Influence of hirudin on the consumption of antithrombin III in experimental DIC

The effect of hirudin and heparin on thrombin-induced consumption of antithrombin III, fibrinogen and platelets was studied in a rat model. Antithrombin III is consumed by tolerated thrombin doses by about 20 per cent. Hirudin and heparin ameliorate the consumption of fibrinogen and platelets at the low thrombin dose used. At high thrombin doses, tolerated only during simultaneous administration of exogenous inhibitors, heparin leads to markedly increased consumption of anti-thrombin III, whereas hirudin does not. With either kind of treatment, the thrombin effect on fibrinogen and platelets is inhibited, however, hirudin acts independently of a cofactor in contrast to heparin.     

Comparison of the inhibition of thrombin by three plasma protease inhibitors.

Human alpha-thrombin is inhibited by the circulating protease inhibitors alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin. Kinetic analyses of the inhibitor thrombin interactions were carried out utilizing either fibrinogen or the synthetic substrate Bz-Phe-Val-Arg-p-nitroanilide as substrates to determine residual thrombin activity. These studies demonstrated that the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin followed second-order kinetics. The rate constants for the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin are 6.51 +/- 0.38 x 10(3), 3.36 +/- 0.34 x 10(5), and 2.93 +/- 0.02 x 10(4) M-1 min-1, respectively. Comparison of the second-order rate constants and the normal plasma levels of the three inhibitors demonstrates that, under the in vitro conditions utilized, antithrombin III is five times and alpha2-macroglobulin is one-third as effective as alpha1-antitrypsin in the inhibition of thrombin.     

Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.

The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

Are factor Xa inhibitors superior to thrombin inhibitors in anticoagulation?

Protease inhibitors are useful tools for increasing the inhibitor potential of plasma. In this context, thrombin inhibitors attracted special interest. However, other clotting enzymes, especially factor Xa, are target enzymes of protease inhibitors besides thrombin. Our studies on structure-activity relationships of benzamidine derivatives resulted in selective inhibitors of thrombin and factor Xa. The use of these inhibitors enabled us to clarify whether the antithrombin activity or the anti-factor Xa activity of a compound is more efficient in anticoagulation. We assessed the concentration-dependent inhibition of the activated partial thromboplastin time by these compounds. If one correlates the inhibitor concentration, which prolonged the clotting time by 60 s, with the dissociation constants one will realize that thrombin inhibition is significantly more efficient in anticoagulation than inhibition of factor Xa.     

Evolution of Serpin Specificity: Cooperative Interactions in the Reactive-Site Loop Sequence of Antithrombin Specifically Restrict the Inhibition of Activated Protein C

Protease cascades and their inhibitors are a common feature of many biological regulatory systems, and the various components of such cascades have been subjected to a long and concerted evolution. We present here evidence that in the coagulation cascade, the sequence of the protease-binding reactive-site loop of antithrombin has evolved such that the majority of its residues has been acquired not for the efficient inhibition of its target proteases, thrombin and factor Xa, but to avoid the inhibition of activated protein C (APC). We substituted residues of the reactive-site loop of antithrombin into α₁-antitrypsin and tested the chimeras against thrombin, factor Xa, and APC. With respect to factor Xa and thrombin, the difference in association rate between the fastest and the slowest inhibitors was 5.5- and 88-fold, respectively. However, with respect to APC the difference was 12,500-fold. While most of the variation in the inhibition rates of thrombin could be accounted for by P2 Gly-to-Pro substitutions, for APC almost every residue had an effect on inhibition. In 22 of 25 direct comparisons of antitrypsin residues with antithrombin residues, either singly or in blocs, the antithrombin residues caused a decrease in the rate of inhibition of APC. The antithrombin residue Asn393, at position P′3, emerged as particularly important for avoiding the inhibition of APC, however, its 190-fold effect was seen only when in conjunction with antithrombin P7 to P′2 residues. Cooperative effects among residues of the reactive-site loop thus emerged as critical for restricting the activity of this sequence against APC. Received: 15 November 1999 / Accepted: 2 August 2000     

Orally active thrombin inhibitors. Part 2: optimization of the P2-moiety

Synthesis and SAR of orally active thrombin inhibitors of the d-Phe-Pro-Arg type with focus on the P2-moiety are described. The unexpected increase in in vitro potency, oral bioavailability, and in vivo activity of inhibitors with dehydroproline as P2-isostere is discussed. Over a period of 24h the antithrombin activity of the most active inhibitors with IC(50)s in the nanomolar range was determined in dogs demonstrating high thrombin inhibitory activity in plasma and an appropriate duration of action after oral administration.     

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.     

Phenolic P2/P3 core motif as thrombin inhibitors--design, synthesis, and X-ray co-crystal structure

Prototypical thrombin inhibitors were synthesized based on a trisubstituted phenol as a core motif. A naphthylsulfonamide analogue showed excellent antithrombin activity. An X-ray co-crystal structure showed the expected interactions.     

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.     

The effect of bovine thrombomodulin on the specificity of bovine thrombin

Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.     

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.     

Serum antithrombin III and alpha-2-antiplasmin concentration in patients with lung carcinoma

Serum antithrombin III and alpha-2-plasmin inhibitor concentrations has been evaluated in 26 patients with lung carcinoma. We observed a twofold decrease in antithrombin III level and no differences between test and control groups in alpha-2-plasmin inhibitor concentrations. Evidently, the decreased antithrombin III level may reflect its consumption because of enhanced plasma thrombin activity, whereas normal alpha-2-plasmin inhibitor may result in no induction of secondary fibrinolysis followed by the stimulation of the coagulation system or no conditions for primary increased fibrinolysis. It seems possible, that the antithrombin III level in the serum may at least partly reflect the tendency for hypercoagulability and spreading of cancer.     

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.     

Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction.

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.     

Pharmacology of benzamidine-type thrombin inhibitors

Findings on the pharmacology of derivatives of benzamidine characterized as potent competitive inhibitors of the clotting enzyme thrombin were presented including data on their toxicity. Bis-benzamidines and amidinophenylalanine amides were shown to exert pharmacodynamic effects in intact animals and isolated preparations (hypotension; influence on smooth muscle reactions to serotonin and histamine). Studies with 14C-4-amidinophenylpyruvic acid and 3H-N alpha-tosyl-(3-amidino)-phenylalanine piperidide on the pharmacokinetics in rabbits showed that benzamidine derivatives are suited, in principle, for use as anticoagulants in vivo. Pharmacokinetic properties of individual thrombin inhibitors have to be taken into account in order to reach and maintain adequate plasma levels. The antithrombotic effect of benzamidine-type thrombin inhibitors in animals experiments is directly related to their antithrombin activity.     

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.     

Design, synthesis and biological evaluation of selective boron-containing thrombin inhibitors.

Based on the structural comparison of the S-1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesised as inhibitors of thrombin. The influence of hydrogen donor/acceptor properties of different residues in the P-1 side chain of these inhibitors on the selectivity profile has been investigated. This study confirmed the structure-based working hypothesis: The hydrophobic/hydrophilic character of amino acid residues 190 and 213 in the neighbourhood of Asp 189 in the S-1 pocket of thrombin (Ala/Val), trypsin (Ser/Val) and plasmin (Ser/Thr) define the specificity for the interaction with different P-1 residues of the inhibitors. Many of the synthesised compounds demonstrate potent antithrombin activity with Boc-D-trimethylsilylalanine-proline-boro-methoxypropylglycine++ + pinanediol (9) being the most selective thrombin inhibitor of this series.     

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.