Serine protease inhibitors (serpins)

of ubiquitously expressed gene leads to nephrotic syndrome . Cell 62 :425-434 . Williams PL, Courtneidge SA, Wagner EF : 1988 . Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene . Cell 52 :121-131 . Woychik RP, Maas RL, Zeller R, Vogt TF,

Leder P: 1990 . Tormins' : proteins deduced from the alternative transcripts of the limb deformity gene . Nature 346 :850-853 . Yee S-P, Mock D, Maltby V, et al. : 1989 . Cardiac and neurological abnormalities in v-fps tmnsgenic mice. Proc Nat] Acad Sci USA 86:5873-5877, TCM

Serine Protease Inhibitors (Serpins)

underlined by the observation that reduced inhibitor levels are key pathogenic factors for the development of specific clinical syndromes (Table 3) . Reduced levels of functional inhibitor result from low concentrations of normal serpins or from the presence of dysfunctional (mutated) proteins (Harpel 1987 ; Huber and Carrell 1989 ; Bock 1991). Serpins can also have deleterious effects, as illustrated by the hemorrhagic lesion caused by infection with the Brighton Red cowpox virus . The viral gene responsible for hemorrhage encodes a protein that exhibits > 30% identity with antithrombin III (Pickup et al . 1986) .

Marc Schapira and Philip A . Patston • Structure-Function Relationship of Serpins

Inhibition of serine proteases by serpins (serpin : serine protease inhibitor) is a key mechanism for the control of proteolysis in thrombosis, shock, and inflammation . The various members of the serpin gene superfamily (a l antitrypsin, ovalbumin, Cl-inhibitor, antithrombin III, a 2 antiplasmin, type-1 plasminogen-activator inhibitor, and so forth) have many characteristics in common . In this article, we review the biochemistry and cell biology of serpins, and we discuss their clinical importance and therapeutic potential . (Trends Cardiovasc Med 1991 ; 1 :146-151)

• Blood Coagulation, Complement Activation, and Fibrinolysis Are Proteolytic Reactions Mediated by Serine Proteases Cleavage of zymogens (for instance, prothrombin and plasminogen) and other protein substrates (fibrinogen, fibrin, and kininogen) by serine proteases is a fundamental mechanism in several host defense reactions, including blood coagulation, complement activation, fibrinolysis, and cell migration. The structurefunction relationship of the proteolytic enzymes involved in these reactions has been studied in detail . The serine proteases involved in blood coagulation, complement, and fibrinolysis are usually two-chain molecules with a noncatalytic, amino-terminal, segment linked by disulfide bonds to a trypsinlike catalytic domain . Noncatalytic segments have a Marc Schapira and Philip A. Patston are at the Hematology Division, Vanderbilt University, Nashville, TN 37232, USA. 146

modular organization (for example, Ca2 tbinding module, fibrin-binding module, or finger or growth-factor module) that determines the binding characteristics and, thereby, the specificity of each particular enzyme (Patthy 1985) . • Serine Proteases Are Inhibited by Serpins Various molecules can inhibit the activity of serine proteases, including both small organic compounds and proteins (Table 1) . The serpin superfamily is a recently identified family of protein protease inhibitors, which comprises a t antitrypsin, antithrombin III, and other inhibitors of related structure (Huber and Carrell 1989) . Although over two dozen different serpins have been identified, only half of them have well-defined inhibitory activity against serine proteases (Table 2) . The physiologic importance of several members of the serpin superfamily (a t -antitrypsin, antithrombin III, Cl-inhibitor, and so on) is

®1991,

Elsevier Science Publishing Co .,

Serpins are single-chain proteins that form bimolecular and inactive complexes with their target enzymes . Each serpin contains one reactive site, which is located close to the carboxy terminus . Studies on the reaction between a 2 antiplasmin and chymotrypsin or trypsin showed that, in the first step of the reaction, there is a thermodynamic equilibrium between free serpnn, free enzyme, and the serpin-enzyme complex (Shich et al. 1989) . Subsequently, irreversible serpin-enzyme complexes are formed. During irreversible enzyme inhibition, the serpin is cleaved at the reactive site peptide bond, which is formed between adjacent amino acid residues termed P t and P t ' . It is believed that irreversible enzyme inhibition involves the formation of a stable ester bond between the carbonyl group of the serpin P t residue and the y-hydroxyl group of the active site serine of the target enzyme . At neutral pH, the covalent serpin-enzyme complex does not undergo (or undergoes very slowly) the deacylation step with subsequent release of cleaved protein, as is normally seen when serine proteases hydrolyze their substrates (Travis and Salvesen 1983) . A more precise definition of the recognition sites between serine proteases and serpins, the nature of the intermolecular bonds, and the conformational changes taking place on association will require determination of the three-dimensional structure of enzyme-serpin complexes (Janin and Chothia 1990) . Inspection of protein and DNA sequences shows that the various serpins

