Preparation of orientated fibrous mats from fibronectin: composition and stability.

The biochemical characteristics of a novel biomaterial, based on the aggregated adhesive protein, fibronectin (Fn), are reported here. Fibrous, orientated mats, formed from solution under directional shear, could be made to incorporate heparin (typically 3.2 mu mg/mg fibronectin). Mats were hygroscopic, doubling their mass by water uptake in less than 10 h from humid air. After an initial rapid loss of protein into physiological solutions over 24 h, mats were stable and not rapidly degraded by fibroblasts. With or without heparin, mats bound basic fibroblast growth factor, which was then released only slowly. Such materials may prove useful models of in vivo tissue Fn function and as clinical implants to organize tissue repair.

Production of artificial-orientated mats and strands from plasma fibronectin: a morphological study.

Fibronectin is an extracellular matrix glycoprotein involved in wound healing. Techniques have been developed for the preparation of orientated fibronectin mats from plasma fibronectin, for use in wound healing and tissue repair. Formation of strands and mats is by self-association and aggregation of fibronectin from solution under a directional shear force. Incorporation of heparin with fibronectin modified the mats. Scanning electron microscopy showed that the mats were composed of well-orientated fibrous fibronectin, with a network of internal interconnecting pores. Single strands of fibronectin were prepared for use as a tissue culture model of cell interaction with aggregated orientated fibronectin mats. Rat tail tendon and human skin fibroblasts were used for assessment of cell interaction with both single strands of fibronectin and mats. Fibroblasts rapidly attached to single strands and became orientated. Dense cultures of fibroblasts growing over single strands became orientated, according to the orientation of the underlying fibronectin. Fibroblasts readily grew on the surface of fibronectin mats and were observed within the porous network.

Preparation of a gelatin-binding conjugate containing gold thiomalate.

Plasma fibronectin has a range of binding sites, for ligands, including denaturated collagen (gelatin). It has been proposed that this activity may be used for nonimmune drug targeting to sites in the extra-cellular matrix such as the targeting of gold thiomalate to rheumatoid joints. In the present study, a novel conjugate has been developed, consisting of the gold thiomalate bound at high density to the gelatin-binding domain of fibronectin, through a polylysine carrier. Isolation and cross-linking of suitable fragments of fibronectin (relative molecular weights 65 and 52 kDa) to polylysine is followed by conjugation to gold thiomalate on a solid phase, gelatin-agarose affinity absorbent. Although gold thiomalate has the ability to inactivate, the protein-gold conjugate produced by this technique retained its gelatin-binding activity.

Ferrocenopapain, an organometallic protein formed by site-specific inactivation of papain using chloroacetylferrocene.

Papain is rapidly, specifically and irreversibly inactivated by chloroacetylferrocene in a time-dependent, two-stage process which involves initial, physical complexation between the enzyme and the organometallic reagent followed by chemical reaction. The kinetics of the process show saturation kinetics with respect to inactivator concentration. The modification occurs with a 1:1 stoichiometry and the degree of loss of enzymatic activity is directly reflected in the loss of thiol groups in the active-site that are accessible to Ellman's reagent. The modification is faster at higher pH. The ferrocenium ion of the modified papain can be generated at low pH by anaerobic treatment with ferric nitrate and shows a 180 percent increased lifetime at pH 3.3 compared to the acetylferrocenium ion under identical conditions. The spectral properties of the ferrocenopapain indicate that the ferrocene is in an unusual environment at the protein active-site and the implications of this are discussed.

A study of platelet fibronectin immunofluorescence in peripheral vascular disease and diabetes mellitus.

Platelets hyperaggregability and hypersecretion fibronectin (Fn) are known to occur in peripheral vascular disease (PVD) and diabetes mellitus (DM) with microangiopathy. To determine whether an increase in platelet membrane bound Fn constitutes to hyperaggregability of platelets, washed platelets from normal subjects and from patients with peripheral vascular disease and patients with diabetes mellitus were examined for the presence of fibronectin (Fn) by means of fluorescein linked antibody to Fn. Platelets from peripheral vascular disease and diabetes mellitus patients tended to aggregate during preparation and apparently exhibited greater fluorescence in platelet "smears" than was observed in smears from controls. In contrast, when washed platelet "smears" were prepared from platelet preparations containing iloprost, an analogue of prostacyclin, platelet aggregates did not form and the 'excess' of fluorescence disappeared from all the three groups. When platelets were stained for Fn fluorescence in suspensions, no fluorescence was observed on the surface of platelets from peripheral vascular disease and diabetes mellitus patients or controls. On stimulation with thrombin washed platelet suspension showed fluorescence for Fn. Platelet activation leads to Fn appearing on platelet surface but this effect cannot be quantified by optical fluorescence microscopy.

Fibronectin concentrations in plasma in peripheral vascular disease.

Fibronectin (Fn) concentrations were measured immunoturbidimetrically in plasma of normal subjects and patients with peripheral vascular disease (PVD) before and after venous compression, which caused Fn concentrations to increase in both normal subjects and PVD patients. Basal Fn concentrations and those after 10-min compression were not significantly different in normal subjects and PVD patients. Five minutes after the release of compression, Fn had consistently declined in normal subjects and reverted to baseline values; in contrast, in PVD patients values either increased further or decreased inconsistently. Thus the Fn concentration at 15 min was significantly (P less than 0.001) greater in PVD patients than in normal subjects. Plasma albumin concentrations, measured in parallel to ensure that changes in Fn concentrations were not nonspecific, increased to a greater extent in normal subjects than in PVD patients and reverted to normal after the removal of compression. The Fn/albumin ratio remained unchanged in normal subjects after venous compression, whereas that in PVD patients increased and remained higher, even after decompression. The sustained increase in plasma Fn concentrations and in the Fn/albumin ratio in PVD patients after venous compression may indicate endothelial injury.