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S T Olson

Publications and source records attributed to S T Olson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin.

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Antithrombin III↗
PubMed1997

Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.

Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54-65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54-65 and Hir54-65 bound to human alpha-thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (>/=200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54-65 and Hir54-65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54-65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.

Antithrombin III↗
PubMed1997

The oligosaccharide side chain on Asn-135 of alpha-antithrombin, absent in beta-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change.

The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.

Antithrombin III↗
PubMed1997

Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme.

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3, 4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B. , Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by alpha1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

Animals↗
PubMed1997

Effect of individual carbohydrate chains of recombinant antithrombin on heparin affinity and on the generation of glycoforms differing in heparin affinity.

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk, et al., 1992, Biochem. J. 286, 793-800; B. Fan et al., 1993, J. Biol. Chem. 268, 17588-17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.

Animals↗
PubMed1997

Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin. Implications for the regulation of pericellular proteolysis.

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into beta-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with beta-anhydrotrypsin, which does not undergo rearrangement of beta-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to beta-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1.plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.

Hydrolysis↗
PubMed1997

Contribution of lysine 60f to S1' specificity of thrombin.

Lys60f has been proposed to limit the S1' substrate binding site specificity of thrombin to small polar P1' residues by occluding the S1' binding pocket, based on the X-ray crystal structure of thrombin. To test this proposal, we prepared a Lys-->Ala (K60fA) mutant of recombinant thrombin and determined whether this mutation enhanced the reactivity of thrombin with a variant inhibitor [antithrombin (AT)-Denver] and a substrate (protein C) containing poorly recognized P1' Leu residues. AT-Denver in the presence of heparin inhibited K60fA thrombin with a second-order association rate constant [k = 4.2 +/- 0.1) x 10(5) M-1 s-1] that was 3.2-fold faster than thrombin [k = (1.3 +/- 0.1) x 10(5) M-1 s-1]. Wildtype AT (P1' Ser) under the same conditions inhibited K60fA thrombin with a 2.5-fold slower rate constant [k = (1.1 +/- 0.1) x 10(7) M-1 s-1] than thrombin [k = (2.8 +/- 0.1) x 10(7) M-1 s-1]. These results indicate an overall 8.3-fold improvement in the recognition of the P1' Leu of AT-Denver by K60fA thrombin over that of wild-type thrombin; i.e., the K60fA mutation partly overcomes the defect in thrombin inhibition produced by the P1' mutation in AT-Denver. Resolution of the two-step reactions of AT and AT-Denver with wild-type and mutant thrombins revealed that the enhanced recognition of P1' Leu in AT-Denver by K60fA thrombin occurs primarily in the second reaction step in which a noncovalent AT-thrombin encounter complex is converted to a stable, covalent complex. Thrombin K60fA activated Gla-domainless protein C (GDPC) approximately 2- and approximately 4-fold faster than thrombin in the presence and absence of thrombomodulin (TM), respectively, consistent with an improved interaction of the Leu P1' residue with the mutant S1' pocket. In contrast, the mutant thrombin clotted fibrinogen (P1' Gly) approximately 3-fold slower than thrombin. Kinetic analysis revealed that the improvement in the catalytic rate of activation of GDPC by K60fA thrombin in the presence of TM was localized in the second reaction step, as reflected by an approximately 2-fold increase in kcat. Direct binding studies showed that the K60fA mutation minimally affected the affinity of thrombin for Na+, indicating that the changes in S1' site-specificity of K60fA thrombin did not result from altering the allosteric transition induced by Na+. We conclude that Lys60f limits the P1' substrate and inhibitor specificity of thrombin by influencing the size and polarity of the S1' site which thereby affects the stability of the transition state for cleavage of the scissile bond in the second reaction step.

Binding Sites↗
PubMed1996

Mechanism of heparin activation of antithrombin. Evidence for reactive center loop preinsertion with expulsion upon heparin binding.

