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†

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 â likely reflects substrate polydispersity (â) 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found â 1, with corresponding 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 â) 0.69, with an “average ” Ka of 3.5 10-5 [(NIHu/L)s]-1. This effect disappeared {â) 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 â) 0.69 effect, FG polydispersit