The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigated on unfractionated fibrinogen samples as a function of CaCl2 concentration. A 50 mM Tris, 104 mM NaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na2 (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mM CaCl2 (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter β likely reflects substrate polydispersity (β = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found β ≈ 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 β = 0.69, with an "average" Ka of 3.5 × 10-5 [(NIHu/L)s]-1. This effect disappeared {β = 0.97, Ka = 2.7 × 10-5 [(NIHu/L)s]-1} 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 Ka versus Ca2+ 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 Ca2+ concentration and I on FPA release. The corresponding Kb plots showed instead that both total depletion and high Ca2+ hampered FPB release. To further investigate the TBC2.5 β = 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 Aα-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 (Ka1/Ka2 ≈ 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 Aα-chains suggests their calcium-dependent involvement in FPA release.
Kinetics of Fibrinopeptide Release by Thrombin as a Function of CaCl 2 Concentration: Different Susceptibility of FPA and FPB and Evidence for a Fibrinogen Isoform-Specific Effect at Physiological Ca 2+ Concentration
FERRI, FABIO;
2003-01-01
Abstract
The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigated on unfractionated fibrinogen samples as a function of CaCl2 concentration. A 50 mM Tris, 104 mM NaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na2 (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mM CaCl2 (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter β likely reflects substrate polydispersity (β = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found β ≈ 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 β = 0.69, with an "average" Ka of 3.5 × 10-5 [(NIHu/L)s]-1. This effect disappeared {β = 0.97, Ka = 2.7 × 10-5 [(NIHu/L)s]-1} 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 Ka versus Ca2+ 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 Ca2+ concentration and I on FPA release. The corresponding Kb plots showed instead that both total depletion and high Ca2+ hampered FPB release. To further investigate the TBC2.5 β = 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 Aα-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 (Ka1/Ka2 ≈ 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 Aα-chains suggests their calcium-dependent involvement in FPA release.
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