Heparin binding domain peptides of antithrombin III: Analysis by isothermal titration calorimetry and circular dichroism spectroscopy

Abstract

The serine proteinase inhibitor antithrombin 111 (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F'23-G'48, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L'30-G148 and K'36-G'48 di splayed attenuated heparin binding activities. In fact, K'36-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K'2'-A'34) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low pM concentrations of heparin induce significant a-helix conformation. K'2'-A'34 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-AT111 interactions.

Affinity-based release of glial-derived neurotrophic factor from fibrin matrices enhances sciatic nerve regeneration

“…The resulting solution was sterilized by filtration through 5.0 and 0.22 µm syringe filters, and the final fibrinogen concentration was determined by measuring absorbance at 280 nm. For the delivery system, a bi-domain peptide (ATIII) based on a modified version of the antithrombin III-heparin binding domain ( (AcG)NQEQVSPK (βA)FAKLAAR-LYRKA, where AcG denotes N-acetyl-glycine and the tranglutaminase substrate is given in italics) [25,44] was synthesized as described previously [42]. Fibrin matrices were also prepared as previously described.…”

Fibrin matrices with affinity‐based delivery systems and neurotrophic factors promote functional nerve regeneration

“…The DS was constructed by incorporating heparin and a bi-domain peptide (Sakiyama et al, 1999;Tyler-Cross et al, 1994), into fibrin matrices during polymerization. The bi-domain peptide was synthesized as described previously (Wood et al, 2009a).…”

“…Our lab has developed an affinity-based DS that sequesters growth factors inside a fibrin matrix (Sakiyama-Elbert and Hubbell, 2000a,b) through the use of bi-domain heparin-binding peptide. One domain within this heparin-binding peptide consists of a transglutaminase substrate (Ichinose et al, 1983;Kimura et al, 1985), to facilitate peptide cross-linking into fibrin matrices, while the other domain consists of a heparin-binding domain (Sakiyama et al, 1999;Tyler-Cross et al, 1994 that allows non-covalent immobilization of heparin to the peptide. As a result, electrostatic interactions between various growth factors with peptidebound heparin effectively immobilizes these neurotrophic factors within the fibrin matrix in a manner similar to growth factor sequestration to heparan sulfate in the extracellular matrix (Yamada, 1983).…”

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated ?-endorphin

“…All syntheses were done at Commonwealth Biotechnologies, Inc., Richmond, VA, following standard automated solid-phase procedures (22), usually using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry, as detailed previously (23)(24)(25)(26). Peptides were labeled with fluorophores by derivatization of the amino group of the appropriate Lys residue on the synthesis resin using fluorescein isothiocyanate.…”