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S T Olson

Publications and source records attributed to S T Olson.

At least 73 records · Page 4Linked to original sources
PubMed1991

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects.

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.

Catalysis↗
PubMed1990

The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen.

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.

Amino Acid Sequence↗
PubMed1989

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction.

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.

Antithrombin III↗
PubMed1989

Binding of heparin to human high molecular weight kininogen.

The binding of heparin to high molecular weight kininogen (H-kininogen) was analyzed by the effect of kininogen in decreasing the heparin-induced enhancement of the rate of inactivation of thrombin by antithrombin. The conditions were arranged so that the heparin-catalyzed antithrombin-thrombin reaction, monitored in the presence of the reversible thrombin inhibitor p-aminobenzamidine, followed pseudo-first-order kinetics and the observed rate constant (kappa obsd) varied linearly with the heparin concentration. In the absence of metal ions, H-kininogen minimally affected kappa obsd, measured at a constant concentration of heparin with high affinity for antithrombin (30 nM), at I = 0.15, pH 7.4 and 25 degrees C. However, at a saturating concentration of Zn2+ (10 microM), kappa obsd was reduced to 50% at approximately 20 nM H-kininogen and to that of the uncatalyzed reaction at greater than or equal to approximately 0.2 microM H-kininogen. Conversely, at a saturating concentration of H-kininogen (0.5 microM), kappa obsd was decreased to 50% at approximately 0.6 microM Zn2+ and to the kappa obsd of the uncatalyzed reaction at greater than or equal to 10 microM Zn2+. Other metal ions were effective in the order Zn2+ approximately Ni2+ greater than Cu2+ approximately Co2+ approximately Cd2+. The single-chain and two-chain forms of H-kininogen and the H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent, whereas low molecular weight kininogen had no influence.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombins↗
PubMed1989

Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-p-guanidinobenzoate.

A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor Xa in primarily the undergraded alpha-form. The chromatography procedure separated factor Xa from factor X, the Russel's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. alpha-Factor Xa was unstable at pH 7.6 and 25 degrees C, undergoing slow proteolytic degradation to functionally heterogeneous products as evidenced by the greater loss of coagulation assay activity compared to activity measured with a chromogenic substrate. The results of monitoring factor Xa degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with proteolysis of the light chain as a major component reaction occurring in parallel with slower proteolysis of the heavy chain. The decreased rates of these reactions at pH 6.0 enabled isolation and storage of factor Xa in greater than or equal to 88% alpha-form and minimized the heterogeneity due to proteolytic degradation. Characterization of the reaction of fluorescein mono-p-guanidinobenzoate (FMGB) with human and bovine factor Xa isolated by SBTI-Sepharose chromatography demonstrated its utility as a sensitive reagent for continuous fluorometric active-site titration. Analysis of the reaction kinetics as a function of FMGB and human factor Xa concentrations in G/2 0.3, pH 7.4, buffer at 25 degrees C indicated that the ratio of acylation to deacylation rate constants was greater than 200 and that the Km for FMGB was 0.06-0.11 microM, predicting pre-steady-state burst amplitudes of greater than or equal to 96-98% of the active-site concentration at FMGB concentrations greater than or equal to 5 microM. Human factor Xa active-site concentrations were consistent with 82-99% active preparations when compared with the protein concentrations determined from the 280-nm absorbance. Concentrations of human alpha-factor Xa as low as 20 nM could be measured with FMGB, indicating a sensitivity approximately 50 times greater than that measured by spectrophotometric active-site titration with p-nitophenyl p'-guanidinobenzoate.

Binding Sites↗
PubMed1988

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin.

