Kinetics of Fibrinopeptide Release by Thrombin as a Function of CaCl2 Concentration: Different Susceptibility of FPA and FPB and Evidence for a Fibrinogen Isoform-Specific Effect at Physiol
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- 作者：Aldo Profumo ; Marco Turci ; Gianluca Damonte ; Fabio Ferri ; Davide Magatti ; Barbara Cardinali ; Carla Cuniberti ; and Mattia Rocco
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：October 28, 2003
- 年：2003
- 卷：42
- 期：42
- 页码：12335 - 12348
- 全文大小：332K
- 年卷期：v.42,no.42(October 28, 2003)
- ISSN：1520-4995
文摘
The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigatedon unfractionated fibrinogen samples as a function of CaCl2 concentration. A 50 mM Tris, 104 mMNaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na2 (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mMCaCl2 (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted withsingle stretched-exponential growth functions, where the stretch parameter 
likely reflects substratepolydispersity ( = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found 
1, withcorresponding normalized rate constants (Ka) 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" Ka of 3.5 × 10-5 [(NIHu/L)s]-1. This effect disappeared{ = 0.97, Ka = 2.7 × 10-5 [(NIHu/L)s]-1} with an increase in the ionic strength I to that of TBC30 with186 mM NaCl (TBCaNa buffer). FPB releases were instead consistent with a nonstretched consecutiveexponential 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 correlationwith 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 depletionand high Ca2+ hampered FPB release. To further investigate the TBC2.5 = 0.69 effect, FG polydispersitywas assessed by Western blot analyses. The thrombin-binding 
'-chain isoform was ~4%, resulting in abound:free thrombin ratio of ~25:75. With regard to the C-terminal ends of the A
-chains, ~45% wereeither intact or lightly degraded, while the remaining ~55% were more degraded. Fitting the % FPArelease data in TBC2.5 with a sum of two exponentials resulted in a faster component and a slowercomponent (Ka1/Ka2 6), with a ratio of ~48:52. While a role for the '-chain isoform cannot be excluded,this good correlation with the C-terminal degradation of the A-chains suggests their calcium-dependentinvolvement in FPA release.
likely reflects substratepolydispersity ( = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found 1, withcorresponding normalized rate constants (Ka) 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" Ka of 3.5 × 10-5 [(NIHu/L)s]-1. This effect disappeared{ = 0.97, Ka = 2.7 × 10-5 [(NIHu/L)s]-1} with an increase in the ionic strength I to that of TBC30 with186 mM NaCl (TBCaNa buffer). FPB releases were instead consistent with a nonstretched consecutiveexponential 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 correlationwith 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 depletionand high Ca2+ hampered FPB release. To further investigate the TBC2.5 = 0.69 effect, FG polydispersitywas assessed by Western blot analyses. The thrombin-binding '-chain isoform was ~4%, resulting in abound:free thrombin ratio of ~25:75. With regard to the C-terminal ends of the A-chains, ~45% wereeither intact or lightly degraded, while the remaining ~55% were more degraded. Fitting the % FPArelease data in TBC2.5 with a sum of two exponentials resulted in a faster component and a slowercomponent (Ka1/Ka2 6), with a ratio of ~48:52. While a role for the '-chain isoform cannot be excluded,this good correlation with the C-terminal degradation of the A-chains suggests their calcium-dependentinvolvement in FPA release.