Characteristics of the association between prothrombin fragment 2 and alpha-thrombin.

The esterolytic activity of bovine alpha-thrombin on the synthetic substrate N-alpha-p-tosyl-L-arninine methyl ester (TosArgOMe) is stimulated when the prothrombin activation fragment, prothrombin fragment 2, is added as previously reported by this laboratory (Heldebrant, C. M., and Mann, K. G. (1973), J. Biol. Chem. 248, 3642). A similar stimulation of beta-thrombin is observed upon addition of prothrombin fragment 2. The binding constant of prothrombin fragment 2 to alpha-thrombin has been determined by the method of Gutfreund ((1972), Enzymes, Physical Principles, Wiley, New York, N.Y., pp 67-71). The dissociation constant is 7.7 X 10(-10)M, and there is one molecule of prothrombin fragment 2 bound per molecule of alpha-thrombin. Prethrombin-2 competes for prothrombin fragment 2, so the enhancement of the esterolytic activity of alpha-thrombin by prothrombin fragment 2 was used as a probe to determine the dissociation constant for the binding of prothrombin fragment 2 to prethrombin 2. The dissociation constant for this association is 1.3 X 10(-10)M. The kinetic parameters for the reaction of alpha-thrombin on TosArgOMe were determined in the absence and presence of prothrombin fragment 2 and are as follows: (a) in the absence of prothrombin fragment 2, Km(app) = 1.92 X 10(-4)M, and k3(app) = 35.8 mol of TosArgOMe/mol of alpha-thrombin s(-1); (b) in the presence of prothrombin fragment 2,Km(app = 1.76 X 10(-4)M, and k3(app) = 60.5 mol of TosArgOMe/mol of alpha-thrombin s(-1). Thus, the stimulatory effect of bovine prothrombin fragment 2 on bovine alpha-thrombin is reflected in k3(app) and not in Km(app). In contrast to the stimulatory effect of prothrombin fragment 2 on the thrombin-catalyzed hydrolysis of TosArgOMe, it inhibits the activity of alpha-thrombin toward N-alpha-benzoyl-L-arginine ethyl ester and N-alpha-benzoyl-L-arginine p-nitroanilide. The inhibition of activity toward these substrates by prothrombin fragment 2 is also reflected in k3(app). Activity toward the nonspecific substrate p-nitrophenyl butyrate was completely inhibited by the addition of prothrombin fragment 2. Prothrombin fragment 2 has no effect on the inhibition of alpha-thrombin activity by the active-site serine inhibitors diisopropyl phosphofluoridate, phenylmethanesulfonyl fluoride, or p-nitrophenyl guanidinobenzoate. Inhibition by the active-site-histidine-modifying inhibitor, N-alpha-p-tosyl-L-arginine chloromethyl ketone, was enhanced by the addition of prothrombin fragment 2. Soybean trypsin inhibitor reduces the stimulation by prothrombin fragment 2, but only at high molar ratios. Prothrombin fragment 2 has no effect on the clotting activity of alpha-thrombin, nor inhibition of this activity by heparin, hirudin, or diisopropyl phosphafluoridate. Bovine prothrombin fragment 2 enhances the esterolytic activity of both human and bovine alpha-thrombin, but human prothrombin fragment 2 does not enhance the esterolytic activity of either human or bovine alpha-thrombin.