Non-Enzymatic Self-Association of Fibrinogen in Solution Studied with 1H NMR Spectrometry

Abstract

Fibrinogen is a blood plasma protein that polymerizes to form the fibrin clot on cleavage of its fibrinopeptides. This work provides quantitative characteristics of the molecular hydrodynamics of fibrinogen in a broad 0.3-60 mg/ml range of concentration and 5-42°C temperature obtained using pulsed-field gradient 1H NMR. Arrhenius plots revealed the activation energy for fibrinogen diffusion Ed = 21.3 kJ/mol at 1.4 mg/ml and 28.4 kJ/mol at 38 mg/ml. The diffusive motion of fibrinogen underwent a remarkable slowdown with concentrations beginning at 1.7-3.4 mg/ml, which deviated from the standard hard-particle behavior, suggesting concentration-dependent intermolecular entanglement. By contrast, diffusivity of fibrinogen variant I-9 with truncated C-terminal portions of the Aα chains was much less concentration-dependent, indicating the importance of intermolecular linkages formed by the αC regions. The remarkable concentration dependence was observed regardless of the absence or presence of the GPRP peptide (inhibitor of fibrin polymerization) and in samples free of fibrin oligomers, confirming that the observed steep decrease in fibrinogen diffusivity was not due to potential contamination by oligomeric fibrin-fibrinogen complexes. Theoretical models combined with all-atom Molecular Dynamics simulations revealed that fibrinogen in solution has a bendable conformation that interpolates between a flexible chain and a rigid rod observed in the crystal. The results obtained illuminate the important role of the αC regions in modulating the fibrinogen molecular shape through formation of weak intermolecular linkages that control the bulk properties of fibrinogen solutions. The results emphasize the importance of fibrinogen self-assembly in modulating the hydrodynamic properties of semi-diluted and concentrated fibrinogen preparations, and point to the potential role of fibrinogen self-assembly in biological and clinical applications.