Thrombin plasticity

Thrombin is the final protease generated in the blood coagulation cascade. It has multiple substrates and cofactors, and serves both pro- and anti-coagulant functions. How thrombin activity is directed throughout the evolution of a clot and the role of conformational change in determining thrombin specificity are issues that lie at the heart of the haemostatic balance. Over the last 20 years there have been a great number of studies supporting the idea that thrombin is an allosteric enzyme that can exist in two conformations differing in activity and specificity. However, recent work has shown that thrombin in its unliganded state is inherently flexible in regions that are important for activity. The effect of flexibility on activity is discussed in this review in context of the zymogen-to-protease conformational transition. Understanding thrombin function in terms of ‘plasticity’ provides a new conceptual framework for understanding regulation of enzyme activity in general. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Sodium ions as the effector of catalytic action of alpha-thrombin

A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of thrombin which can take essential effect on Na(+)-binding site and favor stabilization of the anticoagulant slow-form of thrombin, and of enzyme rational mutants with selective specificity in respect of protein C which display effective and safe anticoagulant and antithrombotic effects in vivo.     

Bacitracin and gramicidin S as biospecific ligands for affinity chromatography of human thrombin

It was shown that the cyclic polypeptide antibiotic bacitracin is a competitive inhibitor of fibrinogen clotting by thrombin. Biospecific adsorbents for isolation of thrombin by gramicidin S and bacitracin attachment to silochrome S-80 modified by gamma-glycido-oxypropyl groups were synthesized. The thrombin yield at pH 7.2 and 8.0 was 76.5-96%, purification--6.2-11.6-fold, specific coagulating activity--940-1750 NIH u./mg protein. At pH 6.1 the enzyme does not practically bind to the adsorbents. In all probability, the differences in thrombin binding are due to conformational changes in the enzyme molecule, when pH changes from 6.1 to 7.2. Possible application of the synthesized adsorbents for obtaining laboratory and commercial preparations of thrombin and their perspective use for purification of other blood plasma serine proteinases possessing a narrow specificity are discussed.     

Mechanism of action of heparin through thrombin on blood coagulation.

It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, T., Blaskó, Gy. and Pálos, L. (1975) Biochim. Biophys. Acta 379, 193-200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.     

How Na+ activates thrombin--a review of the functional and structural data

Thrombin is the ultimate coagulation factor; it is the final protease generated in the blood coagulation cascade and is the effector of clot formation. Regulation of thrombin activity is thus of great relevance to determining the correct haemostatic balance, with dysregulation leading to bleeding or thrombosis. One of the most enigmatic and controversial regulators of thrombin activity is the monovalent cation Na+. When bound to Na+, thrombin adopts a 'fast' conformation which cleaves all procoagulant substrates more rapidly, and when free of Na+, thrombin reverts to a 'slow' state which preferentially activates the protein C anticoagulant pathway. Thus, Na+-binding allosterically modulates the activity of thrombin and helps determine the haemostatic balance. Over the last 30 years, there has been much research investigating the structural basis of thrombin allostery. Biochemical and mutagenesis studies established which regions and residues are involved in the slow-->fast conformational change, and recently several crystal structures of the putative slow form have been solved. In this article, the biochemical and crystallographic data are reviewed to see if we are any closer to understanding the conformational basis of the Na+ activation of thrombin.     

Thrombin is a Na(+)-activated enzyme.