1050-I738/9l/$2 .00

TCM VoL 1, No. 4, 1991



Table 1 . Inhibitors of serine proteases

Organic compounds

Organophosphates Benzamidinc Phenylmethylsulfonyl Fluoride Chloromethylketones Argatroban Cephalosporines Boronic acid derivatives

Protein protease inhibitors

Kunitz-type (soybean trypsin inhibitor, aprotinin, inter-a-trypsin inhibitor, lipoprotein-associated coagulation inhibitor, and amyloid (3 protein precuts''') Kazal-type (ovomucoid) Bowman-Birk-type C(,- Macroglobulin Hirudin Egli n Serpins

Table 2 . Serpin superfamily (partial list)`

Protease inhibitors

Plasma : a t -antitrypsin, antithrombin Ill . Cl-inhibitor. az antiplasmin, PAM, PAI-2, heparin cofactor 11, activated protein-C inhibitor, and a l antichymotrxpsin Cytosolic : horse ncutrophil elastase inhibitor Extracellular matrix : protease nexin 1- inntithrombin III and PAI-1

Hormone-binding proteins

Thyroxine- and cortisol-binding globulin .

Miscellaneous

Ovalbumin, angiolensinogen, uterine milk protein, coo+-pox virus, vaccinia virus, and barley Z protein

At lgnrnent

oC,

mmno- ;rcid sequences nr

20

cr pins 1,, presented in n

fiber a t i (' etch (1989) .

Table 3 . Inherited serpin-deficiency states* Serpin

a,-Antitrypsin Antithrombin III Cl-inhibitor a,-Antiplasmin PAM A

Target enzyme Nentrophil elastase Throttibin, factor Xa Cl, kallikrein, and [act orXIIa Plasmi t t-PA and makinase

comprehensive description

is touu,I

in I Impel

Col . l, Mn . 4 . 1991

Emphysema Thrombosis Hereditary angioedema Hemorrhage Hemorrhage

I I't 7))

contain a homologous region, the serpin module, which spans : 350 amino acid rcsiducs ; the degree of homology between various serpin modules ranges from 25% to , 50% (Huber and Carrell [C11

Clinical manifestation

.

1949 ; Bock 1991) . Some serpins contain additional regions with well-defined functions, For example. antithrombin III, heparin cofactor IL and protease resin I have specific binding sites for heparin '91, E

Isevicr Science Publishing

Co-,

and related glyeosaminoglyeans (Bock 1991) . Similarly, a,-antitnpsin can scavenge reactive oxygen species or release a chemotactic peptide (Travis and Salvesen 1983 ; Banda ei al . 1988), a,antiplasm in coma ins a site for erossl i eking to fibrin (Taniaki and Aoki 1982), and PilM binds to vilronectin (Loskuloff et al . 1959) . The three-dimensional structures of cleaved a.,-antis- psin, cleaved ovalbumin and native ovalbumin have been solved by x-ray crystallography to resolutions ranging h-om 3 .0 to 1 .95 A (Loebermann et al . 1984 ; Weight et al . 1990 ; Stein el al . 1990) . Apart ham one important ditleicncc between the c leaved reactive site, of a t -antitrvpsin and ovalbumin, the snttctures are very similar . Both serpius hake a highly ordered organization ; lot example, 8118,- of the iesid,les of cleaved a,-antitrvpsin are arranged in three IS sheets and eight (,-helices Ihoebcrmallli et al . 1984) . Interestingly, the reactive center of intact ovalbumin has the f'rm of an exposed loop centered he a three

lurtt (x-hehx Ihal contains 12 resualcs boil P,,, to P,' IFieure IAl . Aslim ''•a lbumin Is dcaced beueecu posniuns I' t and F 1 ', the iecvly lurmed earb,rs% venom's remains tree near to the clr,tyage site (Figure I M This contrasts kith the pal tern S'.,11 %kith cleaved rt,antu,-psm, in %%iich the prince loop ending at P I is tnrurpotated into I3 sheet A vyhere it loans --viand s4A (Figure I C1 . 'the nature u! the forces that Iactlnalc insertion of strand s4A into (I-sheet A in tx antW,'psus and prevznt it In ovalhu mm is riot Ostabli'lled . blalur conlorm.,ttonal changes are also detectable upon %leavage of the reactive site region of 'tire inhibitory serpin (toe instance, Cl-inhibitor or antithrombin 11I), but not al angiotensino_en, ud'.ieh, like ovalbumin, does not have inhibitors activity against seine proteases (Brush et al . 1988 ; do .Agos'.ini el al . 1988 ; Get tins 1989 ; Stein rt al 19891 . The extort t,t which the e' nfn-mauonal change seen with inhibitory sr. - pins plans a role, if a ny . i n the inhibition reaction is not knoset an identical conformational change is irnmunologically detectable on Cl-inhibitor envalcntir bored m its target cnzv,oes, or When this supra s cleaved at its reactor site b% nontatget Trine proteases, a reaction that mactisates Cl-inhibitor, hill does not result m enzyme inhibition dc Ago=_lini ct al. I4` 1 '

