Structural and Binding Studies of SAP-1 Protein With Heparin

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (K_D = 158 nm ). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity.     

The anticoagulant and antithrombotic mechanisms of heparin

The molecular basis for the anticoagulant action of heparin lies in its ability to bind to and enhance the inhibitory activity of the plasma protein antithrombin against several serine proteases of the coagulation system, most importantly factors IIa (thrombin), Xa and IXa. Two major mechanisms underlie heparin's potentiation of antithrombin. The conformational changes induced by heparin binding cause both expulsion of the reactive loop and exposure of exosites of the surface of antithrombin, which bind directly to the enzyme target; and a template mechanism exists in which both inhibitor and enzyme bind to the same heparin molecule. The relative importance of these two modes of action varies between enzymes. In addition, heparin can act through other serine protease inhibitors such as heparin co-factor II, protein C inhibitor and tissue factor plasminogen inhibitor. The antithrombotic action of heparin in vivo, though dominated by anticoagulant mechanisms, is more complex, and interactions with other plasma proteins and cells play significant roles in the living vasculature.     

Oversulfated chondroitin sulfate interaction with heparin-binding proteins: New insights into adverse reactions from contaminated heparins

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.     

The anticoagulant activity of heparin: measurement and relationship to chemical structure

For many years the anticoagulant activity of heparin has been estimated by coagulation assays, in which the prolongation of clotting times by heparin is measured under various conditions. More recently, assays have been developed which measure the inhibitory action of heparin on isolated coagulation enzymes, notably Factor Xa and thrombin, using specific amidolytic peptide substrates. The anticoagulant activity of heparin arises primarily from its ability to bind to antithrombin III (AT III), altering the conformation and enhancing the activity of this major protease inhibitor. Passage of heparin through an immobilised AT III column yields two fractions: a high affinity fraction with 300-350 iu mg-1 anticoagulant activity, comprising one-third of the total, and a low affinity fraction with an activity of less than 10 iu mg-1, comprising the remaining two-thirds. Studies in several laboratories have demonstrated that a specific pentasaccharide sequence is required for AT III binding. The authors have shown that the presence or absence of this sequence can be detected by high-field proton NMR, thus providing a semi-quantitative method for a functionally important group. A second major influence on anticoagulant activity is molecular weight distribution. Studies in the authors' laboratory on a series of fractions of 5000-35,000 showed that whereas anticoagulant activity in APTT clotting assays decreased with decreasing molecular weight (Mr), activity in anti-Xa assays was maintained or increased in the low Mr fractions. However, in vivo studies showed that high affinity fragments with anti-Xa activity only were poor antithrombotic agents. It appears that the presence of the AT III binding site alone is not sufficient for full antithrombotic activity; an extra length of polysaccharide chain of at least 15 residues is required. Molecular weight distribution is readily assessed by HPLC, although the lack of suitable reference materials hampers assignment of absolute molecular weights. Important determinants of anticoagulant activity can now be assessed by physicochemical techniques but, at present, these techniques are not precise enough to replace anticoagulant assays as predictors of in vivo behaviour.     

Evidence for antithrombin III involvement in the anticoagulant activity of cellulose sulphate.

1 Cellulose sulphate, like heparin, prolonged the clotting time in partial thromboplastin time (PTT) assays, inhibited the amidolytic activity of thrombin, was without effect on amidolysis catalysed by activated coagulation factor X(Xa), and potentiated the inhibition of both thrombin and Xa by antithrombin III (AT). 2 The anticoagulant activity of cellulose sulphate in PTT assays was, like that of heparin and heparin sulphate, but unlike that of dermatan sulphate, reduced by prior incubation of plasma with antiserum specific for AT. 3 These results, which suggest that the anticoagulant activity of cellulose sulphate is at least partially mediated through AT, are discussed in terms of the structural features of polysaccharides required for AT activation.     

Structural and conformational aspects of the anticoagulant and anti-thrombotic activity of heparin and dermatan sulfate

Heparin and other iduronic acid-containing glycosaminoglycans (GAG) such as dermatan sulfate exert their anticoagulant properties primarily by accelerating the rate of inhibition of the natural protease inhibitors antithrombin III (AT, which inhibits both factor Xa and thrombin) and heparin cofactor II (HCII, which selectively inhibits thrombin). Although AT and HCII are structural homologs, only heparin binds to AT, and HCII has different binding sites for heparin and dermatan sulfate. Whereas the binding site of heparin for AT is a unique pentasaccharide sequence contained in only about one third of the chains of this GAG, HCII-binding sequences of heparin and dermatan sulfate are less specific and contained in practically all the GAG chains. Protein binding and associated biological activities of heparin and dermatan sulfate are modulated by the "plasticity" of their iduronic acid residues due to the availability of up to three equienergetic conformation among which the protein selects the one favouring the most stable complex. Glycol-splitting of nonsulfated uronic acid residues, a device for generating flexible joints along the GAG chains, has different effects on different binding domains. Whereas it inactivates the binding site for AT causing a drop of the anticoagulant activity, it enhances the HCII-associated activity of both heparin and dermatan sulfate.     

