We have examined the initial phase of fibrin formation, thrombin-catalyzed fibrinopeptide cleavage, from adsorbed fibrinogen using surface plasmon resonance and liquid chromatography-mass spectrometry. Fibrinogen adsorption impaired thrombin-fibrinogen interactions compared to the interactions of thrombin with fibrinogen in solution. The properties of the underlying substrate significantly affected the extent and kinetics of fibrinopeptide cleavage, and the conversion of adsorbed fibrinogen to fibrin. Fibrinogen adsorbed on negatively charged surfaces (carboxyl-terminated self-assembled monolayers) released a smaller amount of fibrinopeptides, at a reduced rate relative to those of hydrophobic, hydrophilic, and positively charged surfaces (methyl-, hydroxyl-, and amine-terminated self-assembled monolayers, respectively). Additionally, the conversion of adsorbed fibrinogen to fibrin was comparatively inefficient at the negatively charged surface. These data correlated well with trends previously reported for fibrin proliferation as a function of surface properties. We conclude that thrombin interactions with adsorbed fibrinogen determine the extent of subsequent fibrin proliferation on surfaces.

Fibrin proliferation from both human fibrinogen solutions and platelet-poor plasma was studied quantitatively as a function of substrate surface properties. A quartz crystal microbalance was used to monitor both protein adsorption and fibrin proliferation in real time at hydrophobic, hydrophilic, positively charged, and negatively charged surfaces. Scanning electron microscopy was used to characterize the morphology of the polymerized fibrin layers. The observed changes in mass indicate that fibrinogen adsorption occurs rapidly and mediates subsequent fibrin proliferation. Notably, substrate surface properties significantly affect the ability of adsorbed fibrinogen to promote fibrin proliferation.

The conversion of adsorbed fibrinogen to fibrin in the presence of the enzyme thrombin was studied using surface plasmon resonance (SPR), a quartz crystal microbalance (QCM), sum frequency generation (SFG), atomic force microscopy (AFM), and an elutability assay. Exposure of adsorbed fibrinogen to thrombin resulted in a mass loss at the surface consistent with fibrinopeptide release and conversion to fibrin. Changes in hydration upon conversion of adsorbed fibrinogen to fibrin were determined from comparisons of acoustic (QCM) and optical (SPR) mass adsorption data. Conversion to fibrin also resulted in the adsorbed layer becoming more strongly bound to the surface and more compact. The elutability of adsorbed fibrinogen by Triton X-100, studied with SPR, decreased from 90 +/- 5 to 6 +/- 2% after conversion to fibrin. The height of the adsorbed monolayer, as determined by AFM, decreased from 5.5 +/- 2.2 to 1.7 +/- 0.8 nm. We conclude that thrombin-catalyzed fibrinopeptide release triggers significant changes in fibrinogen conformation beyond peptide cleavage.

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