Fibronectin Increases the Rate of Fibrin Clot Polymerization and Alters Matrix Morphology

Plasma fibronectin is a component of the fibrin clot, a provisional matrix that arrests bleeding but that can also obstruct flow in blood vessels. The goal of this study was to examine the influence of fibronectin on the formation, structural characteristics and composition of reconstituted fibrin clots. Fibrin clots were formed by adding thrombin to 1 mg/ml, 2 mg/ml or 4 mg/ml fibrinogen supplemented with 0-0.4 mg/ml fibronectin. The formation of fibrin clots with varying amounts of fibronectin was monitored by measuring light absorbance during polymerization. Confocal microscopy of fluorescein conjugated fibrinogen was used to determine the structural characteristics of fibrin-fibronectin clots. The composition of the fibrin-fibronectin clots was determined through electrophoresis and immunoblotting of solublized clots. Fibronectin concentration positively correlated with the initial rate of polymerization and with the steady state light absorbance values in fibrin clots. The effect of fibronectin on fibrin clots was saturable; there was no significant difference in the steady state light absorbance value of fibrin-fibronectin clots formed from 2 mg/ml and 4 mg/ml fibrinogen. At fibrinogen concentration of 2 mg/ml and above, an increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin-fibronectin clots. Electrophoresis and immunoblotting showed that fibronectin bound to fibrin clots and formed covalently bonded high molecular weight multimers. These findings are novel because they correlate results from light absorbance studies to microcopy images and demonstrate an influence of fibronectin on fibrin clot structural characteristics. This data is important in developing therapies that destabilize fibrin clots.