The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigated on unfractionated fibrinogen samples as a function of CaCl 2 concentration. A 50 mM Tris, 104 mM NaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na 2 (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mM CaCl 2 (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter likely reflects substrate polydispersity ( ) 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found ≈ 1, with corresponding normalized rate constants (K a ) of 3.8, 4.2, 2.7, and 1.9 × 10 -5 [(NIHu/L)s] -1 . Surprisingly, in TBC2.5 we found ) 0.69, with an "average" K a of 3.} with an increase in the ionic strength I to that of TBC30 with 186 mM NaCl (TBCaNa buffer). FPB releases were instead consistent with a nonstretched consecutive exponential growth function, except in TBC30 where some FPB appeared to be cleaved independently. Log-log plots of K a versus Ca 2+ concentration, Cl -concentration, or I showed a strong linear correlation with only the latter two except in TBCaNa, again suggesting specific effects of the physiological Ca 2+ concentration and I on FPA release. The corresponding K b plots showed instead that both total depletion and high Ca 2+ hampered FPB release. To further investigate the TBC2.5 ) 0.69 effect, FG polydispersity was assessed by Western blot analyses. The thrombin-binding γ′-chain isoform was ∼4%, resulting in a bound:free thrombin ratio of ∼25:75. With regard to the C-terminal ends of the AR-chains, ∼45% were either intact or lightly degraded, while the remaining ∼55% were more degraded. Fitting the % FPA release data in TBC2.5 with a sum of two exponentials resulted in a faster component and a slower component (K a1 /K a2 ≈ 6), with a ratio of ∼48:52. While a role for the γ′-chain isoform cannot be excluded, this good correlation with the C-terminal degradation of the AR-chains suggests their calcium-dependent involvement in FPA release.
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 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 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 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 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.
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