The amidase activity of human alpha-thrombin has been studied at steady state as a function of the concentration of several chloride salts, at a constant ionic strength I = 0.2 M. All kinetic steps of the catalytic mechanism of the enzyme have been solved by studies conducted as a function of relative viscosity of the solution. Among all monovalent cations, Na+ is the most effective in activating thrombin catalysis. This effect is observed with different amide substrates and also with gamma-thrombin, a proteolytic derivative of the native enzyme which has little clotting activity but retains amidase activity toward small synthetic substrates. The specific effects observed as a function of Na+ concentration are indicative of a binding interaction of this monovalent cation with the enzyme. The basis of this interaction has been explored by measurements of substrate hydrolysis collected in a three-dimensional matrix of substrate concentration, relative viscosity, and Na+ concentration, keeping the ionic strength constant with an inert cation such as choline or tetraethylammonium. The data have globally been analyzed in terms of a kinetic linkage scheme where Na+ plays the role of an allosteric effector. The properties of the enzyme change drastically upon binding of Na+, with substrate binding and dissociation, as well as deacylation, occurring on a time scale which is 1 order of magnitude faster. The apparent association constants for Na+ binding to the various intermediate forms of the enzyme have all been resolved from analysis of experimental data and are in the range of 50-100 M-1 at 25 degrees C. Studies conducted at different temperatures, in the range 15-35 degrees C, have revealed the enthalpic and entropic components of Na+ binding to the enzyme. The results obtained from steady-state measurements are supported by independent measurements of the intrinsic fluorescence of the enzyme as a function of Na+ concentration at a constant ionic strength I = 0.2 M, over the temperature range 15-35 degrees C. These measurements are indicative of a drastic conformational change of the enzyme upon Na+ binding to a single site. The energetics of Na+ binding derived from analysis of fluorescence measurements agree very well with those derived independently from steady-state determinations. It is proposed that thrombin exists in two conformations, slow and fast, and that the slow-->fast transition is triggered by binding of a monovalent cation. The high specificity in thrombin activation found in the case of Na+ is the result of its higher affinity compared to all other monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for multiple conformational changes in the active center of thrombin induced by complex formation with thrombomodulin: an analysis employing nitroxide spin-labels

Thrombomodulin (TM) is an endothelial cell surface protein that binds thrombin to form a reversible complex with altered enzyme specificity. The complex rapidly converts protein C to the anticoagulant enzyme activated protein C and has decreased fibrinogen clotting activity. To investigate whether formation of this complex elicits conformational changes in the active center of thrombin, we employed the following fluorosulfonyl spin-label inhibitors: N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-m-(fluorosulfonyl)benzamide (m-V); O-(2,2,6,6-tetramethyl-1-oxy-4-piperidinyl) N-[m-(fluorosulfonyl)phenyl]carbamate (m-VI); N-[4-(fluorosulfonyl)phenyl]-2,2,5,5-tetramethyl-1-oxy-3-pyrroline -3-carboxamide (p-I); N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-p-(fluorosulfonyl)benzamide (p-V). To compare the spectra of the free thrombin with those of the complex, the viscosity of the solution was adjusted with sucrose to give similar tumbling rates (isokylindric spectra) or the macromolecular rotational contribution to the spectra was essentially eliminated with saturated sucrose. Both a buffer-soluble proteolytic derivative of TM and the intact molecule elicited changes in the electron spin resonance signals of many of the labeled thrombins employed. Two of the labels, p-I and p-V, had previously been shown to exhibit decreased mobility when indole derivatives were bound to thrombin. When TM complexes with thrombin, the mobility of the p-I label increases while the mobility of the p-V label decreases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelation between thrombin structure and its stability

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.     

New general approach for determining the solution structure of a ligand bound weakly to a receptor: structure of a fibrinogen Aalpha-like peptide bound to thrombin (S195A) obtained using NOE distance constraints and an ECEPP/3 flexible docking program.

A new approach incorporating flexible docking simulations and NMR data is presented for calculating the bound conformation of a ligand that interacts weakly with an enzyme. This approach consists of sampling directly the conformation of a flexible ligand inside a receptor active site containing surrounding flexible loops. To make this sampling efficient, a ligand-growing procedure has been adopted. Optimization of the ECEPP/3-plus-NOE constraint function is carried out by using a collective variable Monte Carlo minimization technique. Numerous energy minimizations are made possible for such a large system by using a Bezier splines energy grid technique. This new flexible docking approach was applied to determine the structure of a fibrinogen Aalpha-like peptide (7DFLAEGGGVRGPRV20) bound to an active site mutant of thrombin [thrombin(S195A)]. Structure calculations of the bound ligand, using 2D-transferred NOESY distance constraints in the DIANA program, showed that the N-terminal portion of the peptide (D7-R16) involves a chain reversal, whereas the C-terminal portion (G17-V20) adopts a fold that exists in several different orientations. In addition, the ECEPP/3 flexible docking package was used to assess the conformational variability of the ligand and surrounding 60D-insertion loop of thrombin. Amino acid residues (17-20) of the peptide interact with a region of the enzyme that exhibits broad specificity, with a preferred direction between the 60D-insertion loop and Pro37 of thrombin.     

