Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.     

Substitution of antithrombin III by synthetic thrombin inhibitors

Synthetic, low-molecular weight thrombin inhibitors may substitute for antithrombin III in inactivating thrombin in vitro and in vivo. They are superior to heparin as antithrombotic agents in antithrombin deficiency or consumption. Studies in experimental animals show the effectiveness of synthetic thrombin inhibitors in immunologic antithrombin depletion, thrombin-induced consumption and CCl4-induced liver failure.     

Kinetics of thermal denaturation of antithrombin III

Purified antithrombin III (AT III), a single-chain human plasma glycoprotein, molecular weight 58,000 daltons, and one of the major serine protease inhibitors, was heated in the 60-70 degrees C range for inactivating possible contaminations by hepatitis B virus (HBV). Loss of inhibitory activity, unfolding of tertiary structure, and the rate of aggregate formation of AT III were monitored experimentally during heatig. Sucrose and sodium citrate were demonstrated to stabilize the protein. From the rate data the calculated activation energies (E) showed E(tert. struct.) < E(biol. act.) < E(aggreg.) indicating the order (lower activation energy process first) in which heat causes these changes in the protein molecule. The activation energy corresponding to denaturation of HBV was estimated to be at least fourfold lower than that associated with the unfolding of the tertiary structure of the protein. Purified AT III, thus stabilized and pasteurized, should be therapeutically effective, and the risk for transmission of hepatitis B should be decreased significantly.     

Ostrich antithrombin III: kinetics and mechanism of inhibition of ostrich thrombin

A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.     

Potentiation by calcium ions of the antithrombin III inhibition of thrombin

Calcium ions potentiated heparin-modulated antithrombin III inhibition of amidolysis catalysed by thrombin. Potentiation by calcium ions of heparin-independent antithrombin III inhibition of thrombin activity appeared to contribute to this effect. These results suggest a complex modulatory role for calcium ions in proteinase-catalysed reactions influenced by anti-proteinases and glycosaminoglycans.     

Conformational changes accompanying the binding of antithrombin III to thrombin.

The conformational aspects of the binding of antithrombin III to thrombin were investigated by difference spectroscopy, circular dichroism, and optical rotatory dispersion. The CD and ORD studies indicate an increase of 6--8% in alpha-helix content at the expense of the beta structure, while the results from difference spectroscopy showed an increased exposure of approximately seven tyrosine residues. In the presence of heparin there is a slightly greater increase in helicity which is accompanied by exposure of an average of two tryptophan and one tyrosine residues. These spectral results indicate that the thrombin-antithrombin III complex formed in the presence of heparin differs in its conformation from that produced in its absence.     

荧光标记脂肪酶的固定化及其稳定性

将标记有荧光探针FITC(异硫氰基荧光素)的脂肪酶固定化,通过测定活性和荧光光谱,探究各种因素对固定化后荧光标记脂肪酶性质的影响,并分析活性、构象和荧光光谱三者之间的联系。研究结果表明:在固定化脂肪酶过程中,聚乙二醇400二丙烯酸酯能形成合理的网格结构,使酶活较高;配体诱导酶的催化构象,使酶活性提高到未诱导酶的2倍以上;配体抽提能使脂肪酶活性中心得到释放从而提高催化活力。固定化脂肪酶的稳定性大大提高,在90℃、强酸强碱下固定化酶仍保有原酶70%、60%以上的活性;用盐酸胍、脲等溶解变性剂浸泡15d后,酶活性仍然可以保持初始活性的70%以上。荧光光谱能较好地反映脂肪酶的活性和构象变化,最适pH和温度下脂肪酶的荧光强度最低,在溶解变性剂中,荧光强度随时间延长而逐渐降低,这表明不同条件下脂肪酶构象经历的去折叠过程不同。     

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor

-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human -thrombin shows multiple surface exosites for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). _T ^–-Thrombin and thrombin Quick I (Arg 67 Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. -Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (M_r = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5phosphate modifiedthrombin.     

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.     