Oligosaccharide mapping of low molecular weight heparins: structure and activity differences

Low molecular weight heparins from a variety of commercial sources were examined. These had been prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical beta-elimination, enzymatic beta-elimination, and chromatographic fractionation. The molecular weight and polydispersity of these low molecular weight heparins showed greater differences than were observed for typical commercial heparin preparations. Considerable differences were also observed in the antithrombin III mediated anti factor Xa activity, the heparin cofactor II mediated antifactor IIa activity, and the USP activity of these low molecular weight heparins. An oligosaccharide-mapping technique (comparable to the peptide mapping of proteins) was applied to these low molecular weight heparins in an effort to understand the structural features responsible for their activity differences. Heparin lyase from Flavobacterium heparinum was first used to depolymerize the low molecular weight heparin into its constituent oligosaccharides. The oligosaccharides present in the resultant mixture were identified and quantitated by using standard oligosaccharides of defined structure on gradient polyacrylamide gel electrophoresis and strong anion exchange high pressure liquid chromatography. Six of the oligosaccharide products have been identified and represent nearly 90 wt % of heparin's mass. Even though all the low molecular weight heparins showed these six oligosaccharide components, their content in each varied greatly, accounting for 20 to over 90% of their mass. The antithrombin III mediated anti factor Xa activities of the low molecular weight heparins correlated only poorly to the concentration of a hexasaccharide containing a portion of heparin's antithrombin III binding site. The heparin cofactor II mediated antifactor IIa activity, however, could not be correlated to these six oligosaccharides of known structure nor to the molecular weight or charge density of these low molecular weight heparins. The low molecular weight heparins prepared by different methods each showed a new distinctive oligosaccharide in their maps. Their isolation and structural characterization, which included two-dimensional NMR and fast atom bombardment mass spectrometry, indicated that these unusual oligosaccharides result from end-sugar modification during chemical depolymerization. Both gel electrophoresis and high-pressure liquid chromatography mapping techniques showed a greater structural diversity between low molecular weight heparins than had previously been observed between similarly analyzed commercial heparins.     

Synthetic heparin derivatives as new anticoagulant drugs

The journey towards a detailed mechanistic understanding of the anticoagulant action of heparin has resulted in synthetic mimetics with improved pharmacodynamic profiles. Inspired by the ternary complex formation of heparin with antithrombin III and thrombin, the active pentasaccharide fondaparinux has been succeeded by several clinical candidates, such as SR123781, that have tailor-made factor Xa and thrombin inhibitory activities combined with less aspecific binding (e.g. binding to platelet factor 4 involved in thrombocytopenia). Novel compounds with both antithrombin III-mediated inhibition of factor Xa and direct thrombin inhibition are emerging. Org42675 is one such compound, balancing dual inhibition of factor Xa and thrombin in one anticoagulant drug, with excellent pharmacokinetic properties and strong inhibitory activity toward clot-bound thrombin.     

Factor Xa inhibitors

Summary Factor Xa is the serine protease that activates prothrombin to yield thrombin. Inhibitors of factor Xa play a crucial role in curtailing thrombin generation. Two key factor Xa inhibitors that are found in blood are antithrombin III and tissue factor pathway inhibitor. Inhibition of factor Xa is a mechanism that is also exploited by certain hematophagous animals to facilitate feeding. Evaluation of tick anticoagulant peptide (TAP) and leech-derived antistasin (ATS) using animal models of thrombotic disorders has confirmed that specific blockade of factor Xa activity is an effective antithrombotic strategy. Several laboratories are currently pursuing low-molecular weight synthetic factor Xa inhibitors for use as anticoagulants in the treatment and/or prevention of thrombosis.     

