High affinity binding of human and bovine thrombins to p-chlorobenzylamido-epsilon-aminocaproyl agarose.

Human thrombin (EC 3.4.21.5) binds tightly to p-chlorobenzylamido-epsilon-aminocaproyl agarose, and is not eluted by 2 M NaCl at pH 8. Its zymogen, human prothrombin, does not bind to the same absorbent. 2 M NaCl partially elutes DFP-treated thrombin. For native human and bovine thrombins, protein and activity are quantitatively eluted by 25% dioxane, and upon rechromatography the active human enzyme exhibits the same binding properties. Equally tight binding of human thrombin occurs with derivatives of the m- and p-chlorobenzylamines. With the o-chloro derivative or benzylamine itself insolubilized to epsilon-aminocaproyl agarose, thrombin is eluted by high ionic strength. Bovine trypsin and bovine factor Xa bind less tightly than thrombin to p-chlorobenzylamido-epsilon-aminocaproyl agarose, being eluted by high ionic strength. It is proposed that the specific thrombin adsorption is related to a secondary binding site of high affinity and with hydrophobic properties. This site is not available in the zymogen. Furthermore, the less specific protease, trypsin, and the more specific protease, factor Xa, lack this binding site.     

The heparin-accelerated neutralisation of bovine α and β thrombins by antithrombin III

The heparin-accelerated neutralisation of bovine α and β thrombins has been examined using a peptide substrate H-d-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. α and β thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of α and β thrombin. This work is therefore unabel to confirm reports that α and β thrombins have different heparin sensitives.     

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.     

Characterization of the adenosine deaminase-adenosine deaminase complexing protein binding reaction

Glutaraldehyde-fixed membranes from rabbit kidney cortex were used to characterize binding of monomeric adenosine deaminase to the adenosine deaminase complexing protein. With the use of bovine adenosine deaminase it was shown that enzyme binding is a saturable, high affinity process. The K value for binding of the bovine enzyme was 11 nM. Maximum enzyme binding and rate of binding to a constant amount of membrane did not vary significantly from pH 5.0 to 9.5. Metal ions, with the exception of Hg2+, sulfhydryl reagents, and other proteins had little or a slightly stimulatory effect on maximum binding. Mercuric ion inhibited binding. Using biotinylated bovine adenosine deaminase it was shown that purified rabbit, human, and monkey enzymes compete for binding sites on fixed membranes. The K values for the rabbit and human enzymes were 9 and 6 nM, respectively. Mouse or guinea pig adenosine deaminase did not bind to the membranes or compete with the biotinylated bovine enzyme for binding sites. The retention of characteristics required for binding by enzymes from rabbit, human, monkey, and calf tissues argues for biologic significance of the adenosine deaminase-complexing protein interaction. The basis for the apparent failure of rodent adenosine deaminase to bind to complexing protein remains to be determined.     

The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding

The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (deltaK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (alpha-NAPAP) to the wild-type (WT) and deltaK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free deltaK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the deltaK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A-B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.     

Purification and characterization of ostrich prothrombin

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu(2+)-chelate Sepharose chromatography. Ostrich prothrombin exhibited a M(r) of 72,800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin beta-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.     

The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction.

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.     

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.     

Structural and immunological similarities between high molecular weight zinc ion-dependent p-nitrophenylphosphatase and fructose-1,6-bisphosphate aldolase from bovine liver

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.     

Evolution of prothrombin: Isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.The nucleotide sequences reported in this paper have been submitted to the EMBL/Genbank database under the following secession numbers: M 81391 for Gallus gallus, M 81393 for Eptatretus stouti.Correspondence to: R.T.A. MacGillivray     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human alpha-, beta- and gamma-thrombin; a kinetic and thermodynamic study

Kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human alpha-, beta- and gamma-thrombin have been determined, between 5 and 45 degrees C, at pH 7.5. BPTI-binding properties to human thrombins have been analyzed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates, with particular reference to the bovine beta-trypsin/BPTI system. The observed binding behaviour of BPTI to human alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme/inhibitor contact region(s).     