Heparin (H) was previously shown to accelerate the inactivation of alpha-thrombin (T) by antithrombin III (AT) primarily by promoting the initial binding of thrombin and AT in a ternary T.AT.H complex intermediate without significantly influencing the subsequent product formation step (Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895). In the present study, the protein-heparin interactions which contribute to the assembly of the ternary complex intermediate and their linkage were quantitated by equilibrium binding and stopped-flow kinetic studies at pH 7.4, I 0.3, 25 degrees C, using p-aminobenzamidine (P) as a fluorescence probe. Equilibrium binding studies of the AT.H and T.H binary complex interactions monitored by the 40% enhancement in AT fluorescence or the 16-18% quenching of thrombin-bound p-aminobenzamidine fluorescence, respectively, indicated a 100-fold greater affinity of AT for heparin (KAT,H 0.23 microM) as compared to thrombin for heparin (KT,H 35-42 microM). Consistent with this large difference in affinities, rapid kinetic studies indicated that assembly of the ternary complex occurred predominantly as a bimolecular association between the AT.H binary complex and free thrombin. Thus, under pseudo-first order conditions ([AT]o, [H]o much greater than [T]o much less than [P]o), the observed thrombin inactivation rate constant (kobs) exhibited a saturable dependence on [AT]o or [H]o when [H]o much less than KT,H, reflecting a KAT,H (0.25 microM) similar to that directly determined by equilibrium binding. Moreover, competitive inhibition of the reaction by T.H binary complexes was indicated from the hyperbolic decrease in kobs produced by heparin with either high or low affinity for AT or active-site blocked thrombin at concentrations comparable to KT,H. This behavior was consistent with values of KT,H (27-51 microM) similar to those determined directly from equilibrium binding measurements. Comparison of the affinities of the binary protein-heparin interactions with the affinity of thrombin for AT.H complex in the ternary complex measured previously, indicated that the affinity of either protein for heparin was enhanced about 10-fold by the prior binding of the other protein to heparin. This linkage of the protein-heparin interactions implies that the ternary complex will be assembled at thrombin, AT, and heparin concentrations considerably lower than those predicted from previous reaction models which fail to account for this linkage.

Algorithms↗
PubMed1987

Acceleration of surface-dependent autocatalytic activation of blood coagulation factor XII by divalent metal ions.

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations, Divalent↗
PubMed1986

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. The reaction step limiting heparin turnover in thrombin neutralization.

The intrinsic protein fluorescence quenching which accompanies the heparin-accelerated inhibition of thrombin (T) by antithrombin III (AT) was resolved into a heparin-independent component associated with formation of the product T-AT complex and a component associated with an AT conformational change linked to heparin dissociation. To determine whether dissociation of heparin from the product T-AT complex limits the rate at which heparin can turn over catalytically, the kinetics of protein fluorescence quenching during the reaction of thrombin with AT X heparin complex (AT X H) were investigated by stopped-flow fluorimetry under pseudo-first order conditions ([AT X H]o much greater than [T]o). Both fluorescence components were quenched in a single exponential reaction with a hyperbolic dependence of the first order rate constant (kobs) on [AT X H]o. An indistinguishable hyperbolic dependence of kobs on [AT X H]o was measured by displacement of p-aminobenzamidine from the T active site, with both signals extrapolating to a limiting rate constant of 5 s-1. These results indicate that heparin dissociation occurs concomitant with T-AT complex formation at the limiting 5 s-1 rate constant. In reasonable agreement with this value, a kcat of 2.3 s-1 was determined for heparin turnover at catalytic concentrations. We conclude that formation of the T-AT complex is the primary rate-limiting step in heparin catalytic turnover and that this reaction is accompanied by a change in conformation of the AT component resulting in facile heparin dissociation.

Animals↗
PubMed1986

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate.

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

Anions↗
PubMed1985

Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin.

The stoichiometry of antithrombin III (AT) inhibition of alpha-thrombin (T) has been investigated in the presence and absence of heparin as a function of ionic strength by quantitative titration of enzyme active sites. In contrast to the ionic strength-independent stoichiometry of 1.0 mol of AT/mol of T observed in the absence of heparin, the presence of high-affinity heparin (HAH) resulted in an ionic strength-dependent increase in the apparent stoichiometry of inhibition from a molar ratio of 1.1 AT/T at an ionic strength of 0.3 to 9.8 mol of AT/T when the ionic strength was lowered to 0.01. Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products revealed that the increased AT/T stoichiometry was due to preferential formation of a specific proteolytically cleaved form of AT that was indistinguishable from the previously characterized reactive site-cleaved AT (ATM). Using high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to quantitate ATM, the cleaved inhibitor was shown to be formed rapidly and concomitant with the stable thrombin-antithrombin complex (TAT) and quantitatively accounted for the apparent increase in reaction stoichiometry at low ionic strength in the presence of HAH. The levels of HAH required to produce maximum ATM were catalytic at mu greater than or equal to 0.15, but became stoichiometric as the ionic strength decreased below 0.1. Substantially less ATM was produced in the presence of low-affinity heparin, while a low molecular weight HAH, virtually inactive in accelerating T inhibition by AT, was unable to promote significant ATM formation. These results indicate competition between substrate and inhibition reactions of AT with T which are affected by an ionic strength-dependent heparin interaction. A reaction mechanism accounting for these observations is proposed.

