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

Aldo Profumo, Marco Turci, Gianluca Damonte, Fabio Ferri, Davide Magatti, Barbara Cardinali, Carla Cuniberti, Mattia Rocco

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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.

Original languageEnglish
Pages (from-to)12335-12348
Number of pages14
JournalBiochemistry
Volume42
Issue number42
DOIs
Publication statusPublished - Oct 28 2003

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Fibrinopeptide B
Fibrinopeptide A
Thrombin
Fibrinogen
Protein Isoforms
Kinetics
Polydispersity
Binding energy
Buffers
Growth
Ionic strength
Edetic Acid
Osmolar Concentration
Rate constants
Western Blotting
Calcium
Degradation
Substrates

ASJC Scopus subject areas

  • Biochemistry

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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. / Profumo, Aldo; Turci, Marco; Damonte, Gianluca; Ferri, Fabio; Magatti, Davide; Cardinali, Barbara; Cuniberti, Carla; Rocco, Mattia.

In: Biochemistry, Vol. 42, No. 42, 28.10.2003, p. 12335-12348.

Research output: Contribution to journalArticle

Profumo, Aldo ; Turci, Marco ; Damonte, Gianluca ; Ferri, Fabio ; Magatti, Davide ; Cardinali, Barbara ; Cuniberti, Carla ; Rocco, Mattia. / 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. In: Biochemistry. 2003 ; Vol. 42, No. 42. pp. 12335-12348.
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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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T1 - Kinetics of Fibrinopeptide Release by Thrombin as a Function of CaCl 2 Concentration

T2 - Different Susceptibility of FPA and FPB and Evidence for a Fibrinogen Isoform-Specific Effect at Physiological Ca 2+ Concentration

AU - Profumo, Aldo

AU - Turci, Marco

AU - Damonte, Gianluca

AU - Ferri, Fabio

AU - Magatti, Davide

AU - Cardinali, Barbara

AU - Cuniberti, Carla

AU - Rocco, Mattia

PY - 2003/10/28

Y1 - 2003/10/28

N2 - 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.

AB - 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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