A heparin-induced conformational change is required to convert antithrombin from a slow to a fast inhibitor of factor Xa. It has been proposed [van Boeckel et al. (1994) Nat. Struct. Biol. 1, 423-425] that the reactive center residue P14 is inserted into beta-sheet A in native antithrombin and is displaced from the beta-sheet by heparin binding, thereby altering the conformation of the reactive center and making it a better target for factor Xa binding. To test this hypothesis, we have characterized a P14 serine --> tryptophan antithrombin variant. From changes in tryptophan fluorescence upon heparin binding, increased affinity for heparin, and partial activation of the variant against factor Xa, we conclude that the proposed mechanism of heparin activation is correct with respect to loop expulsion and that it may consequently be possible to create more highly activated antithrombin variants through suitable hinge region substitutions.

Antithrombin III↗
PubMed1996

Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase.

Fluorescent analogs of the proteinase zymogen, plasminogen (Pg), which are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg activation. The active site induced non-proteolytically in Pg by streptokinase (SK) was inactivated stoichiometrically with the thioester peptide chloromethyl ketone. N alpha-[(acetylthio)acetyl]-(D-Phe)-Phe-Arg-CH2Cl; the thiol group generated subsequently on the incorporated inhibitor with NH2OH was quantitatively labeled with the fluorescence probe, 2-((4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid; and the labeled Pg was separated from SK. Cleavage of labeled [Glu]Pg1 by urokinase-type plasminogen activator (uPA) was accompanied by a fluorescence enhancement (delta Fmax/Fo) of 2.0, and formation of 1% plasmin (Pm) activity. Comparison of labeled and native [Glu]Pg1 as uPA substrates showed that activation of labeled [Glu]Pg1 generated [Glu]Pm1 as the major product, while native [Glu]Pg1 was activated at a faster rate and produced [Lys]Pm1 because of concurrent proteolysis by plasmin. When a mixture of labeled and native Pg was activated, to include plasmin-feedback reactions, the zymogens were activated at equivalent rates. The lack of potential proteolytic activity of the Pg derivatives allowed their interactions with SK to be studied under equilibrium binding conditions. SK bound to labeled [Glu]Pg1, and [Lys]Pg1 with dissociation constants of 590 +/- 110 and 110 and 11 +/- 7 nM, and fluorescence enhancements of 3.1 +/- 0.1 and 1.6 +/- 0.1, respectively. Characterization of the interaction of SK with native [Glu]Pg1 by the use of labeled [Glu]Pg1 as a probe indicated a approximately 6-fold higher affinity of SK for the native Pg zymogen compared to the labeled Pg analog. Saturating levels of epsilon-aminocaproic acid reduced the affinity of SK for labeled [Glu]Pg1 by approximately 2-fold and lowered the fluorescence enhancement to 1.8 +/- 0.1, whereas the affinity of SK for labeled [Lys]Pg1 was reduced by approximately 98-fold with little effect on the enhancement. These results demonstrate that occupation of lysine binding sites modulates the affinity of SK for Pg and the changes in the environment of the catalytic site associated with SK-induced conformational activation. Together, these studies show that the labeled Pg derivatives behave as analogs of native Pg which report functionally significant changes in the environment of the catalytic site of the zymogen.

Enzyme Precursors↗
PubMed1995

Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.

The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes.

Animals↗
PubMed1995

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals↗
PubMed1995

Kinetic characterization of the proteinase binding defect in a reactive site variant of the serpin, antithrombin. Role of the P1' residue in transition-state stabilization of antithrombin-proteinase complex formation.