Antithrombin III↗
PubMed1984

Kinetics of the conformational alterations associated with nucleophilic modification of alpha 2-macroglobulin.

The effect of nucleophilic modification of alpha 2-macroglobulin (alpha 2M) with methylamine on the kinetics of sulfhydryl exposure was investigated. The generated sulfhydryl groups were detected with 4,4'-dithiodipyridine. The bimolecular rate constant for sulfhydryl exposure was determined to be 11.6 +/- 0.8 M-1 s-1 at 30 degrees C and pH 8.0. Treatment of alpha 2-macroglobulin with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in alpha 2-macroglobulin upon treatment with methylamine. It was found that the conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of two thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the two dimeric units present in alpha 2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 +/- 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 +/- 2.0) X 10(-3) s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of alpha 2-macroglobulin, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on alpha 2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.

Fluorescent Dyes↗
PubMed1982

p-Aminobenzamidine as a fluorescent probe for the active site of serine proteases.

p-Aminobenzamidine is weakly fluorescent in neutral aqueous buffer, with excitation and emission maxima at 293 and 376 nm, respectively. Binding to trypsin results in a blue shift of the emission peak to 362 nm, and 50-fold fluorescence enhancement, while binding to thrombin causes a shift to 368 nm and a 230-fold fluorescence enhancement. This phenomenon is due to hydrophobic interactions, as evidenced by the similar properties observed when p-aminobenzamidine is dissolved in solvents of decreasing polarity. The absorbance spectrum of p-aminobenzamidine is red-shifted by formation of a complex with proteases, with the major difference peak appearing at 317 nm and 323 nm for trypsin and thrombin, respectively. The difference extinction coefficients were 6000 M-1 cm-1 for trypsin complex and 13,300 M-1 cm-1 for thrombin complex at the peak wavelengths. Titration of trypsin and thrombin with the probe indicated one binding site per molecule, with dissociation constants equal to the kinetically determined inhibition constants. The KD values for trypsin and thrombin were 6.1 and 65 microM, respectively. An important potential use of this probe is in studies of inhibitor and substrate binding by rapid reaction kinetic techniques. Using this probe to study the interaction of thrombin with antithrombin III yielded a bimolecular rate constant of 8.0 x 10(3) M-1 s-1, which compares favorably with the value of 8.7 x 10(3) M-1 s-1 obtained from discontinuous assays of the rate of thrombin neutralization.

Amidines↗
PubMed1981

Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction.

The kinetics of high affinity heparin binding to human antithrombin III has been studied by stopped flow fluorimetry, using the 40% antithrombin fluorescence enhancement resulting from this interaction. At mu 0.15, pH 7.4, and 25 degrees C, the observed pseudo-first order rate constant varies hyperbolically with heparin concentration with a limiting rate constant of 440 +/- 90 s-1, demonstrating that heparin binding is a two-step process involving a conformational change in antithrombin III. An identical dependence is produced when antithrombin is varied, consistent with a symmetrical mechanism in which heparin binding induces a conformational change in antithrombin rather than perturbing an equilibrium between two conformational states of the protein. The rate constant for dissociation of the antithrombin-heparin complex is 1.1-1.5 s-1 at mu 0.15, as determined from the ordinate intercept at low heparin concentrations or by dissociation of the antithrombin-heparin complex with iodide. Observation of a single pseudo-first order binding rates over a 400-fold heparin concentration range with no detectable lags is compatible with the initial binding step being in rapid equilibrium with a KD of 4.3 +/- 1.3 X 10(-5) M at mu 0.15. Variation in ionic strength primarily affects the KD for the initial binding step with little effect on the conformational change rate constants, implying that binding involves ionic interactions. Calculation of the overall dissociation equilibrium constant from these rate parameters agrees with the directly determined value of 7.2 +/- 1.9 X 10(-8) M at mu 0.15. A major function of the conformational change is, thus, to increase the affinity of heparin for antithrombin III greater than 300-fold. The implications of these findings for the mechanism of the heparin-catalyzed inhibition of coagulation proteases by antithrombin III are discussed.

Antithrombin III↗
PubMed1980

Influence of click sound pressure direction on brain stem responses in children.

Brain stem auditory responses were evoked by stimulation with condensation (C) and rarefaction (R) clicks separately. The latencies of the first five waves of the response were compared in response to the two sound pressure directions. Responses to R clicks were significantly shorter than responses to C clicks for wave 4, in the absence of consistent response differences in the other waves.

Acoustic Stimulation↗