Interaction of heparin and antithrombin III. The role of O-sulfate groups

A synthetic pentasaccharide corresponding to the sequence involved in heparin for binding and activation of antithrombin III contains eight sulfate groups. The role of some of them in the interaction with the protein has been demonstrated through the study of fragments obtained from heparin. An approach based on the total chemical synthesis of heparin fragments allows us to provide new information on the O-sulfate groups borne by the iduronic acid and the glucosamine units that constitute the reducing-end disaccharide of the above pentasaccharide sequence. Although not strictly necessary for a weak interaction to take place, these two sulfates co-operate to express maximal activity. This suggests that they belong to a secondary sub-region of interaction with antithrombin III, the primary one being accounted for by other critical parts of the structure and particularly the trisaccharide sequence placed at the non-reducing end of the pentasaccharide.     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding affinity of heparin for thrombin

The present study has shown that calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction. The initial rate of thrombin (4.0 nM) inhibition by antithrombin III (200 nM) in the presence of heparin (2.5 ng/ml) decreased from 3.6 nM/min (in the absence of calcium) to 0.12 nM/min in the presence of 10 mM calcium. In the absence of heparin, the initial rate of thrombin inhibition by antithrombin III was not affected by calcium. The heparin-catalyzed antithrombin III/thrombin reaction is described by the general rate equation for a random-order, bireactant, enzyme-catalyzed reaction (M. J. Griffith (1982) J. Biol. Chem. 257, 13899-13902). As such, the reaction is saturable with respect to both thrombin and antithrombin III. The apparent kinetic parameters for the heparin-catalyzed antithrombin III/thrombin reaction were determined in the presence and absence of calcium. The apparent heparin/antithrombin III dissociation constant values were not measurably different in the presence of 0, 1.0, and 3.0 mM calcium. The apparent heparin/thrombin dissociation constant value increased from 7.0 nM, in the absence of calcium, to 10 and 30 nM in the presence of 1.0 and 3.0 mM calcium, respectively. The maximum reaction velocity, at saturation with respect to both proteins, was not affected by calcium. It is concluded that calcium binds to functional groups within the heparin molecule which are required for thrombin binding.     

Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [³H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.     

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.     

Microscale fluorescent thermal stability assay for membrane proteins

Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).     

Dependence of antithrombin III and thrombin binding stoichiometries and catalytic activity on the molecular weight of affinity-purified heparin

Heparin was fractionated by affinity chromatography on immobilized antithrombin III followed by gel filtration on Sephadex G-100. Eighteen fractions were obtained ranging in molecular weight from 9,700 to 34,300 as determined by sedimentation equilibrium. The binding stoichiometries of antithrombin III and thrombin interactions with the heparin of these fractions were measured, using changes in intrinsic and extrinsic fluorescence. Catalytic activity also was measured for each of the heparin fractions. As the molecular weight of heparin varied from about 10,000 to 30,000, the average number of antithrombin and thrombin sites/heparin molecule varied from 1.0 to 2.1 and 2.4 to 6.8. In addition, the molar specific activity increased 5.7-fold, an increase which correlated directly with the product of the number of antithrombin III and thrombin molecules bound. Thus as the number of bound molecules increased with increased molecular weight, the rate of reaction/bound antithrombin III increased in proportion to the number of bound thrombin molecules and vice versa. This can be explained by assuming that heparin functions as a template for both proteins, that all bound thrombin and antithrombin III molecules are accessible to each other, and that the rate at which a bound molecule reacts is proportional to the number of molecules of its interacting counterpart bound. These observations and conclusions are similar to those of Hoylaerts et al. (Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Biol. Chem. 259, 5670-5677), who demonstrated that the rate at which single molecules of antithrombin III, covalently attached to heparin, react increases as the thrombin binding capacity (chain length) of heparin increases.     

Effect of heparin modification on its activity in enhancing the inhibition of thrombin by antithrombin III

Studies were conducted to determine the effect of modifying specific functional groups of heparin on its antithrombin III-enhancing activity. The derivatives employed were heparin methyl ester, heparinylglycine and N-desulfated heparin. The carboxyl-modified derivatives increase the rate of inhibition of thrombin by antithrombin III, although not to the same extent as heparin. N-Desulfated heparin is devoid of any activity. Heparin methyl ester is more potent than heparinylglycine in activating antithrombin III, as exhibited by its immediate effect on the thrombin-fibrinogen reaction. However, heparinylglycine is the more effective of the two, in increasing the rate of thrombin deactivation by antithrombin III. The results indicate that although free carboxyl groups of heparin are not crucial for its binding to antithrombin III, they are important for the combination of the latter with thromobin. In contrast, N-sulfates are critical for the interaction of heparin with antithrombin III.