Early events in the polymerization of fibrin

The early events in the thrombin-induced formation of fibrin have been studied by the use of stopped-flow multiangle laser light scattering (SF-MALLS). This technological advancement has allowed the recovering, as a function of time with a resolution of about 0.5 sec, of the mean square radius of gyration (Rg2)z and of the molecular weight Mw, and to place an upper bound to the values of the mass/unit length ML. The ionic strength, pH and salt type conditions investigated were all close to physiological, starting with a 50 mM Tris, 104 mM NaCl, pH 7.4 buffer (TBS), to which either 1 mM EDTA-Na2 or 2.5 mM CaCl2 were also added. Fibrinogen was 0.2-0.3 mg/ml and rate-limiting concentrations of thrombin were used (0.05-0.25 NIH units/mg fibrinogen). By plotting (Rg2)z and ML versus Mw on log-log scales, runs proceeding at different velocities and under different solvent conditions could be compared and confronted with model curves. It was found that: (1) within this thrombin range, the mechanism of association does not depend on its concentration, nor on the buffers employed; (2) the (Rg2)z versus Mw curves could all be reasonably fitted with a bifunctional polycondensation scheme involving semiflexible worm-like, double-stranded, half-staggered polymers with persistence length between 200-600 nm, provided that a ratio Q = 16 between the rate of release of the two fibrinopeptides A was employed; (3) the ML versus Mw data seemed more compatible with lower Q values (4 < Q < 8), but their uncertainty prevented a better assessment of this issue; the formation of fibrinogen-fibrin monomer complexes may also play a role in the polymer distributions; (4) in the very early stages (e.g., when Mw < 7 × 105), the (Rg2)z versus Mw data were fitted well only in TBS and at the lowest thrombin concentration, suggesting that a transient, either sequential or concurrent fast second mechanism, involving longer and thinner polymers, may be at work.