To elucidate the role of the P1' residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394-->Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and 7-fold slower, respectively, than those of native AT, consistent with an altered specificity of the variant inhibitor for its target proteinases. AT-Denver inhibited thrombin and Factor Xa with nearly equimolar stoichiometries and formed SDS-stable complexes with these proteinases, indicating that the diminished inhibitor activity was not due to an enhanced turnover of the inhibitor as a substrate. Binding and kinetic studies showed that heparin binding to AT-Denver as well as heparin accelerations of AT-Denver-proteinase reactions were normal, consistent with the P1' mutation not affecting the heparin activation mechanism. Resolution of the two-step reaction of AT-Denver with thrombin revealed that the majority of the defective function was localized in the second reaction step and resulted from a 190-fold decreased rate constant for conversion of a noncovalent proteinase-inhibitor encounter complex to a stable, covalent complex. Little or no effects of the mutation on the binding constant for encounter complex formation or on the rate constant for stable complex dissociation were evident. These results support a role for the P1' residue of antithrombin in transition-state stabilization of a substrate-like attack of the proteinase on the inhibitor-reactive bond following the formation of a proteinase-inhibitor encounter complex but prior to the conformational change leading to the trapping of proteinase in a stable, covalent complex. Such a role indicates that the P1' residue does not contribute to thermodynamic stabilization of AT-proteinase complexes and instead favors a kinetic stabilization of these complexes by a suicide substrate reaction mechanism.

Antithrombins↗
PubMed1995

Mechanism of acceleration of antithrombin-proteinase reactions by low affinity heparin. Role of the antithrombin binding pentasaccharide in heparin rate enhancement.

The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (< 0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity (KD 19 +/- 6 microM) and 5-6-fold smaller fluorescence enhancement (8 +/- 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to approximately 100-fold higher polysaccharide concentrations and with a approximately 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. 1H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.

Antithrombins↗
PubMed1995

Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system. Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA. Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful. This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function. PAI-1 (0.7 microM) was incubated with peptide (55 microM) at 37 degrees C. At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate. The T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 of 98 +/- 18 min. The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis. However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated. Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1. These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro. These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.

Amino Acid Sequence↗
PubMed1994

Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.

Base Sequence↗
PubMed1994

Surface-induced alterations in the kinetic pathway for cleavage of human high molecular weight kininogen by plasma kallikrein.

We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO4) and sulfatides. Using a combination of SDS-polyacrylamide gel electrophoresis, Western blotting with polyclonal antibodies that specifically recognize the COOH terminus of the bradykinin moiety, and high pressure liquid chromatography analyses of the cleavage reaction, we have identified two intermediates in the formation of bradykinin from intact kininogen and demonstrated that alternative cleavage pathways are followed in the absence and presence of surfaces. The COOH-terminal bradykinin cleavage occurred first both in the absence and presence of DxSO4, producing a 103-kDa HK intermediate consisting of disulfide-linked heavy and light chains that retained the kinin moiety. In the presence of DxSO4, this was followed exclusively by the NH2-terminal bradykinin cleavage and release of kinin with no apparent change in molecular mass. Subsequently, a slower cleavage of an 8-kDa peptide from the amino terminus of the HK light chain occurred to form a 95-kDa end product. In contrast to this sequential cleavage pattern, NH2-terminal bradykinin and light chain cleavages occurred randomly in the absence of DxSO4, resulting in the production of an additional 95-kDa intermediate that retained bradykinin but had lost the 8-kDa peptide from the HK light chain. Comparison of the relative rates of the three kallikrein cleavages in the absence and presence of DxSO4 indicated that the surface enhanced the rates of both bradykinin cleavages 2-4-fold, but inhibited the light chain cleavage rate approximately 10-fold, thereby accounting for the change from a partially random to a sequential cleavage pattern in the presence of the surface. Steady-state kinetic analysis revealed that DxSO4 enhanced the kcat/KM for bradykinin release by the rate-limiting NH2-terminal bradykinin cleavage by approximately 2-fold due exclusively to an increase in kcat. Sulfatides appeared to produce the same effects on the pattern of HK cleavages as DxSO4. Blocking of the nonactive site, i.e. exosite, interaction between kallikrein and HK with excess prekallikrein or a synthetic peptide containing the region of HK that interacts with the kallikrein exosite significantly reduced the rate of bradykinin release as well as HK cleavages detected by SDS-polyacrylamide gel electrophoresis either in the absence or presence of DxSO4, indicating that the exosite interaction facilitates bradykinin cleavage.

Blood Proteins↗
PubMed1994

Engineering plasminogen activator inhibitor 1 mutants with increased functional stability.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.

Base Sequence↗