Macromolecular specificity determinants on thrombin for fibrinogen and thrombomodulin

The endothelial cell surface membrane protein thrombomodulin binds thrombin with high affinity and acts as both a cofactor for protein C activation and an inhibitor of fibrinogen hydrolysis. We have previously shown that bovine thrombomodulin is a competitive inhibitor of fibrinogen binding to thrombin but has no effect on thrombin activity toward tripeptide substrates or antithrombin III. Hence, thrombomodulin and fibrinogen may share macromolecular specificity sites on thrombin which are distinct from the active site. In this investigation, we have studied the interaction of thrombin-thrombomodulin with fibrinogen and various thrombin derivatives. We show that fibrinogen is a competitive inhibitor of thrombomodulin binding to thrombin, with a Kis = 10 microM. Thrombin derivatives (bovine (pyridoxal phosphate)4-thrombin and human thrombin Quick I), which bind fibrinogen with much reduced affinity, are shown to also interact with thrombomodulin with greatly reduced affinity. These results are consistent with the hypothesis that thrombomodulin and fibrinogen share macromolecular specificity sites on thrombin.     

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.     

Modifications in the aminoether side chain of clomiphene influence affinity for a specific antiestrogen binding site in MCF 7 cell cytosol

Studies on the binding of a series of clomiphene derivatives to a specific high affinity antiestrogen binding site in MCF 7 cell cytosol revealed that substitutions in the aminoether side chain influenced affinity for this site. Removal of the aminoether side chain completely eliminated binding while changes in length of the C chain, and changes in the number and size of the substituents on the terminal amino group modified binding affinity. Conversion of the ether linkage to an amine also markedly reduced affinity. It is concluded that the aminoether side chain which is essential for antiestrogenic activity is a major structural determinant of the binding of synthetic triphenylethylene antiestrogens to the specific antiestrogen binding site.     

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.     

Prostacyclin-stimulating drugs: new prospects

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The PGI2-stimulating activity of SKF 525-A was characterized by specific structural requirements: activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI2 by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI2 and inhibition of platelet TxA2 represents an original pharmacological profile: SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.     

Characterization of the residues involved in the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor

Haemadin is a 57-amino acid thrombin inhibitor from the land-living leech Haemadipsa sylvestris, whose structure has recently been determined in complex with human alpha-thrombin. Here we communicate the effect of ionic strength on the kinetics of the inhibition of human alpha-thrombin by haemadin, by using thrombin mutants modified in exosite II. Data analysis has allowed both the ionic and nonionic binding contributions to be ascertained, with the nonionic component being virtually the same for all of the thrombins that have been examined, while the ionic binding energy contributions varied from molecule to molecule. In the case of the native human alpha-thrombin-haemadin complex, ionic interactions contribute -17 kJ/mol to the Gibbs free energy of binding, this being the equivalent of up to six salt bridges. These salt bridges make up 20% of the total binding energy at zero ionic strength, and this has been attributed to the C-terminal tail alone. In addition, the contributions of the N-terminal and C-terminal regions of haemadin to its tight binding have been ascertained by using derivatives of both haemadin and thrombin. Limited proteolysis using formic acid produced haemadin cleaved between residues 40 and 41, removing the majority of the C-terminal tail. This truncated haemadin displayed a 20000-fold reduced affinity for thrombin, and was no longer a tight binding inhibitor. A form of thrombin in which the active site serine has been blocked by diisopropyl fluorophosphate binds to haemadin, but with a 72000-fold reduced affinity, indicating that the N-terminus is more important than the C-terminus for strong binding.     

The amino acid sequence of bovine cardiac tamponin-C. Comparison with rabbit skeletal troponin-C.

The amino acid sequence of bovine cardiac troponin-C has been determined. The protein chain is composed of 161 amino acid residues and its amino terminal is acetylated. There are 55 replacements and 2 additional amino acids when compared with rabbit skeletal troponin-C. Cardiac troponin-C probably contains 3 calcium binding sites, one less than rabbit skeletal troponin-C. The difference in amino acid sequence is largely due to the difference in tissue, not to the difference in species.     

Interaction of thrombin with sucrose octasulfate

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.     

Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity.

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.