Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358

alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.     

Physiologic inhibition of human activated protein C by alpha 1-antitrypsin

The plasma antithrombotic enzyme activated protein C (APC) has two major plasma inhibitors. One is heparin-dependent, has been characterized, and is known as protein C inhibitor. The second inhibitor was isolated based on its heparin-independent ability to inhibit and complex with APC. The purified inhibitor had the amino acid composition and NH2 terminus of alpha 1-antitrypsin and reacted with monoclonal antibodies to alpha 1-antitrypsin. The inhibitor was greater than 95% pure alpha 1-antitrypsin as judged by electroimmunoassay, inactivation of trypsin, and electrophoresis in two gel systems. To identify the second major plasma inhibitor of APC, immunoblot studies of enzyme-inhibitor complexes were made to compare APC addition to normal plasma and to plasma deficient in protein C inhibitor or alpha 1-antitrypsin. The results showed that alpha 1-antitrypsin is the second major plasma APC inhibitor. Given the association rate constant of alpha 1-antitrypsin for APC of 10 M-1 s-1 and its plasma concentration of approximately 40 microM, it accounts for approximately half of the heparin-independent APC inhibitory activity of plasma. Based on immunoblot analysis plasmas of 15 patients with intravascular coagulation contained APC-alpha 1-antitrypsin complexes suggesting that this inhibition reaction occurs in vivo. Thus, alpha 1-antitrypsin is a major physiologic inhibitor of APC.     

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity

Hydrogen peroxide is a component of cigarette smoke known to be essential for inactivation of alpha(1)-antitrypsin, the primary inhibitor of neutrophil elastase. To establish the molecular basis of the inactivation of alpha(1)-antitrypsin, we determined the sites oxidized by hydrogen peroxide. Two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation was known to cause loss of anti-elastase activity. The other, methionine 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-elastase activity, an effect not previously recognized. The equal susceptibility of methionine 358 and methionine 351 to oxidation was confirmed by mass spectrometry. To verify this finding, we produced recombinant alpha(1)-antitrypsins in which one or both of the susceptible methionines were mutated to valine. M351V and M358V were not as rapidly inactivated as wild-type alpha1-antitrypsin, but only the double mutant M351V/M358V was markedly resistant to oxidative inactivation. We suggest that inactivation of alpha(1)-antitrypsin by oxidation of either methionine 351 or 358 provides a mechanism for regulation of its activity at sites of inflammation.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.     

Chymotrypsin inhibitory activity of normal C1-inhibitor and a P1 Arg to His mutant: evidence for the presence of overlapping reactive centers.

C1-inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1-inhibitor, normal rabbit C1-inhibitor, and P1 Arg to His mutant human C1-inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate-resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1-inhibitor (3.4 x 10(4) compared with 7.3 x 10(3)). The carboxy-terminal peptide of normal human C1-inhibitor, derived from the dissociated protease-inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with alpha 2-antiplasmin, C1-inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg-specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike alpha 2-antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1-inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.     

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.     

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.     

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.     

Plasma protease inhibitors in premature infants: influence of gestational age, postnatal age, and health status

In newborn infants, the influence of gestational age (GA), postnatal age (PA), and health status on the plasma protease inhibitors alpha 2-macroglobulin (alpha 2-M), alpha 1-antitrypsin (alpha 1-AT), C1 esterase inhibitor (C1E-INH), alpha 2-antiplasmin (alpha 2-AP), and antithrombin III (AT-III) was investigated. Inhibitor levels were measured by radial-immunodiffusion and expressed as a percentage of pooled plasma from adults (mean +/- SEM). In total, 54 premature infants (28-36 weeks gestation) were classified at birth as healthy (N = 22) (IV fluids, antibiotics only) or sick (N = 32) (all other support, but excluding infants with disseminated intravascular coagulation (DIC] and studied on Days 1 and/or 7 of life. Healthy term infants (N = 18) and infants with DIC (N = 10) were studied on Day 1 only. All inhibitors except C1E-INH increased with increasing gestational age (P less than 0.01). In healthy premature infants all inhibitor levels reached the normal adult range by 1 week of age. In contrast, at 1 week of age, sick infants had lower levels of alpha 2-M and alpha 2-AP, and higher levels of alpha 1-AT compared to healthy infants (P less than 0.01). The presence of DIC depressed all of the inhibitors on Day 1 except alpha 1-AT when compared to healthy controls (P less than 0.01). Thus, gestational age, postnatal age, and health status all significantly influenced the levels of these plasma protease inhibitors.     

