Heat denaturation of fibrinogen to develop a biomedical matrix.
Native and heat denatured fibrinogen are the basis for various matrices used to establish hemostasis as well as for constructing biomedical devices. For example, fibrin microbeads (FMB) prepared by a heated ( approximately 70 degrees C) oil emulsion process were reported to be attractive to mesenchymal-type cells, such as fibroblasts, endothelial and smooth muscle cells, and useful for isolating mesenchymal stem cells from bone marrow. Here, we examined the solution properties of fibrinogen subjected to heat (47-60 degrees C). Fibrinogen exhibited maximal stability of pH(max stab) = 6.8. At physiologically relevant concentrations, Ca(II) stabilized and Zn(II) destabilized fibrinogen against heat denaturation. Scanning electron micrographs (SEM) of precipitated, heat denatured, fibrinogen showed globular structures ( approximately 400 nm diameter), composed of aggregates of >3000 fibrinogen monomers. Monoclonal antibodies (MAb) to various regions of fibrinogen, as well as two polyclonal antibody (Ab) to haptotactic peptides (Haptides) equivalent to or near the C-termini of beta and gamma-chains (beta(463-483) and gamma(372-391/411)), were used to monitor epitopic changes of fibrinogen bound to and heated on plastic ELISA plates. The pattern of altered Ab binding indicated that fibrinogen heat denaturation on plastic exposed the C-terminal epitope gamma(397-411) as well as Haptide epitopes (beta(463-483) and gamma(372-391)). Immuno-staining of FMB prepared by a heated (below 75 degrees C) oil emulsion process, also presented many exposed Haptide epitopes, which probably helped to attract cells. Our results indicated that moderately heat-denatured fibrinogen, in the form of FMB, could be used for cell culturing and biomedical applications.
Marx
Gerard
G
Hapto Biotech Ltd., Jerusalem, Israel.
Mou
Xiaode
X
Hotovely-Salomon
Anna
A
Levdansky
Lilia
L
Gaberman
Elena
E
Belenky
Dimitry
D
Gorodetsky
Raphael
R
D001672:Biocompatible Materials / Q000737:chemistry* / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005340:Fibrinogen / Q000737:chemistry* / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008968:Molecular Conformation; D010446:Peptide Fragments / Q000494:pharmacology; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D001672:Biocompatible Materials; D002413:Cations, Divalent; D000939:Epitopes; D010446:Peptide Fragments; D005340:Fibrinogen