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 physiological Ca2+ concentration.

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 beta likely reflects substrate polydispersity (beta = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found beta approximately 1, with corresponding normalized rate constants (K(a)) of 3.8, 4.2, 2.7, and 1.9 x 10(-5) [(NIHu/L)s](-1). Surprisingly, in TBC2.5 we found beta = 0.69, with an "average" K(a) of 3.5 x 10(-5) [(NIHu/L)s](-1). This effect disappeared [beta = 0.97, K(a) = 2.7 x 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 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 beta = 0.69 effect, FG polydispersity was assessed by Western blot analyses. The thrombin-binding gamma'-chain isoform was approximately 4%, resulting in a bound:free thrombin ratio of approximately 25:75. With regard to the C-terminal ends of the Aalpha-chains, approximately 45% were either intact or lightly degraded, while the remaining approximately 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) approximately 6), with a ratio of approximately 48:52. While a role for the gamma'-chain isoform cannot be excluded, this good correlation with the C-terminal degradation of the Aalpha-chains suggests their calcium-dependent involvement in FPA release.