Insight into the molecular basis of coagulation proteinase specificity by mutagenesis of the serpin antithrombin

Specific cleavage of factor V at several P1Arg sites is critical for maintenance of hemostasis. While cleavage by procoagulant proteinases fXa and thrombin activates the cofactor, its cleavage by the anticoagulant proteinase activated protein C (APC) inactivates it. Antithrombin (AT), a specific serpin inhibitor of both thrombin and factor Xa, but not APC, was used as a model system to investigate molecular determinants of APC specificity in the inactivation reaction. Two mutants were prepared in which the P2 or the P3-P3' residues of the reactive site loop of the serpin were replaced with the corresponding residues of the APC cleavage site in factor V spanning residues 504-509 (Asp(504)-Arg-Arg-Gly-Ile-Gln(509)). Kinetic analysis showed that the reactivities of mutants were impaired by approximately 2-3 orders of magnitude with both factor Xa and thrombin, but improved by approximately 2 orders of magnitude with APC. The saturable dependence of the observed first-order rate constants on the concentrations of AT in complex with approximately 70-saccharide high-affinity heparin revealed that changes in the reactivity of the 504-509 mutant with proteinases are primarily due to an effect in the second reaction step in which a noncovalent serpin-proteinase encounter complex is converted to a stable, covalent complex. These results suggest that the P3-P3' residues of the APC cleavage site in factor Va, particularly P2Arg, confer specificity for the anticoagulant proteinase by improving the reactivity of the catalytic pocket with the transition state of the substrate in the second step of the reaction.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

The heparin binding site of protein C inhibitor is protease-dependent

Protein C inhibitor (PCI) is a member of the serpin family of protease inhibitors with many biological functions and broad inhibitory specificity. Its major targets in blood are thrombin and activated protein C (APC), and the inhibition of both enzymes can be accelerated by glycosaminoglycans, including heparin. Acceleration of thrombin and APC inhibition by PCI requires that both protease and inhibitor bind to the same heparin chain to form a bridged Michaelis complex. However, the position of the heparin binding site of APC is opposite to that of thrombin, and formation of the bridged complexes must require either radical reorientation of the proteases relative to PCI or alternate heparin binding modes for PCI. In this study, we investigate how heparin bridges thrombin and APC to PCI by determining the effect of mutations in and around the putative heparin binding site of PCI. We found that heparin binds PCI in a linear fashion along helix H to bridge thrombin, consistent with our recent crystal structure (3B9F), but that it must rotate by approximately 60 degrees to engage Arg-229 to bridge APC. To gain insight into the possible modes of heparin binding to PCI, we solved a crystal structure of cleaved PCI bound to an octasaccharide heparin fragment to 1.55 angstroms resolution. The structure reveals a binding mode across the N terminus of helix H to engage Arg-229 and align the heparin binding site of APC. A molecular model for the heparin-bridged PCI.APC complex was built based on mutagenesis and structural data.     

An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center

Horse leukocyte elastase inhibitor rapidly forms stable, equimolar complexes with both human leukocyte elastase and cathepsin G, porcine pancreatic elastase, and bovine alpha-chymotrypsin. Formation of the inhibitor-pancreatic elastase complex results in peptide bond cleavage at the reactive site of the inhibitor so that a small peptide fragment representing the carboxyl-terminal sequence of the inhibitor is released. Sequence analysis of both this peptide, as well as that of an overlapping peptide obtained by enzymatic inactivation of native inhibitor with either Staphylococcus aureus metalloproteinase, Pseudomonas aeruginosa elastase, or cathepsin B, yields data which indicate that the reactive site encompasses a P1-P1' Ala-Met sequence. However, unlike the human endothelial plasminogen activator inhibitor, which also has a Met residue in the P1' position, oxidation of the horse inhibitor only slightly reduces its association rate constant with either of the elastolytic enzymes tested or with chymotrypsin. Comparison of the amino acid sequence at or near the reactive site of the horse inhibitor (P2-P18') with members of the serpin superfamily of proteinase inhibitors indicates that it not only belongs in this class but also represents the first example of a functionally active intracellular serpin.     

Assessment of the Interaction Between Urokinase and Reactive Site Mutants of Protein C Inhibitor

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3 Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA–PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3 interact with PCI rather than uPA, consistent with P3 variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.     

Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin.

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.     

The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity

Antithrombin, the principal physiological inhibitor of the blood coagulation proteinase thrombin, requires heparin as a cofactor. We report the crystal structure of the rate-determining encounter complex formed between antithrombin, anhydrothrombin and an optimal synthetic 16-mer oligosaccharide. The antithrombin reactive center loop projects from the serpin body and adopts a canonical conformation that makes extensive backbone and side chain contacts from P5 to P6' with thrombin's restrictive specificity pockets, including residues in the 60-loop. These contacts rationalize many earlier mutagenesis studies on thrombin specificity. The 16-mer oligosaccharide is just long enough to form the predicted bridge between the high-affinity pentasaccharide-binding site on antithrombin and the highly basic exosite 2 on thrombin, validating the design strategy for this synthetic heparin. The protein-protein and protein-oligosaccharide interactions together explain the basis for heparin activation of antithrombin as a thrombin inhibitor.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.     

Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin

Thrombomodulin (TM), or its epidermal growth factor-like domains 456 (TM456), enhances the catalytic efficiency of thrombin toward both protein C and protein C inhibitor (PCI) by 2-3 orders of magnitude. Structural and mutagenesis data have indicated that the interaction of basic residues of the heparin-binding exosite of protein C with the acidic residues of TM4 is partially responsible for the efficient activation of the substrate by the thrombin-TM456 complex. Similar to protein C, PCI has a basic exosite (H-helix) that constitutes the heparin-binding site of the serpin. To determine whether TM accelerates the reactivity of thrombin with PCI by providing a binding site for the H-helix of the serpin, an antithrombin (AT) mutant was constructed in which the H-helix of the serpin was replaced with the same region of PCI (AT-PCIH-helix). Unlike PCI, the H-helix of AT is negatively charged. It was discovered that TM456 slightly (<2-fold) impaired the reactivity of AT with thrombin; however, it enhanced the reactivity of AT-PCIH-helix with the protease by an order of magnitude. Further studies revealed that the substitution of Arg35 of thrombin with an Ala also resulted in an order of magnitude enhancement in reactivity of the protease with both PCI and AT-PCIH-helix independent of TM. We conclude that TM enhances the reactivity of PCI with thrombin by providing both a binding site for the serpin and a conformational modulation of the extended binding pocket of thrombin.     

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor

-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human -thrombin shows multiple surface exosites for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). _T ^–-Thrombin and thrombin Quick I (Arg 67 Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. -Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (M_r = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5phosphate modifiedthrombin.     

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3-P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.     

Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.     

Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites

Blood coagulation factor IXa has been presumed to be regulated by the serpin, antithrombin, and its polysaccharide activator, heparin, but it has not been clear whether factor IXa is inhibited by the serpin with a specificity comparable to that for thrombin and factor Xa or what determinants govern this specificity. Here we show that antithrombin is essentially unreactive with factor IXa in the absence of heparin (k(ass) approximately 10 M(-1) s(-1)) but undergoes a remarkable approximately 1 million-fold enhancement in reactivity with this proteinase to the physiologically relevant range (k(ass) approximately 10(7) M(-1) s(-1)) when activated by heparin in the presence of physiologic levels of calcium. This rate enhancement is shown to derive from three sources: (i) allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (300-500-fold), (ii) allosteric activation of factor IXa by calcium ions (4-8-fold), and (iii) heparin bridging of antithrombin and factor IXa augmented by calcium ions (130-1000-fold depending on heparin chain length). Mutagenesis of P6-P3' reactive loop residues of antithrombin further reveals that the reactivity of the unactivated inhibitor is principally determined by the P1 Arg residue, whereas exosites outside the loop which are present on the activated serpin and on heparin are responsible for heparin enhancement of this reactivity. These results together with our previous findings demonstrate that exosites are responsible for the unusual specificity of antithrombin and heparin for three clotting proteases with quite distinct substrate specificities.     

Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.     

alpha(1)-Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1

Coagulation and complement proteinases are activated in sepsis, and one approach to therapy is to develop proteinase inhibitors that will specifically inhibit these proteinases without inhibiting activated protein C, a proteinase that is beneficial to survival. In this study, we made mutants of the serpin alpha(1)-PI, designed to mimic the specificity of C1-inhibitor. The P3-P2-P1 residues of alpha1-PI were changed from IPM to LGR and PFR, sequences preferred by C1s and kallikrein, respectively. Inhibition of C1s, kallikrein, factor XIIa, and activated protein C was assessed by SDS-PAGE, and by determination of the k(app) and SI. alpha(1)-PI-LGR inhibited C1s with a rate of 7790 M(-1)s(-1), but only minimal inhibition of C1 in a hemolytic assay was observed. Kallikrein, factor XIIa, and activated protein C were inhibited with rates of 382,180 M(-1)s(-1), 10,400 M(-1)s(-1), and 3500 M(-1)s(-1), respectively. alpha(1)-PI-PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4' residue of alpha(1)-PI-LGR Pro to Glu reduced the activity with C1s, consistent with the idea that C1s requires hydrophobic residues in this region of the serpin for optimal interaction. The data provide insight into the requirements for kallikrein and C1s inhibition necessary for designing inhibitors with appropriate properties for further investigation as therapeutic agents.     

A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP*

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1′ in rodents and P1′-P2′ in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3′ reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5′ residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.     

Importance of the P2 glycine of antithrombin in target proteinase specificity, heparin activation, and the efficiency of proteinase trapping as revealed by a P2 Gly --> Pro mutation.

A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic studies revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex.