Antithrombotic activity of a novel C-terminal hirudin analog in experimental animals.
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Amino Acid Sequence↗
Biomedical subjects
J L Krstenansky
Publications and source records attributed to J L Krstenansky.
Antithrombotic activity of a novel C-terminal hirudin analog in experimental animals.
A synthetic hirudin55-65 C-terminal fragment analog was evaluated for anticoagulant activity and in models of experimental thrombosis in mice and rats. Intravenous injection caused dose-related inhibition of thrombin and anticoagulation in rat blood samples, protection from thromboembolism in mice and inhibition of stasis-induced venous thrombosis in rats. Antithrombotic effectiveness corresponded with anticoagulant activity. Anephric animals exhibited longer duration of activity than normal animals suggesting the kidney as a major route of elimination.
Amino Acid Sequence↗
Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes.
The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
1,2-Dipalmitoylphosphatidylcholine↗
Positional effects of sulfation in hirudin and hirudin PA related anticoagulant peptides.
C-Terminal fragment analogues of the leech protein hirudin or the related protein hirudin PA block thrombin's cleavage of fibrinogen. Three series of synthetic peptides were synthesized to study the effects of sulfation in hirudin-derived peptides. Potency of hirudin analogues increased with p-(amino)Phe63, p-(aminosulfonate)Phe63, and p-(sulfate)Tyr63 substitution in place of Tyr63. Sulfation of Tyr56, which in hirudin is normally Phe, resulted in a loss of 1 order of magnitude in potency. The sulfation of Tyr64 of the hirudin PA related analogue resulted in increased potency as for the hirudin analogue. However, in this series the p-(amino)Phe64 and p-(amino-sulfonate)Phe64 did not have increased potency. In addition to these positional effects, replacing all the Glu residues with (O-sulfato)Ser yielded an analogue with full antithrombin potency.
Amino Acid Sequence↗
Structure-function relationships of the C-terminal functional domain of hirudin and its variants.
The C-terminal functional domain of hirudin, hirudin variant 1 (residues 55-65), binds to a non-catalytic site on thrombin. In doing so, it is capable of inhibiting the procoagulant actions of thrombin. In terms of free energy of binding, this domain, which comprises 17% of the total sequence of the protein, contributes approximately half of the binding energy of the whole protein to thrombin. This situation also appears to hold true for the known variants of hirudin, some of which differ in the functional nature of their C-terminal regions. Extensive structure-function studies on this domain yield insights into the differences and similarities in the modes of thrombin interaction of hirudin and its variants. In particular, hirudin and hirudin PA have a similar and somewhat interchangeable structure-activity relationships (SAR) profile that indicates that they interact with thrombin in a similar manner. Hirullin P18, a 62 amino acid member of the hirudin family and isolated from Hirudinaria manillensis, is substantially different in sequence and its SAR, which shows that, although it seems to utilize the same non-catalytic binding domain as hirudin, it must utilize a different mode of interaction with thrombin.
Amino Acid Sequence↗
Centrally truncated neuropeptide Y analog acts as an agonist for Y1 receptors on SK-N-MC cells.
The similarity of neuropeptide Y (NPY) to pancreatic polypeptide (PP), whose X-ray crystallographic structure is known, has allowed computer-assisted molecular modelling of NPY and predictions of its three-dimensional structure. Utilizing these techniques, Krstenansky et al. (Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 4377-4381) reported that a centrally truncated analog of porcine NPY, [D-Cys7-Aoc8-17-Cys20]pNPY, which was designed to maintain the tertiary structure of the native molecule, bound to sites on membranes from mouse brain with even higher affinity than native NPY. As brain membranes may represent a heterogeneous mixture of receptor subtypes, we decided to characterize the activity of this analog on a defined cell line. SK-N-MC cells are a human epithelioma cell line with high-affinity receptors of the Y1 subtype which are coupled to inhibition of adenylate cyclase. (D-Cys7-Aoc8-17-Cys20]pNPY bound to receptors on SK-N-MC cells, but in contrast to membranes from mouse brain, with a lower affinity than pNPY. Furthermore, [D-Cys7-Aoc8-17-Cys20]pNPY was able to inhibit isoproterenol-stimulated cAMP production in these cells. Therefore, it appears that the central amino acids deleted from this analog are not involved in NPY binding, and biological activity can be maintained by conservation of the tertiary structure of NPY around the binding surface.
