Inactivation of alpha- and beta- thrombin by antithrombin-III and heparin.

Inactivation of alpha- and beta-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b). It was found that the inactivation rates of alpha- and beta-thrombin by antithrombin were different, namely alpha-thrombin was more sensitive to antithrombin than beta-thrombin. Heparin facilitated the complex formation between alpha-thrombin and antithrombin-III, whereas beta-thrombin inactivation was only slightly affected. Furthermore, heparin protected alpha-thrombin against the inactivating effect of heat, while beta-thrombin lost its activity during the heat treatment. These findings suggest that the formation of beta-thrombin in blood circulation may have an important role in thrombosis predisposition.     

Effect of calcium ion on the interaction between thrombin and heparin. Thermal denaturation

Heat inactivation of thrombin at 54 degrees C followed first order kinetics with a rate constant of 1.0 min-1 approximately. Addition of heparin resulted in protection against thermal denaturation and, at the same time, rendered denaturation kinetics more complex. Analysis of the biphasic curve of heat inactivation in the presence of heparin revealed that the rate constants of the second phase changed systematically with heparin concentrations. Namely, at 4.5 x 10(-6)M, 9 x 10(-6)M, 1.8 x 10(-5)M and 3.6 x 10(-5)M heparin concentrations, the rate constants were 0.27 min -1, 0.17 min-1, 0.11 min-1 and 0.06 min-1, respectively. Sulfate as well as phosphate ions displayed also enzyme protection against heat inactivation, however, the same effect was obtained already at a heparin concentration, lower by three orders of magnitude. The kinetics of enzyme denaturation was not affected by calcium ions, whereas in the presence of heparin the inactivation rate of thrombin changed, i.e. calcium ions abolished the biphasic character of time course of thermal denaturation. Thus, the data suggest that calcium ions contribute to the effect of heparin on thrombin.     

Effect of a synthetic thrombin-inhibitor MD805 on the reaction between thrombin and plasma antithrombin-III

MD805, a synthetic thrombin-inhibitor, effectively retarded the time-dependent inactivation of thrombin which was generated endogeneously or added exogeneously in human plasma. The kinetical study of the time-dependent inactivation indicated that the type of inhibition was competitive and the obtained Ki of MD805 for thrombin was 3 × 10⁻⁸M. MD805 also inhibited the formation of thrombin-ATIII complex. These results indicated that the active site of thrombin was involved in the reaction between thrombin and ATIII, and that MD805 competed with ATIII for thrombin in exactly the same manner as it competed with fibrinogen or synthetic peptide substrates. As a result, MD805 would serve as a protective agent for ATIII from being consumed, in addition to its potent thrombin-inhibitory activity without the aid of ATIII. By contrast, heparin accelerated the time-dependent inactivation rate of thrombin and the formation of thrombin-ATIII complex, which indicates that heparin accelerates the consumption of ATIII.     

Heparin-like effect of polymethacrylic acid on the reaction between thrombin and antithrombin-III

The effect of polymethacrylic acid on the reaction between thrombin and antithrombin-III was investigated. It was found that polymethacrylic acid accelerated the inactivation rate of thrombin by antithrombin-III, whereas this polyanion did not affect thrombin activity. It is suggested that polymethacrylic acid may simulate the action of heparin on thrombin and antithrombin-III.     

Effect of heparin and thrombin on platelet adherence to the surface of rabbit aorta

Platelet adherence to both the damaged and undamaged surfaces of rabbit aorta in perfusion experiments in vitro was inhibited by heparin. The preparation of the vessel segments for these perfusion studies required 30 to 60 minutes because of the need to tie off all of the small vessels. In other experiments in which damaged or undamaged vessel segments were everted on a probe, the preparation of the vessels could be done more quickly and heparin did not inhibit platelet adherence. However, exposure of either the damaged or undamaged surfaces used in the latter type of experiment to thrombin markedly promoted platelet adherence to the surfaces. Heparin inhibited this effect. It seems likely that during the preparation of the aortas for the perfusion studies, thrombin generation occurred in the vessels. Thus, in preparing aortic segments for studies of platelet adherence, thrombin generation must be avoided. Since thrombin enhanced platelet adherence to undamaged endothelial surfaces, it may be that thrombin formation in vivo could cause platelet adherence to vessel walls from which endothelial cells have not been lost.     