Role of induced fit in enzyme specificity: a molecular forward/reverse switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.     

Murine thrombin lacks Na+ activation but retains high catalytic activity

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

Interaction of human alpha-thrombin with organic ligands of ionic nature

Investigations results of human thrombin interaction with organic ligands of ion nature containing nonpolar groups are presented. It is shown that electrostatic interaction is the basic one under enzyme binding, while hydrophobic binding is only additional function in the reaction enzyme-ligand, this fact is confirmed by the absence of interaction between thrombin and rivanol which has a positive charge side by side with cumbrous hydrophobic group. New data are presented about the ligand specificity of binding sites of thrombin active centre. The importance of relative arrangement of hydrophobic ligand groups for interaction with enzyme is shown. It is supposed that thrombin binding with organic ligands occurs owing anionic site of beta-domain of active thrombin centre with the major aminoacids arginine and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.). It is shown that the compounds containing negative group SO3 and have some cunbours hydrophobic groups interact more intensively with the enzyme. Thus, rosseline--with symmetrical hydrophobic nucleus (four benzene rings)--is the most efficient ligand for the binding with thrombin. The obtained investigation results evidence for bacteriostatical and stabilizing effect of low-molecular asobenzene ligands on rather labile thrombin molecules.     

The specificity of induced conformational changes. The case of yeast glyceraldehyde-3-phosphate dehydrogenase.

The specificity of induced conformational changes and of the probes used to detect them has been investigated in yeast glyceraldehyde-3-phosphate dehydrogenase. Cyanylation of the active-site SH groups in two of the four identical subunits of glyceraldehyde-3-phosphate dehydrogenase has no effect on reactivity of the unmodified SH groups toward the cyanylating reagent (2-nitro-5-thiocyanogenzoic acid, NTCB) but results in total loss of catalytic activity. Cyanylation of the dicarboxamidomethylated enzyme was four orders of magnitude slower than with the unmodified enzyme in contrast to cyanylation of the dicyanylated enzyme. Cyanylation by NTCB as well as alkylation by iodoacetate and acylation with beta-(2-furyl)acryloyl phosphate are enhanced in the presence of NAD+ while alkylation by iodoacetamide is inhibited by NAD+. In the absence of NAD+, hydrolysis of the acylated enzyme is faster than phosphorolysis while the reverse is true in the presence of NAD+. NAD+ accelerates hydrolysis of the 3-phosphoglyceroylated enzyme about 60-fold but decreases the rate of hydrolysis of the furylacryloylated enzyme by a factor of 17. Other examples of the specificity of the induced conformational changes and the probes are described. The conformational changes induced by NAD+ make the protein specifically reactive toward its physiological substrates and less reactive toward extraneous competing compounds.     

An ensemble view of thrombin allostery

Abstract Thrombin is the central protease of the coagulation cascade. Its activity is tightly regulated to ensure rapid blood clotting while preventing uncontrolled thrombosis. Thrombin interacts with multiple substrates and cofactors and is critically involved in both pro- and anticoagulant pathways of the coagulation network. Its allosteric regulation, especially by the monovalent cation Na+, has been the focus of research for more than 30 years. It is believed that thrombin can adopt an anticoagulant ('slow') conformation and, after Na+ binding, a structurally distinct procoagulant ('fast') state. In the past few years, however, the general view of allostery has evolved from one of rigid structural changes towards thermodynamic ensembles of conformational states. With this background, the view of the allosteric regulation of thrombin has also changed. The static view of the two-state model has been dismissed in favor of a more dynamic view of thrombin allostery. Herein, we review recent data that demonstrate that apo-thrombin is zymogen-like and exists as an ensemble of conformations. Furthermore, we describe how ligand binding to thrombin allosterically stabilizes conformations on the continuum from zymogen to protease.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.     

Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Specificity of serine proteases for cleavage sites on proatrial natriuretic factor

An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of ³⁵S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF_103–126, which cross reacts with proANF, ANF_99–126 and ANF_103–126, and a newly prepared antisera to synthetic ANF_99–105, which uniquely recognizes ANF_99–126, but not proANF or ANF_103–126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF_99–126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF_103–126. This cleavage activity of serum is blocked by leupeptin (40 μg/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF_99–126 while kallikrein cleaves only at the dibasic site to produce ANF_103–126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.