Animals↗
The C-terminal binding domain of hirullin P18. Antithrombin activity and comparison to hirudin peptides.
Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin P18(41-62) [corrected] possesses an antithrombin potency similar to that of acetyl-desulfatohirudin(45-65). Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P18(54-61) and hirudin(59-65) have substantial differences, the C-terminal functional domain represented by hirullin P18(50-61) appears to be comparable to hirudin(55-65) in terms of its functional role in antithrombin activity.
Amino Acid Sequence↗
Characterization of a monoclonal antibody specific to the amino terminus of the alpha-chain of human fibrin.
A peptide, Gly-Pro-Arg-Val-Val-Glu, corresponding to the first six residues of the amino terminus of the alpha-chain of human fibrin (desAA-fibrin) was prepared by solid-phase peptide synthesis. The peptide was covalently linked to keyhole-limpet hemocyanin (KLH) and used as an immunogen for preparing monoclonal antibodies. A monoclonal antibody specific to the hexapeptide, but not to KLH or fibrinogen, was produced. The antibody did not bind to thrombin-mediated clots prepared from either plasma or purified fibrinogen. However, immunoreactivity was detected when fibrin (prepared from fibrinogen) was solubilized with 8 M urea. In contrast, a monoclonal antibody specific to the amino terminus (Gly-His-Arg-Pro-Leu-Asp-Lys) of the beta-chain of fibrin recognized the epitope in clots. These results indicate that thrombin cleavage of fibrinogen produces a structural change in the amino terminal domain of the alpha-chain that makes it inaccessible to antibody interaction. In addition, our study suggests that the potential clinical application of monoclonal antibodies to localize fibrin-rich thrombi must take into account the final structure of clots.
Amino Acid Sequence↗
Lipid and membrane interactions of neuropeptide Y.
The interactions of neuropeptide Y with dimyristoylphosphatidylcholine and cell membranes were examined by several physical techniques to probe the potential role of its putative C-terminal amphipathic alpha-helix. Neuropeptide Y binding was demonstrated by a rapid release of entrapped 6-carboxyfluorescein and a rapid decrease in the turbidity of dimyristoylphosphatidylcholine liposomes. In addition, an increase in tyrosine fluorescence intensity and an increase in the anisotropy of diphenylhexatriene in dimyristoylphosphatidylcholine liposomes was observed. In isolated, aortic smooth muscle cell membranes, the anisotropy of diphenylhexatriene increased as a function of added neuropeptide Y. The concentration range (low microM) over which neuropeptide Y increases the polarization of diphenylhexatriene in cell membranes is similar to the range in which it inhibits isoproterenol-stimulated cAMP accumulation. This inhibition is not affected by pertussis toxin, nor does neuropeptide Y cause the release of preloaded [3H]adenine from cells into the medium. These data suggest that neuropeptide Y contains an amphipathic alpha-helical region which interacts with lipids in much the same way as the amphipathic alpha-helical regions of the plasma apolipoproteins and that the inhibition of isoproterenol-stimulated cAMP accumulation at low microM concentrations of peptide may be the result of an alteration in the cell membrane bilayer structure.
Animals↗
Development of MDL 28,050, a small stable antithrombin agent based on a functional domain of the leech protein, hirudin.
MDL 28,050 is a decapeptide antithrombin agent that inhibits alpha-thrombin-induced fibrin clot formation by binding to a non-catalytic site on alpha-thrombin. It is the result of chemical and structural optimization of a functional domain of the leech anticoagulant, hirudin. In contrast to the contention that the polyanionic nature of this C-terminal functional domain governs its interaction with alpha-thrombin, systematic study of this region has shown the importance of the lipophilic residues for providing the functionality necessary for potent binding to alpha-thrombin. The development of MDL 28,050 and other effective antithrombin agents are outlined through the description of the structure-activity relationships (SAR) for these peptides. These peptides are effective in a variety of in vitro and in vivo models of thrombosis.