人工骨材料磷酸钙及硫酸钙在腱-骨愈合中的作用

背景：有关促进腱-骨愈合的方法文献报道很多,主要方法就是给腱-骨间隙添加一些刺激物质,以此促进腱骨的愈合。磷酸钙盐作为生物活性材料具有骨传导性,已广泛应用于临床骨缺损的替代和填充。而硫酸钙作为人工材料,具有潜在的骨诱导活性。 目的：在自体腘绳肌肌腱移植重建膝关节前交叉韧带过程中,观察人工骨材料磷酸钙及硫酸钙促进腱-骨愈合的效应。 方法：选用36条雄性成熟比格犬,先行切断双侧膝关节前交叉韧带,取同侧后肢趾长屈肌腱作为移植物,采用悬吊式固定重建前交叉韧带。按随机数字表法分为3组,磷酸钙组于股骨腱骨隧道中注入磷酸钙,硫酸钙组注入硫酸钙,空白组韧带重建结束后不添加任何填充物。分别于重建后1,2,3,4,6个月取材行大体观察、组织学和生物力学观测。 结果与结论：前交叉韧带重建后1,2,3,4个月时,磷酸钙组及硫酸钙组腱骨界面纤维连接明显强于空白组,而磷酸钙组、硫酸钙组差异无显著性意义。6个月时,各组愈合程度相似。生物力学方面,重建后1个月时,磷酸钙组及硫酸钙组腱骨界面的抗拉脱强度均高于空白组(P < 0.05),而磷酸钙组、硫酸钙组差异无显著性意义(P > 0.05)。提示磷酸钙及硫酸钙均能促进腱-骨愈合,两者之间无明显差异。     

Studies on the binding of heparin to prothrombin and thrombin and the effect of heparin-binding on thrombin activity

Heparin binds with high affinity to thrombin, active as well as inactive, but not to prothrombin. The heparin-binding site therefore must be unmasked or created during prothrombin activation. The binding of heparin to thrombin has no effect on the amidase activity of the enzyme, as measured with a chromogenic substrate. All heparin molecules bind to thrombin with similar affinity, regardless of their affinity for antithrombin. The binding of heparin to antithrombin has been shown previously to be correlated with anticoagulant activity. The lack of a similar correlation between the binding of heparin molecules to thrombin and their anticoagulant activity demonstrates that heparin cannot function by binding only to thrombin, and in this manner facilitate the reaction of the latter with antithrombin. Binding of heparin to antithrombin is thus an essential feature of the anticoagulant activity of the polysaccharide.     

Comparison of the molecular mass dependency of heparin stimulation of heparin cofactor II:thrombin interaction to antithrombin III:thrombin interaction

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).     

Effect of rabbit thrombomodulin on thrombin inhibition by antithrombin in the presence of heparin

Thrombomodulin acts as a cofactor for protein C activation by thrombin (PC activation cofactor activity) and inhibits thrombin-induced fibrinogen clotting (direct anticoagulant activity). In addition, rabbit thrombomodulin has been shown to promote thrombin inactivation by antithrombin (AT-dependent anticoagulant activity). However, a non-acidic form (i.e. non-retarded on ion-exchange chromatography) of thrombomodulin generated by limited proteolysis retained only the PC activation cofactor activity. The acidic form (retarded on ion-exchange chromatography) of thrombomodulin is now shown to prevent the rapid inactivation of thrombin by antithrombin in the presence of heparin, presumably by preventing the formation of the ternary thrombin-AT-heparin complex. This effect was not observed with non-acidic thrombomodulin. When submitted to chondroitinase digestion, thrombomodulin was converted into an essentially non-acidic form that lacked both the AT-dependent and the direct anticoagulant activities but showed a PC activation cofactor function indistinguishable from that of native thrombomodulin. This chondroitinase-digested form did not prevent the catalytic effect of heparin on the inhibition of thrombin by AT. It is concluded that the acidic domain of rabbit thrombomodulin, a chondroitin (dermatan) sulfate glycosaminoglycan, interacts with a site of the thrombin molecule that is not involved in the protein C activation cofactor function, but is essential to the cleavage of fibrinogen or binding of heparin.     

Structural features of heparin and their effect on heparin cofactor II mediated inhibition of thrombin

Heparins from different species and tissues show similar levels of ATIII and HCII mediated anti-IIa activities. On fractionation, chains containing predominantly ATIII or HCII activities could not be separated. Oligosaccharide mapping demonstrates that the concentration of an oligosaccharide comprising a portion of heparin's ATIII binding site in a particular heparin fraction correlates with ATIII mediated anti-IIa activity, but does not correlate with HCII mediated anti-IIa activity. These results suggest that ATIII and HCII do not share a common binding site. Partial enzymatic depolymerization of heparin resulted in large oligosaccharides which could be purified and partially characterized. Although oligosaccharides of degree of polymerization (dp) 18 and 20 showed significant ATIII and HCII mediated anti-IIa activities no separation of these activities resulted. These data suggest however that a minimum chain length of dp18 was required for HCII mediated anti-IIa activity.     