A peptide derivative (1-70 fragment) of protein SV-IV accelerates human blood coagulation in vitro by selective inhibition of the heparin-induced antithrombin III activation process

The effect of two peptide derivatives of the rat SV-IV (SV-IV/A: 1–70 fragment; SV-IV/B: 71–90 fragment) on human blood coagulation was investigated. The SV-IV/A fragment was found to possess the same procoagulant activity of the native protein, whereas SV-IV/B retained only a very small fraction of the activity. The results obtained strongly suggest that the procoagulant activity of SV-IV/A is due, like SV-IV, to a selective inhibition of the antithrombin III (AT III) activation process induced by heparin, an essential cofactor of AT III. The main data supporting this hypothesis are the following: I) the concentration of active serum AT III decreases when SV-IV/A is present in the clotting system; 2) the plasma treatment with SV-IV/A reduces the concentration of AT III, but not that of other plasma serine protease inhibitors; 3) the recalcification time (RT) of the plasma treated with a rabbit anti-AT III polyclonal antiserum is not modified by SV-IV/A; 4) the presence of SV-IV/A in a reaction mixture containing pure fibrinogen, α-thrombin, heparin. and AT III neutralizes the thrombin inhibition induced by AT III; 5) the concentration of the thrombin-AT III complexes, occurring in sera obtained from CaCl₂-coagulated plasma, is markedly reduced in the presence of SV-IV/A; 6) appropriate concentrations of heparin neutralize the inhibitory effect of either SV-IV/A or SV-IV on the AT III activity in vitro.     

Purification and characterization of an antithrombin III inactivating enzyme from the venom of the African night adder (Causus rhombeatus).

A serine proteinase was isolated from the venom of the night adder (Causus rhombeatus) by fast protein liquid chromatography (anion-exchange, gel filtration and hydrophobic interaction). The protein (termed CR-serpinase) had an estimated mol. wt of 45,500 as determined by SDS-PAGE, pI of 4.7 and a carbohydrate content of 18.9%. Incubation of CR-serpinase with purified human antithrombin III at a molar ratio of 1:66 resulted in a loss of more than 90% of the initial AT III activity within 10 min. The reaction was dependent on heparin. In SDS-PAGE inactivation of human antithrombin III was correlated with the occurrence of two cleavage products. The cleavage site in the antithrombin III molecule was determined to be Arg 393-Ser 394 by amino-terminal sequencing. CR-Serpinase had no thrombin-like activity since no fibrinogen conversion was induced and had no procoagulant activity. CR-Serpinase activity was not inhibited by antithrombin III-heparin and was not decreased by a 10-min preincubation in normal human plasma. Inactivation of antithrombin III by CR-serpinase appeared to be very specific.     

The anticoagulant activity of dermatan sulphates: evidence against the involvement of antithrombin III.

Anticoagulant activity of dermatan sulphates is unaffected by antiserum specific for antithrombin III (AT III) unless the glycosaminoglycan preparation contains demonstrable heparin. 2 Only dermatan sulphate preparations of considerable heparin content potentiate AT III inhibition of thrombin, factor Xa and plasmin. 3 These data suggest that dermatan sulphates exert anticoagulant activity which, unlike that of heparin, is largely or totally independent of AT III.     

Mechanism of poly(acrylic acid) acceleration of antithrombin inhibition of thrombin: implications for the design of novel heparin mimics

The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive approximately 2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K(D) of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics.     

New antithrombotic oligosaccharides]

In the early eighties, following breakthroughs in oligosaccharide chemistry, the total chemical synthesis of pentasaccharides has been achieved, representing the antithrombin binding domain of heparin (the active site). The selective inhibitors of coagulation factor Xa thus obtained represent a new type of antithrombotic drugs. In a further step, based on the knowledge of the mechanism of antithrombin activation by heparin, oligosaccharides (pentadeca- to eicosasaccharides), comprising an antithrombin binding domain prolonged by a thrombin binding domain, were designed and synthesised in the Sanofi group. These compounds inhibit both factor Xa and thrombin, in the presence of antithrombin. Endowed with the full anticoagulant activity of heparin but devoid of undesired non-specific interactions, particularly with platelet factor 4 (PF4), they might represent "the ideal heparin-like antithrombotic".     

Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of clot-associated (-derived) thrombin in cell proliferation induced by fibrin clots in vitro

Thrombin is a potent mitogenic agent. Clot-associated thrombin retains its amidolytic and pro-aggregant activity. We therefore studied the ability of fibrin clots to induce proliferation in CCL39 cells (Chinese hamster lung fibroblasts), in the absence and presence of the thrombin inhibitors PPACK, recombinant hirudin (rHV2 Lys47) and heparin:antithrombin III. Fibrin clots incubated for 48 h with CCL39 cells led to significant cell proliferation, which was dependent on the concentration of thrombin used to prepare the clots. Thus, clots prepared with 91 nmol l(-1) thrombin produced a similar proliferation (231+/-21%) to that obtained with 50 nmol l(-1) thrombin in solution (213+/-29%). Rabbit plasma clots led to a 499+/-41% increase in cell number under identical conditions. Fibrin clot-induced cell proliferation was inhibited by all three thrombin inhibitors with no difference in IC(50) values compared to those obtained against thrombin in solution, suggesting that cell proliferation be due to thrombin leaching from the clots. We found a time-dependent increase in thrombin release from the clots attaining a plateau at 24 h (approximately 61% of the total thrombin used in clot formation). Clots separated from the cells using porous cell culture chamber inserts led to similar proliferation to that of clots in contact with the cells. Thus fibrin-clot induced CCL39 proliferation is due to thrombin released from the clots.     