Amino Acid Sequence↗
Examination of the role of the amphipathic alpha-helix in the interaction of neuropeptide Y and active cyclic analogues with cell membrane receptors and dimyristoylphosphatidylcholine.
To test the potential importance of the putative C-terminal amphipathic alpha-helical region of neuropeptide Y (NPY) in receptor binding, the interactions of porcine NPY and several peptide analogues with lipid and cell membrane receptors were compared. Cyclic analogues were designed to constrain the N- and C-terminal regions of the peptide and to retain the folded conformation of NPY predicted from its sequence analogy with pancreatic polypeptide and its similar spectral behavior. The three cyclic peptides were [Cys2, 8-aminooctanoic acid5-24, D-Cys27]-NPY (C2-NPY), [Cys5, 8-aminooctanoic acid7-20, D-Cys24]-NPY (C5-NPY), and [D-Cys7, 8-aminooctanoic acid8-17, Cys20]-NPY (C7-NPY). All of the peptides bind with high affinity to pig spleen membranes, but only NPY and [Glu16, Ser18, Ala22, Leu28,31]-NPY (ESALL-NPY) bind quantitatively to dimyristoylphosphatidylcholine (DMPC) liposomes. C7-NPY and NPY20-36 bind with moderate affinity to liposomes, but only NPY and C7-NPY bind with high affinity to mouse brain receptors. Thus, lipid binding and receptor binding are not correlated in this series of peptides, and binding to the pig spleen receptor appears to require only the C-terminal region of the peptide. Simple lipid binding, as in NPY20-36, is insufficient for binding to the mouse brain receptor, suggesting that the N-terminal region of the peptide is required for high-affinity binding to this receptor. Data from fluorescence, differential scanning calorimetry, and liposome clearing experiments suggest that, although the interaction of NPY with lipid is consistent with formation of an amphipathic alpha-helix, a simple amphipathic alpha-helical model for the interaction with the high-affinity NPY receptor is insufficient to explain the data.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
C-terminal modifications of neuropeptide Y and its analogs leading to selectivity for the mouse brain receptor over the porcine spleen receptor.
Neuropeptide Y (NPY) is known to bind to at least two types of receptors (Y1 & Y2). One type (Y2) is able to bind and undergo activation by both NPY and its C-terminal fragments with good potency while the other (Y1) requires the full length of NPY for good potency. For most NPY analogs that have been examined, potency for the Y2 system (porcine spleen) is greater than or equal to that for the Y1 system (mouse brain), since the Y2 system is generally less selective. However, modifications of NPY and its analogs at position 34 can lead to materials with some Y1 selectivity. For example, [Pro34]-pNPY binds to mouse brain with an affinity of 0.14 nM. Its affinity for porcine spleen is 140 nM. [His34]-pNPY was also found to be Y1 selective (19-fold), but not to the degree of the [Pro34] analog (1000-fold). The Pro34 modification in the Y2 selective C-terminal fragment NPY (20-36) converted it into an essentially non-selective analog. The selectivity from the Pro34 substitution results from a loss of Y2 binding potency along with little effect on the Y1-receptor binding. Therefore, Y1 and Y2 receptors have differing requirements for the C-terminal region of NPY in addition to their different requirements for NPY's N-terminus.
Amino Acid Sequence↗
Signal epitopes in the three-dimensional structure of neuropeptide Y. Interaction with Y1, Y2, and pancreatic polypeptide receptors.
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Amino Acid Sequence↗
Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine.
The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)
Acrylamides↗
Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site.