Purification of thrombin by affinity chromatography on immobilized heparin

Bovine thrombin has been purified from commercial crude thrombin preparations by affinity chromatography on immobilized heparin. This procedure was shown to be superior to affinity chromatography on matrix-linked synthetic low-molecular-weight thrombin inhibitors in that the purified enzyme could be conveniently eluted by a salt gradient and also was of higher purity. The degree of purification of the affinity chromatography step was 30 times and the purified material had a specific activity of about 2000 NIH units/mg. Dodecyl sulphate-polyacrylamide gel electrophoresis indicated the presence of the native two-chain form of thrombin as well as of several forms of thrombin with specific proteolytic cleavages in the B-chain. These modified forms were similar to those also found in thrombin preparations obtained from crude commercial thrombin by other methods. Affinity chromatography on heparin-agarose was shown to be useful also for the purification of thrombin from a prothrombin activation mixture.     

凝血酶对原代培养海马神经元游离钙浓度的影响

目的研究凝血酶对原代培养海马神经元内游离Ca^2 水平的影响。方法采用原代培养大鼠海马神经元方法，用钙离子指示剂Fura-2双波长荧光检测凝血酶对海马神经元内游离钙浓度的影响。结果(1～40)U／ml凝血酶可使海马神经元内游离Ca^2 水平显著升高，且呈剂量依赖性。凝血酶受体激活肽可明显升高细胞内游离[Ca^2 ]i。当胞外Ca^2 为1．3mmol／L时，40U／ml凝血酶可使海马神经元游离[Ca^2 ]i明显增加；而当胞外Ca^2 为0．0mmol／L时，40U／ml凝血酶不影响海马神经元游离[Ca^2 ]i。预先加入MK-801可显著降低凝血酶的升钙作用。结论凝血酶使神经细胞内游离Ca^2 浓度异常升高的作用机制可能是通过激活凝血酶受体，继而激活NMDA受体门控的Ca^2 通道介导胞外Ca^2 大量内流。     

凝血酶对原代培养海马神经元游离钙浓度的影响

目的研究原代培养的海马神经元内游离Ca2 水平及凝血酶的影响.方法大鼠海马神经元进行体外原代培养,用钙离子指示剂Fura-2双波长法测定海马神经元内游离[Ca2 ]i及不同浓度的凝血酶作用后细胞内[Ca2 ]i.结果原代培养的海马神经元生长旺盛,密度高,符合实验要求.在胞外Ca2 浓度为0.0 mmol/L时,静息状态下海马神经元游离[Ca2 ]i为(79.83±18.78)nmol/L.当胞外Ca2 浓度为1.3 mmol/L时,海马神经元游离[Ca2 ]i为(106.41±22.53)nmo1/L.(1～40)U/ml凝血酶可使海马神经元内游离Ca2 水平显著升高,与对照组相比均有显著性差异(P＜0.01).随凝血酶浓度的增加,胞内游离[Ca2 ]i之呈剂量依赖性增加.结论凝血酶可使原代培养的海马神经元内游离Ca2 浓度明显升高.     

Decreased binding of heparin to antithrombin following the interaction between antithrombin and thrombin

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.     

Effect of metabolic inhibition with piperonyl butoxide on rodent sensitivity to tri-ortho-cresyl phosphate

The effect of metabolic interference on the onset of organophosphate neuropathy in rats was examined. Long-Evans hooded, male rats were exposed by gavage to single, weekly doses of tri-ortho-cresyl phosphate (TOCP) (1160 mg/kg). Others were pretreated with 50 mg/kg of the mixed-function oxidase inhibitor, piperonyl butoxide (PiPB), 1 h before administering TOCP. The animals were killed after three treatments and their spinal cords, various peripheral nerves, and livers were examined microscopically. Rats treated with PiPB in combination with TOCP showed significantly more damage to both central (spinal cord) and peripheral nervous tissues than those treated with TOCP alone. These data indicate that PiPB can potentiate TOCP-induced neuropathy in rats and suggest that the rodent's resistance to organophosphate neuropathy may have a hepatic basis.     

Antithrombin III and heparin inactivation in thrombin involving reactions

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.