Inhibition of antithrombin by protein SV-IV normalizes the coagulation of hemophilic blood

The aim of the study was to evaluate the effect of the protein Seminal Vesicle Protein No. 4 (SV-IV), a potent inhibitor of antithrombin III (antithrombin), on the coagulation of blood obtained from patients affected by hemophilia A. In the coagulating blood of these patients, the antithrombin/thrombin ratio was found to be markedly higher (about 44) than in normal individuals (about 4. 4). This high ratio was related to the low efficiency of thrombin-generating reactions induced by the factor VIII deficiency and to the high levels of free (not bound to serine proteases) antithrombin present in the hemophilic serum (antithrombin concentration was the same in normal and hemophilic plasma). The elevated concentration of free antithrombin in hemophiliacs was primarily a consequence of a reduced consumption caused by the scarce availability in the hemophilic serum of factors Xa and IIa, which are serine proteases possessing strong binding affinity for antithrombin. Addition of SV-IV to coagulating hemophilic blood reduced markedly the serum antithrombin and thrombin-antithrombin complexes, normalizing, as a consequence, the clotting time and other coagulation parameters. Similar results were obtained by using appropriate concentration of factor VIII.     

Crystal structures of thrombin and thrombin complexes as a framework for antithrombotic drug design

Summary The wealth of structural information now available on thrombin, its precursors, its substrates and its inhibitors allows a rationalization of its many roles. -Thrombin exhibits an unusually deep and narrow active-site cleft, formed by loop insertions that are characteristic of thrombin. This canyon structure is one of the prime causes for the narrow specificity of thrombin. As a result of the conjunction of amino acid residues with similar properties such as charge or hydrophobicity, thrombin can be divided up into a number of functional regions. The apposition of the active site to a hydrophobic pocket (the apolar binding site) on one side and a basic patch (the fibrinogen recognition exosite) on the other allows for a fine-tuning of enzymatic activity, as seen for fibrinogen. These two sites are also optimally used by the leech-derived inhibitor hirudin, allowing the very tight binding observed; thrombin inhibition is effected by blocking access to the active site. Interactions with antithrombin III are tightened with the help of heparin, which binds to a second basic site (the heparin binding site). Non-proteolytic cellular properties are attributed to the rigid insertion loop at Tyr^60A. The observed rigidity of the thrombin molecule in its complexes makes thrombin ideal for structure-based drug design. Thrombin can be inhibited either at the active site or at the fibrinogen recognition exosite, or both. Structural information shows that binding at the former is enhanced by good fit of aromatic moieties to the aryl and S2 binding sites (the apolar binding site). Binding at the fibrinogen recognition exosite is facilitated by negatively charged groups. The unpredictable nature of inhibitor binding underlines the importance of experimental monitoring of structures of thrombin inhibitors in the drug design process.     

Heparin and heparinoids impair adrenaline and platelet-activating factor but not thrombin-induced inhibition of adenylate cyclase and stimulation of GTP hydrolysis in human platelet membranes

Summary The effects of heparins and heparinoids were studied on adenylate cyclase and GTPase activities in human platelet membranes. Inhibition of adenylate cyclase by adrenaline and platelet activating factor was completely abolished by heparin at 1 /ml. At similar concentration heparin blocked the stimulation of high affinity GTPase(s) by these hormonal factors. In contrast, heparin (up to 30 g/ml) did not abolish adenylate cyclase inhibition and stimulation of GTP hydrolysis by thrombin in the absence of antithrombin III. In the presence of antithrombin III, thrombin action on adenylate cyclase was blocked by unfractionated and high molecular weight heparin at 0.1 g/ml. Low molecular weight heparins and pentosanpolysulfate were less or not effective. In contrast, all high and low molecular weight heparins tested were almost equally potent in inhibiting adrenaline-induced inhibition of adenylate cyclase in the absence of antithrombin III. The data indicate that heparins discriminate platelet activating factor and adrenaline-induced inhibition of adenylate cyclase from the inhibitory action of thrombin and delineate different structural requirements for the interaction of heparins with the adenylate cyclase system and antithrombin III.Abbreviations G_i the inhibitory guanine nucleotide binding regulatory protein of the adenylate cyclase - PAF platelet activating factor Send offprint requests to K. Aktories     

Characterization of a thrombin-like serine protease, Kangshuanmei, isolated from the venom of a Chinese snake, Agkistrodon halys brevicaudus stejneger

An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.