Hirudin is a 65 amino acid anticoagulant peptide produced in the leech. The single polypeptide is cross-linked by three disulfide linkages in the NH2 terminal half of the molecule. A peptide corresponding to the COOH terminus (residues 45-65) was synthesized utilizing lysine 47 as a specific residue to conjugate to thyroglobulin as a carrier for raising antibodies in mice. Using an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the major antigenic domain(s) was located between residues 52-65. The COOH terminal residues Ile-59, Tyr-63, and Leu-64 are crucial for maintaining the antigenic structure. The NH2 terminal region (residues 45-52) that is proximal to the carrier protein, however, was not immunoreactive. A possible mechanism by which antibodies recognize the COOH terminal region of the synthetic peptide and the strategy for raising such antibodies are discussed.
Animals↗
C-terminal peptide alcohol, acid and amide analogs of desulfato hirudin54-65 as antithrombin agents.
Analogs of the antithrombin peptide hirudin54-65 with C-terminal modifications have been synthesized in order to examine the requirements for alpha-thrombin inhibition. The C-terminal residue, Gln65, could be replaced with L-amino acids or amino alcohols with neutral or charged hydrophilic side chains without greatly affecting the peptide's antithrombin potency as determined by inhibition of thrombin-induced clot formation in human plasma in vitro. Derivatives with D- or L-amino carboxamides at position 65 had significantly reduced potency, but still retained activity. Deletion of residue 65 with conversion of residue 64 to the amide or alcohol derivative resulted in a three-fold loss of potency. In addition to these results the solid-phase synthesis of peptide alcohols via direct displacement of p-nitrobenzhydrylideneisonitroso resin attached peptides with the desired C-terminal amino alcohol is reported.
Acids↗
Short model peptides having a high alpha-helical tendency: design and solution properties.
Secondary structure is not typically observed for small peptides in solution. Several of the properties of alpha-helical peptides are known which lead to the stabilization of the structure. The utilization of all the known factors important for alpha-helical stabilization in the design of model alpha-helical peptides (MAP) is reported. The peptides are based on the repeating eleven amino acid sequence, Glu-Leu-Leu-Glu-Lys-Leu-Leu-Glu-Lys-Leu-Lys (MAP1-11). The CD spectra of these peptides give evidence for more alpha-helical content than has been reported for any short peptide (less than 18 amino acids) to date. This alpha-helical tendency does not require the presence of lipid or reduced temperature. For instance, Suc-[Trp9]MAP9-3'' amide (5), a seventeen amino acid peptide has 100% and 80% alpha-helical contents at 1.7 x 10(-4) M and 1.7 x 10(-5) M, respectively. Suc-[Trp9]MAP2-11 amide (3), merely ten amino acids in length, is 51% alpha-helical at 1.7 x 10(-4) M in 0.1 M phosphate buffer at room temperature. In the presence of lipid or trifluoroethanol, the alpha-helical content of these peptides is increased. This series of peptides demonstrates the complimentarity of various secondary structure design principles and the extent to which structure can be induced in small linear peptides.
Amino Acid Sequence↗
Human hepatoma (HepG2) cells secrete a single 65 K dalton triglyceride lipase immunologically identical to postheparin plasma hepatic lipase.
A triacylglycerol lipase was isolated from the culture medium of HepG2 human hepatoma cells and its properties were compared to hepatic triglyceride lipase (H-TGL) from human postheparin plasma. The HepG2 cell enzyme bound to heparin-Sepharose, was eluted with 1 M NaCl and was not inhibited by 1 M salt. Western-blotting of the fractions from the heparin-Sepharose column with a monoclonal antibody prepared against postheparin plasma H-TGL and which binds to an epitope in the carboxyl-terminus of H-TGL gave a single immunoreactive protein band of 65 kDa. This finding of immunochemical identity was confirmed with polyclonal antibodies prepared against synthetic peptides of H-TGL corresponding to amino acid residues 82-94 near the amino-terminus and residues 468-477, the carboxyl-terminus of the enzyme. We conclude that HepG2 cells secrete a single triacylglycerol lipase with molecular weight properties and immunological characteristics identical to post-heparin plasma H-TGL.
Antibodies, Monoclonal↗