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.

Characterizing fibrin glue performance as modulated by heparin, aprotinin, and factor XIII.

We describe the performance of fibrin glue (FG) as modulated by heparin, aprotinin, or factor XIII levels. In vitro tests and a rat kidney excision model demonstrated that the hemostatic efficacy of fibrin was not modulated by aprotinin. Overlapping rat skin sections demonstrated that adhesion strength (AS) was proportional to the area of overlap as well as to fibrinogen levels. AS was not modulated by exogenous heparin or aprotinin and was independent of the endogenous factor XIII in fibrinogen. SDS-PAGE developed by Coomassie or Western blots with anti-gamma chain antibody confirmed that normal skin sections contain adequate trans-glutaminase to maximally cross-link normal, as well as XIII-depleted, fibrin. Fibrin glue (FG) sprayed onto rat skin incision wounds with a dual channel spray applicator acted in 2 phases: initially (day 1), compared to wounds stapled without or treated with only thrombin, FG significantly increased breaking strength. In the second phase of wound healing (after day 3), all groups achieved increased but equivalent breaking strength. FG containing aprotinin (to 3000 U/m; Immuno, Behringwerke, Germany) exhibited initial tissue bonding strength equivalent to fibrin without aprotinin, but histological examination showed delayed fibrinolysis and a concomitant slower regeneration of granulation tissue. Thus, our data indicated that aprotinin was not particularly beneficial to wound healing and that the endogenous factor XIII level in the fibrinogen did not contribute significantly to skin bonding. Rather, the tissue supplied adequate trans-glutaminase activity required to crosslink fibrin to itself and to the tissue.