Search

Amidolytic activity of thrombin for Boc-Val-Pro-Arg-MCA was inhibited by CaCl2 at relatively high concentrations. Kinetic analysis showed neither competitive nor noncompetitive inhibition by CaCl2 of thrombin. The nonspecific interaction of thrombin with calcium ions prolonged fibrinogen-clotting and neutralization by antithrombin III (AT III) in the absence of heparin. However, calcium ions had no effects on rate of thrombin inactivation in dilute solution and affinity of the enzyme for Sepharose 6B. The inverse effects of calcium ions on the thrombin activities in vitro and on generation of the enzyme in plasma ex vivo depended mostly on the ion concentration.

Thrombin-clotting time of fibrinogen was delayed by inositol hexasulfate (IHS). Clotting time of the recalcified plasma was also delayed by IHS. Moreover, when IHS was intravenously injected into anesthetized male rabbits (0.11 mmoles/kg), it delayed clotting time of the recalcified plasma. IHS prevented generations of factor Xa and thrombin in the recalcified plasma. It is probable that IHS inhibits the clot-formation of the recalcified plasma in competition with calcium ions.

Less than a stoichiometric amount of bovine chymotrypsin was progressively inactivated by a bovine AT III preparation. The rate of inactivation was not influenced by a synthetic substrate. Since chymotrypsin has low affinity for heparin, the polysaccharide only doubled the inactivation of the enzyme by the AT III preparation. At a low salt concentration, about two-thirds of the chymotrypsin was instantaneously inactivated by the AT III preparation in the presence of heparin. These observations indicate that inactivation of chymotrypsin is due to AT III itself, not to another contaminating inhibitor(s).

Inactivation of thrombin was enhanced by dextran sulfate (DS) coated on polypropylene tubes with plastic adhesive. Long interaction of thrombin with DS was not essential to enhance the enzyme inactivation. Albumin inhibited the inactivation of thrombin by DS-coated tubes, but had no effect after interaction of the enzyme with DS. NaCl, especially, sodium ions, inhibited the inactivation by DS-coated tubes. Enzyme inactivation occurred at 37 degrees C, but not at 4 degrees C. Thus, the inactivation was due not only to ionic interaction with DS, but also to intramolecular perturbation of thrombin.

Heparin with low affinity (LA-heparin) to antithrombin III (AT III) enhanced the rate of inactivation of thrombin by AT III. The enhancement of the rate was saturable with AT III and was proportional to the LA-heparin concentration. Although the rate-enhancement in the presence of LA-heparin decreased with increase in NaC1 concentration, it was comparable with that in the presence of high affinity-heparin (HA-heparin) in the absence of NaC1. Inactivation of thrombin by AT III in the presence of dextran sulfate (DS) was also sensitive to NaC1 concentration. These findings indicate that free AT III is favorable for binding to the complexes of thrombin and highly sulfated polysaccharides having low affinities to AT III in the absence of NaC1.

A pico-molar concentration of thrombin was significantly inactivated by antithrombin III (AT III). The pseudo-first order rate constant, k'app, and the second order rate constant, k", were inversely proportional to the concentration of thrombin. The k'app and k" values increased and decreased, respectively, with increase in the concentration of AT III. All of these results indicated that the rate of the thrombin/AT III reaction was determined with the molar ratio of the two rather than their absolute concentrations.

Amidolytic activity of thrombin increased proportionally with NaCl concentrations up to 1 M NaCl, due to an increase in the turnover rate with no change in the Km of the enzyme. Neutralization of thrombin by antithrombin III (AT III) was maximum in 0.1 to 0.15 M NaCl, but completely absent at high NaCl concentrations. Moreover, ratio of rates of the amidolytic activity of thrombin to of the enzyme/AT III reaction was not constant at several pHs. The rate of the thrombin/AT III reaction appeared to be inversely correlated with the enzyme's stability. Therefore, it was concluded that some factor(s) other than the active site of the thrombin molecule was more important in its primary interaction with AT III.

The rate of the thrombin/antithrombin III (AT III) reaction was decreased in the presence of free polybrene or protamine. The reaction rate was also decreased in protamine-coated tubes and tubes on which polybrene was absorbed nonspecifically. The reaction was also prevented when either thrombin or AT III was transferred into noncoated tubes after instantaneous contact with protamine-coated tubes or tubes with polybrene. These facts suggest that the thrombin/AT III reaction rate is determined with concentrations of reactive protein species.

Dextran sulfate and polybrene were immobilized on polypropylene tubes with plastic adhesive. Serum albumin was also immobilized on the tube by heat-denaturation. In the those tubes thrombin was protected from inactivation in the presence of 0.1 M NaCl remarkably. Cholesterol coated on the tube also protected the enzyme from inactivation. The results indicate that amounts of thrombin protected by these four coating materials corresponded to the amount of thrombin inactivated on surface of the tube. At least half of the thrombin was also inactivated within 20 sec at 37 degrees C in the presence of quartz sand, supporting a significant contribution of a solid surface to temperature-dependent inactivation of enzyme in dilute solution.

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin. Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme. These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.

Proteolysis of fibrinogen by bovine trypsin and chymotrypsin was enhanced by heparin, dextran sulfate (DS) and polyvinyl sulfate (PVS) in the presence of 0.1 M NaCl. Decrease in intrinsic fluorescence of fibrinogen with time was also enhanced by DS and PVS in the absence of NaCl, but not in the presence of 0.1 M NaCl. Thus, increase in susceptibility of fibrinogen to proteases in the presence of three polysulfate was more sensitive than time-dependent conformational change of the substrate protein.

At high thrombin concentration, loss of enzyme activity was proportional to the initial enzyme concentration. At low thrombin concentration, loss of activity followed first order kinetics, indicating mainly denaturation (inactivation) of the enzyme. Dextran sulfate stimulated thrombin denaturation, while serum albumin and polyethylene glycol 6000 (PEG) protected the enzyme from denaturation. These results indicate that the enzyme molecule is stabilized by weak interactions with albumin and PEG, but destabilized by strong interaction with dextran sulfate.

Of four Tris-salts tested (chloride, sulfate, phosphate and acetate), chloride caused complete elution of antithrombin III (AT III) from a heparin-Sepharose column and sulfate caused partial elution. AT III was also partially eluted from the column with sodium acetate, but not Tris acetate. On the other hand, thrombin was eluted from the column with the Tris-salts in the order chloride greater than sulfate greater than acetate, but was not eluted with Tris-phosphate. Thrombin was also eluted from the column with sodium acetate. These findings indicate that the affinity of heparin to AT III was influenced only by strongly electronegative ions, whereas its affinity to thrombin was affected by both strongly electropositive and strongly electronegative ions.

Dextran sulfate (DS)-enhanced inactivation of alpha-thrombin was faster at pH 6 than at pH 8 in the absence of NaCl. Substrate protected only partly thrombin from inactivation by DS at pH 6. Some amidolytic activity of alpha-thrombin inactivated by DS was restored time-dependently by the addition of NaCl. Moreover, beta and gamma-thrombin, and other intermediates were not formed from alpha-thrombin with DS-enhanced inactivation. All these results indicate that autolysis of alpha-thrombin in dilute solution is not responsible for enhancement of reversible inactivation of alpha-thrombin by DS.