Kinetics of fibrinopeptide release by thrombin as a function of CaCl2 concentration:: Different susceptibility of FPA and FPB and evidence for a fibrinogen lsoform-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 CaCl2 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 CaCl2 (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter P likely reflects substrate polydispersity (beta = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found beta approximate to 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 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 K-b plots showed instead that both total depletion and high Ca2+ 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 similar to4%, resulting in a bound:free thrombin ratio of similar to25:75. With regard to the C-terminal ends of the Aalpha-chains, similar to45% were either intact or lightly degraded, while the remaining similar to55% 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 approximate to 6), with a ratio of similar to48:52. 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.