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Abstract
A fibrinogen-fibronectin complex (γγ’pdFI-pdFN) was purified from normal human plasma using a sequence of cryoprecipitation, ammonium sulfate fractionation, and DEAE Sepharose chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition showed both a 1:1 stoichiometric ratio of fibrinogen (FI) to fibronectin (FN) as well as a stoichiometric ratio of 1:1 of γγ to γγ’. The γγ’pdFI-pdFN complex was non-covalent in nature as it was disrupted by affinity adsorption to Gelatin Sepharose where pdFN bound strongly and the disrupted γγ’pdFI fell through the chromatographic column. Surprisingly, the purified γγ’pdFI-pdFN complex was more broadly thermally stable than plasma FI (pdFI) preparations not containing plasma FN (pdFN) and was stable at physiologic pH, ionic strength and temperature.
The complex appeared as a compact species that was distinctly larger than pdFN alone when analyzed by high pressure size exclusion chromatography (HPSEC). Dynamic light scattering (DLS) showed that the native γγ’pdFI-pdFN complex is a more compact form at low ionic strength but adopt and extended conformation in high salt and denaturing conditions. DLS also showed that FN decreased the degree of polydispersity and hydrodynamic radius of both γγ and γγ’ FI, indicating that FN interact with both subspecies.
The clottability of the native γγ’pdFI-pdFN complex and mixtures of FN with unfractionated FI and FI subspecies was evaluated by Thromboelastography (TEG) assay. The γγ’pdFI-pdFN complex had appreciably enhanced clotting strength than comparable mixtures of FI and FN. FN had not effect on the polymerization rates of fibrin clots. FN, however, showed greater influence on the shear strength of fibrin clots even in the absence of factor FXIII mediated crosslinking. The maximal amplitude and shear strength increased over the entire range of FN concentrations for clots made from unfractionated FI and γγ’FI. FN had mixed effect on the rigidity of clots made from γγFI. While high concentrations of FN enhanced the maximal amplitude and shear modulus, low concentrations decreased both factors.
The fibrin clots made from γγ’pdFI-pdFN showed a biological activity of human fibroblast and human umbilical vein endothelial cells (HUVEC) recruitment and adhesion in vitro exceeding that of fibrin made from equimolar concentration of pdFI and pdFN.