Comprehensive assessment of the hemostasis system in the participants of the Mars-500 experiment

Hemostasis has been studied in the course of long-term (520 days) isolation in hermetic chamber. Measured parameters included activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (ТT); concentrations of fibrinogen (FBG), plasminogen (PG), Willebrand factor (WF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (TPA), and thrombomodulin (ТМ); activities of the coagulation cascade factors II, V, VII, X, VIII, IX, XI, and XII, antithrombin III (ATIII), protein С (PC), С1-inhibitor (С1), α₂-antiplasmin (АP), TPA and TFPI. The investigation revealed a diversity of changes in plasma FBG concentration, slower blood coagulation in the intrinsic pathway and in final stage, and a relative rise in the activities of ATIII and PC-inhibited factors. The remaining parameters exhibited different trends.     

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.     

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.     

Interaction of type 1 plasminogen activator inhibitor with the enzymes of the contact activation system

The interaction between type 1 plasminogen activator inhibitor (PAI-1), a serine protease inhibitor, and the three serine proteases generated during contact activation of plasma was studied using functional and immunologic approaches. Incubation of Factor XIIa, Factor XIa, and plasma kallikrein with either purified PAI-1 or platelet-derived PAI-1 resulted in the formation of sodium dodecyl sulfate-stable complexes as revealed by immunoblotting techniques. Functional assays indicated that Factor XIa and, to a lesser extent, Factor XIIa and plasma kallikrein neutralized the ability of purified PAI-1 to bind to immobilized tissue-type plasminogen activator (t-PA). Immunoblotting demonstrated that these enzymes also neutralized the ability of PAI-1 to form complexes with fluid-phase t-PA. Clot lysis assays employing purified proteins and 125I-fibrinogen were used to investigate the profibrinolytic effect of these contact activation enzymes. At enzyme concentrations that did not result in direct activation of plasminogen, only Factor XIa was capable of stimulating the lysis of clots supplemented with both t-PA and PAI-1. As a consequence of their interactions with PAI-1, the amidolytic activity of Factor XIIa, Factor XIa, and plasma kallikrein was neutralized by this inhibitor in a time-dependent and concentration-dependent manner. Minimum values estimated for the apparent second-order rate constant of inhibition were 1.6 x 10(4), 2.1 x 10(5), and 6.0 x 10(4) M-1 s-1 for Factor XIIa, Factor XIa, and plasma kallikrein, respectively. These data define new reactions between coagulation and fibrinolysis proteins and suggest that a major mechanism for stimulation of the intrinsic fibrinolytic pathway may involve neutralization of PAI-1 by Factor XIa.     

A positively charged loop on the surface of kallistatin functions to enhance tissue kallikrein inhibition by acting as a secondary binding site for kallikrein

Kallistatin is a serine proteinase inhibitor (serpin) that specifically inhibits tissue kallikrein. The inhibitory activity of kallistatin is abolished upon heparin binding. The loop between the H helix and C2 sheet of kallistatin containing clusters of basic amino acid residues has been identified as a heparin-binding site. In this study, we investigated the role of the basic residues in this region in tissue kallikrein inhibition. Kallistatin mutants containing double Ala substitutions for these basic residues displayed a 70-80% reduction of association rate constants, indicating the importance of these basic residues in tissue kallikrein inhibition. A synthetic peptide derived from the sequence between the H helix and C2 sheet of kallistatin was shown to suppress the kallistatin-kallikrein interaction through competition for tissue kallikrein binding. To further evaluate the function of this loop, we used alpha1-antitrypsin, a non-heparin-binding serpin and slow tissue kallikrein inhibitor as a scaffold to engineer kallikrein inhibitors. An alpha1-antitrypsin chimera harboring the P3-P2' residues and a sequence homologous to the positively charged region between the H helix and C2 sheet of kallistatin acquired heparin-suppressed inhibitory activity toward tissue kallikrein and exhibited an inhibitory activity 20-fold higher than that of the other chimera, which contained only kallistatin's P3-P2' sequence, and 2300-fold higher than that of wild-type alpha1-antitrypsin. The alpha1-antitrypsin chimera with inhibitory characteristics similar to those of kallistatin demonstrates that the loop between the H helix and C2 sheet of kallistatin is crucial in tissue kallikrein inhibition, and this functional loop can be used as a module to enhance the inhibitory activity of a serpin toward tissue kallikrein. In conclusion, our results indicate that a positively charged loop between the H helix and C2 sheet of a serpin can accelerate the association of a serpin with tissue kallikrein by acting as a secondary binding site.     

Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli

Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.     

Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases

Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1-inhibitor, and alpha 2-antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 1-antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino-terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy-terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis--one near the amino-terminal end and another in the exposed reactive site loop of the inhibitor.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.