Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa.
Bi-domain peptides with a factor XIIIa substrate in one domain and a bioactive peptide in another domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa. The cross-linking characteristics were determined for two bi-domain peptides with factor XIIIa substrates based on fibrinogen, dYRGDTIGEGQQHHLGG-NH2, and dLRGDGAKDV-NH2, as well as one bi-domain peptide with a substrate sequence based on alpha2-plasmin inhibitor, dLNQEQVSPLRGD-NH2, and another with a nonbiological, oligolysine substrate, dLRGDKKKKG-NH2 (substrate domains in italic). Each of these peptides was able to cross-link into the fibrin gels during coagulation, with the peptide containing the factor XIIIa substrate based on alpha2-plasmin inhibitor being incorporated at levels in excess of 8 mol/mol fibrinogen. The structural characteristics of these peptide-modified gels proved to be the same as those for a native fibrin gel. The bioactivity of the incorporated active factors was tested in a neuronal culture model with day 8 chicken dorsal root ganglia using two bioactive sequences, RGD and DGEA, and one inactive control sequence, RDG. Each of these peptides influenced the extension of neurites from the ganglia as expected, indicating that the incorporated factors retained their activity. With the use of soluble competitive inhibitors, it was shown that this effect was due to the covalently incorporated peptides. Through exploiting the role of factor XIIIa in coagulation, we have developed a method by which to impart the character of nonfibrin proteins, such as extracellular matrix proteins, to fibrin, a biological material with many potential therapeutic and academic applications.
Schense
J C
JC
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Hubbell
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002642:Chick Embryo; D003094:Collagen / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005337:Fibrin / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D005782:Gels; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D011503:Transglutaminases / Q000737:chemistry*
D003432:Cross-Linking Reagents; D005782:Gels; D010455:Peptides; D005337:Fibrin; D005340:Fibrinogen; D003094:Collagen; D011503:Transglutaminases