethod offers further advantages, e.g. quantitation by light scattering densitometry, and hence seems suitable for clinical investigations.

[The influence of cytostatic and immunosuppressive methyl hydrazones on myelo- and lymphopoiesis in vitro (author's transl)]. / Einfluss von cytostatischen und immunosuppressiven Methylhydrazonen auf die Myelo- und Lymphopoese in vitro.

The cytostatic and immunosuppressive N'-methyl-N'-beta-chloroethylbenzaldehyde hydrazones B1 and B2 inhibit the colony growth of mouse bone marrow cells and PHA-stimulated human lymphocytes in vitro in a dose-dependent manner. In the presence of B1, however, in contrast to B2, the inhibition of 3H-thymidine (3H-Tdr) uptake by the bone marrow cells and lymphocytes is insignificant. Two further benzaldehydrazones CyB4 and EB4 show little or no influence both on clony growth and nucleoside uptake. On the other hand, CyB4 inhibits the 3H-Tdr uptake by ConA- or LPS-stimulated mouse spleen cells to a gretaer degree than does B1 or B2, although CyB4 unlike B1 or B2 does not display any immunosuppressive effects in the mouse. These findings demonstrate that the 3H-Tdr method is less sensitive than the colony assays and is hence only of limited value as a measure of the vitro proliferation of mammalian cells treated with cytostatics.

Screening for specific calf thymus inhibitors (chalones) of T-lymphocyte proliferation.

Using agar colony assays with truly proliferating stimulated human T-lymphocytes and mouse granulocytes, two ultrafiltrate fractions were obtained from calf thymus which preferentially inhibited lymphocyte colony growth: Fraction I in the molecular range 1000-10,000 proved to be stable upon heating, prolonged storage and lyophilization, whereas Fraction II in the molecular range 10,000-30,000, was found to be unstable. Fraction I was also extracted with Tween 80 and cetyltrimethylammonium bromide. Chromatography of Fraction I on Biogel P6 and DEAE-cellulose further increased its specificity of inhibition for lymphocyte colony growth and revealed an estimated molecular weight of below 1400. Its inhibitory activity was found to be reversible and unlikely to result from spermine. Thus the properties of fraction I meet the requirements of a T-lymphocyte chalone as an endogenous non-cytotoxic and reversible inhibitor of T-lymphocyte proliferation.

The influence of spermine, spermidine and various sera on T-lymphocyte and granulocyte colony growth in vitro.

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the dose-response relationships of spermine and spermidine, fetal calf (FCS), calf (CS), horse (HS) and human AB serum (ABS), and of the polyamines in the presence of selected does of the sera were analyzed. In contrast to earlier observations using [3H]thymidine uptake masurements it was found that the polyamines as well as the sera themselves inhibit T-LC and GC growth in the presence of autologous HS and ABS, respectively. The polyamines also inhibit in the presence of additional HS and ABS, yet most effectively with added FCS and CS. Thus the previously reported species-specificity of the serum-dependent polyamine inhibition has here been shown to be more quantitative than qualitative. These studies stress the significance of assays utilizing truly dividing cells and broad dose-response relationship in order to assess specific biological effects in vitro.

Function of individual E. coli 30 S ribosomal proteins as determined by in situ immunospecific neutralization: a tentative classification.

The accessibility of each 30S subunit protein to their cognate antibodies (IgG or Fabs) having been previously well established, the effect of their in situ specific neutralization by monovalent IgG fragments (FabI) are reported for five reactions: 1) T4 and R17 RNA directed protein synthesis: 2) polyphenylalanine synthesis: 3) enzymatic Phe-tRNA binding in the presence of 30S + 50W subunits: 4) fMet-tRNAf binding to the 30S subunit in the presence of initiation factors IF1, IF2, IF3; 5) coupling with lambda plac DNA transcription of the initial translation step (i.e., interaction of IF3 activated 30S subunits with nascent mRNA, in the absence of tRNA). According to evident similarities in their inhibition pattern concerning the five reactions tested, 30S subunit proteins can be classified in five categories which are discussed in terms of functional topography.

Screening for granulopoiesis inhibitors (chalones) by different assays.

The suitability of various granulocyte chalone sources was examined; for this purpose rat ascites fluid and the conditioned media of ascites and bone marrow cells were fractionated by ultrafiltration and Sephadex gel filtration. To evaluate different assay systems, the ability of the fractions to inhibit the growth of granulocytic and T-lymphocytic colonies in agar capillaries, as well as to inhibit the uptake of [3H]thymidine in bone-marrow cells, T- and B-lymphocytes, was tested and compared. Three granulocyte colony inhibiting fractions were obtained that contained apparent chalone activities, but showed different elution parameters with molecular weights well below 10 000. Comparison of the test systems revealed that the granulocyte colony assay may detect inhibitors different from those found by the [3H]thymidine bone-marrow assay; the validity of the latter test is seriously questioned, however. The need for precisely defined assays to screen for the apparently various inhibitors is emphasized by these studies.