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1.
The inhibition of thrombin and plasmin by antithrombin III was studied in the presence of heparin conjugated to Sepharose. When either enzyme was adsorbed to the heparin conjugate in quantities insufficient to occupy all the affinity sites, subsequent passage of antithrombin III through the column invariably produced complete inhibition as measured by the response to the chromogenic substrate S2160. However, when the loading sequence was reversed (i.e. adsorbing non-saturating quantities of antithrombin III before the enzyme), antithrombin III only insignificantly inhibited either enzyme over a 20-hour period. A second load of antithrombin III following the thrombin load resulted in complete inactivation. When thrombin was rendered incapable of binding to heparin by cyclohexanedione treatment and reacted with Sepharose-heparin to which antithrombin III was adsorbed, the protease inhibitor exhibited ‘progressive’ antithrombin activity. These studies may indicate that thrombin and plasmin possess higher affinities for heparin than for the antithrombin III-heparin complex. Furthermore antithrombin III reacts more readily with the enzyme-heparin complex than with heparin. If the relative affinities of heparin, thrombin and antithrombin III in plasma are similar to those observed under these affinity chromatographic conditions, then the present data are consistent with the view that heparin promotes the inactivation of thrombin and plasmin by augmenting their reaction with antithrombin III. An allosteric effect of heparin on thrombin is apparent from the 50% increase in esterase activity of thrombin on α-N-benzoyl-L-arginine ethyl ester which is observed in the presence of optimal concentrations of heparin. 相似文献
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Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca(2+)-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Antithrombin III (At-III) purified from human plasma was incubated with thrombin (T), activated factor X (Xa) or mixtures of T and Xa. The amount of enzymes inactivated by the amount of At-III in one ml of normal plasma was determined. - At-III which had inactivated one enzyme, was incapable of inactivating the other. Inactivation of Xa and T by At-III was found to proceed like a second order reaction. The inactivation rate was highly dependent on the At-III concentration, the half-time values of the enzyme activities decreasing with increasing At-III concentration. With excess inhibitor, variations in the concentration of enzyme had little influence on the inactivation rate. Bovine Xa was more rapidly inactivated than bovine T. In contrast, human T was more rapidly inactivated than human Xa. 相似文献
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The ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assays. There was good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical. For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma. 相似文献
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E Marciniak 《Thrombosis and haemostasis》1977,38(2):486-493
The inhibitory capacity of antithrombin III (AT III) was measured by a quantitative method independent of the velocity of inhibition. When AT III was in excess of thrombin in plasma or in purified system the capacity of inhibitor decreased quantitatively in proportion to the amount of thrombin neutralized. Heparin present in reaction together with thrombin invariably induced a more extensive utilization of inhibitor than thrombin alone. The extent of this additional loss of inhibitory capacity was to a limited degree related to the concentration of heparin. Heparin itself was neutralized in thrombin-AT III reaction losing its anticoagulant property in proportion to the amount of thrombin bound by inhibitor. This quantitative neutralization of heparin occurred not only when the anticoagulant participated in thrombin-AT III binding but also when heparin was added to a medium containing a preformed thrombin-AT III complex. These results suggest that acceleration of binding and increased utilization of binding capacity are the two regular effects of heparin on thrombin-involving reactions of AT III. Both of these effects may be abolished by quantitative binding of heparin to thrombin-AT III complex. 相似文献
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The effect of human thrombomodulin on the inactivation of thrombin by human antithrombin III 总被引:1,自引:0,他引:1
The effect of human thrombomodulin (TM) on the inactivation of thrombin by human antithrombin III (ATIII) was evaluated in comparison with that produced from rabbits. Human TM did not accelerate the thrombin inhibition by ATIII but rabbit TM enhanced the activity of ATIII. Also inclusion of human TM at increasing concentration suppressed the thrombin inhibitory activity of ATIII. The intensity of ATIII activity in the presence of heparin (0.01U/ml) was also diminished by the human TM. However, this ATIII- heparin cofactor activity recovered with the addition of a 10-fold amount of heparin (0.1U/ml). In SDS-polyacrylamide gel electrophoresis and immunoblotting analysis, we found a complex formation of ATIII with both human and rabbit TM (and further confirmed their presence with isoelectrofocusing electrophoresis- data not shown). These results indicate that human TM is substantially different from rabbit TM. Our results suggest that human TM show the crucial role on protein C activation system via thrombin. 相似文献
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J Z Mortensen 《Thrombosis research》1984,33(5):511-515
A family with inherited AT-III deficiency is presented. This variant is unlike those formerly described, as the plasma concentration is high, the thrombin inhibition with heparin is slow, and factor Xa inhibition is decreased. 相似文献
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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. 相似文献
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The influence of increasing concentrations of heparin of different molecular mass (Mr) has been compared in potentiation of the rate of heparin cofactor II:thrombin interaction and of antithrombin III:thrombin interaction. Unfractionated and fractionated heparin showed a concentration dependent ascending and descending limb of stimulation of the rate for both inhibitors. Unfractionated heparin and fractions of 16.5 KDa or less showed a peak acceleration of the rate of interaction of thrombin with both inhibitors at 0.3 X 10(-6) M heparin although the observed maximum rate at this peak decreased with fall in Mr. For both inhibitors two high Mr fractions showed peak stimulation at a lower heparin concentration (0.3 X 10(-7) M) and approximately two-fold greater increase in rate than that observed with unfractionated heparin. Potentiation of heparin cofactor II inhibitory activity differed from that of antithrombin III in that it was reversed by lower ionic strength and was not reversed by a heparin pentasaccharide with high affinity for antithrombin III. It is proposed that differences in the profiles of stimulation by high Mr fractions to those of lower Mr are related to higher binding affinities for the inhibitor permitting maximal binding of heparin before the descending part of the slope due to saturation of thrombin (according to the template hypothesis). 相似文献
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Numerous plants were tested for inhibitors of human Hageman factor fragment (HFf), plasma kallikrein, urinary kallikrein, plasmin, thrombin, porcine pancreatic kallikrein, and bovine Factor Xa, trypsin, and chymotrypsin. Pumpkin seeds and iris bulbs contain trypsin inhibitors which specifically inhibit HFf. Flower bulbs—especially those of tulip, lily, hyacinth, and calla—are hitherto unrecognized rich sources of inhibitors with different inhibitory spectrums and physicochemical properties. 相似文献
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A complex formed between bovine antithrombin and bovine thrombin was shown to elute from a heparin-agarose column at a lower ionic strength than free antithrombin. Moreover, it was demonstrated that less heparin remained attached to the antithrombin-thrombin complex than to free antithrombin, when mixtures of high-affinity heparin and either complex or antithrombin were separated by gel electrophoresis or gel chromatography under identical conditions. These results suggest that the interaction between antithrombin and thrombin leads to a decreased affinity of heparin for the antithrombin moiety of the complex. This decrease may be an essential feature of the proposed function of heparin as a catalyst in the antithrombin-thrombin reaction. 相似文献
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QUESTION: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties? APPROACH: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex. RESULTS: The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (Kd=1.86+/-0.13 microM) binds 11 times tighter than factor Xa (Kd=20.2 +/-1.5 microM). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain). CONCLUSION: The data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT. 相似文献
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To investigate the physiologic function of heparin cofactor II (HCII), endothelial cells from human umbilical vein were incubated in vitro for 20 min with 0.5 NIH U/ml thrombin in the presence of HCII or antithrombin III (ATIII), and prostacyclin production determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Although ATIII at 20 mInh.U/ml slightly but significantly inhibited thrombin-induced prostacyclin production, neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) at 1 U/ml accelerated the inhibitory effect of ATIII. HCII at 10 and 20 mInh.U/ml did not decrease thrombin stimulation of prostacyclin production in the presence or absence of UFH or LMWH. However, HCII caused a marked decrease in the thrombin-stimulated prostacyclin prostacyclin production in the presence of 2 mg/ml dermatan sulfate (DS). The significant inhibition by HCII occurred when the DS concentrations were 0.2 microgram/ml and higher. From these results we suggest that HCII may prevent a prostacyclin-induced inhibition of platelet aggregation for hemostasis when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of DS. 相似文献
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Alan D. Cardin Roger L. Barnhart Kathleen R. Witt Richard L. Jackson 《Thrombosis research》1984,34(6):541-550
The heparin-binding properties of human plasma apolipoproteins B-100 and E (apoB-100 and E) of low density lipoproteins (LDL), thrombin, and antithrombin-III (AT-III) were investigated. A highly reactive heparin (HRH) to apoB-100 was isolated by chromatography of crude heparin on a column of LDL immobilized to Affi-Gel 10. This HRH showed a high, Ca2+-dependent precipitating activity towards LDL; 1 μg HRH uronic acid precipitated 50–70 μg LDL-protein. HRH was fractionated further by chromatography on a column of AT-III bound to concanavalin A-Sepharose. The unretained fraction of heparin (HRH1)had a low affinity for AT-III. The bound heparin (HRH2) had a high affinity for AT-III and precipitated LDL in the presence of Ca2+. To assess further their heparin-binding properties, the proteins were subjected to gradient-gel electrophoresis under denaturing conditions, transferred to nitrocellulose by electrophoresis, and then assayed for their ability to bind [125I]-labeled HRH2. Autoradiographic analysis showed that thrombin, apolipoproteins E and B-100, and the AT-III-thrombin covalent complex bound HRH2. Denatured AT-III did not bind HRH2, indicating that its heparin recognition site may depend on conformation. 相似文献
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The regulation of plasmin, miniplasmin, and streptokinase-plasmin complex (SkPm) was studied in vitro in the presence of unfractionated porcine intestinal heparin using purified plasma proteinase inhibitors. Heparin enhanced the reaction of antithrombin III (AT) with plasmin (up to 40-fold with 20 units/ml). The rate of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) and by alpha 2-macroglobulin (alpha 2M) was not changed by heparin (0.5-100 units/ml); the rank-order of plasmin-inhibitory activity remained alpha 2AP greater than alpha 2M greater than AT. The reaction of miniplasmin with AT was studied also. The second order rate constant was 9.2 x 10(2) M-1s-1 without heparin and 2.6 x 10(4) M-1s-1 in the presence of 20 units/ml heparin. Heparin did not affect the rank-order of miniplasmin-inhibitory activity; it remained alpha 2M greater than alpha 2AP greater than AT. While the reaction of AT with SkPm was negligible, heparin stimulated this reaction dramatically. The SkPm-inhibitory activity of alpha 2AP was not changed by heparin. When plasma concentrations of alpha 2AP (1.05 microM) and AT (4.76 microM) were compared, AT inhibited greater amounts of SkPm in the presence of more than 5 units/ml of heparin. The increased SkPm-inhibitory activity of AT in heparin did not result from SkPm dissociation, and heparin did not decrease the rapid rate of streptokinase association with plasmin. These studies demonstrate that heparin can affect the regulation of fibrinolysis at multiple levels of the enzyme cascade. 相似文献