1050-1738/91/$2.00

147





Whereas wild-type a 1 -antitypsin Met358 is an inhibitor of elastase (but not of thrombin), a 1 -antitrypsin Arg 358 is a potent inhibitor of thrombin, but does not react with elastase (Table 4) . This observation is confirmed by experiments with recombinant a 1 -antitrypsin Arg 358 (obtained by site-directed mutagenesis of cloned a 1 -antitrypsin cDNA), which show that the mutated serpin exhibits the inhibitory characteristics of a 1 antitrypsin Pittsburgh against thrombin and also inhibits Arg-specific serine proteases of the kallikrein-kinin and fibrinolytic systems (Courtney et al. 1985 ; Patston et al . 1990) . Secondary sites of interaction have an important role in the rapid and efficient inhibition of serine proteases by serpins . PAM [the natural inhibitor of tissue plasminogen activator (t-PA)], Cl-inhibitor (the natural inhibitor of Cls and kallikrein), and a 1 -antitrypsin A1a 357 Arg35 8 (an a 1 -antitrypsin double mutant) have the same P 1 and P2 residues (Arg and Ala, respectively) . Therefore, these serpins can be used to define further the determinants of serpin reactivity . Comparison of the rate constants obtained for the inhibition of t-PA, Cis, or kallikrein by these serpins demonstrates that other factors, in addition to the structure of the reactive site, are required for efficient enzyme inhibition . Indeed, t-PA is rapidly inhibited by PAM, weakly by the double mutant, and remains unaffected by Cl-inhibitor. In contrast, Cis is efficiently inhibited by Cl-inhibitor, weakly by the double mutant, and not at all by PAI-1 (Table 4) . Support for a role of secondary sites of interaction for the rapid inhibition of t-PA is also provided by the observation that t-PA deletion

Figure 1 . Schematic ribbon drawing showing the reactive site region and sheet A of ovalbumin and a,-antitrypsin . (A) Structure of native ovalbumin (Stein et al . 1990). Four of the strands composing l;-sheet A are shown in green, the region between P, 5 and P, is in red, and the region between P 1 ' and the carboxy terminus is in blue . In intact ovalbumin, the 12 residues comprised between P, o and P=' form a three-turn a-helix . (B) Structure of ovalbumin cleaved at residue P 1 (Wright et al. 1990) . After proteolysis at the P,-P, ' peptide bond, the a-helix found in native ovalbumin is disrupted, and the newly formed carboxy terminus stays next to the cleavage site. (C) Structure of a 1 -antitrypsin cleaved at residue P, (Loebcrmann et al . 1984) . After proteolysis at the P iP 1 ' peptide bond, the protein loop between P 75 and P 1 undergoes considerable refolding, since it is incorporated into P-sheet where it forms a new strand (s4A) . Whereas the distance between C and N in an intact peptide bond is 1 .3 A, P 1 (Met15B) and P 1 ' (Ser359 ) in cleaved a,-antitrypsin are separated by 67 A . The extent to which the structure of the reactive center of native a 1 -antitrypsin is similar to the corresponding region in ovalbumin is not known. • The Inhibitory Specificity of Serpins

conclusion is derived from the study of a patient with a lifelong bleeding disorder caused by increased inhibition of thrombin (Owen et al . 1983) . The patient had a mutated form of a 1 -antitrypsin (a 1 antitrypsin Pittsburgh) with a Met-*Arg substitution at P 1 (position 358), which caused dramatic changes in the inhibitory spectrum (Owen et al . 1983) .

Role of the Reactive Site and Secondary Sites of Interaction The amino-acid residue located at P 1 (the central position of the reactive center) has an important role in determining the inhibitory specificity of serpins. This

Table 4 . Reactivity of serpins Enzyme inhibition Serpln

Reactive site structure Pi

a 1 -AntitrypsinMet 358 a 1 -AntitrypsinArg358 a 1 -AntitrypsinAla 357 Arg358 PAI-I Cl-inhibitor Antithrombin III (+heparin)

P1

Elostate

Thrombin

t-PA

+++ ++ ++

+++

Cls

Kallikrein

++

++ ++

'

Ile-Pro-Met-Ser-Ile-Pro

+++

Ile-Pro-Arg-Ser-Ile-Pro

+

I1e-Ala-Arg-Ser-Ile-Pro Ser-Ala-Arg-Met-Ala-Pro

ND ND

Val-Ala-Arg-Thr-Leu-Leu

-

Ala-Gly-Arg-Ser-Leu-Asn

+++

ND: not determined.

148

©1991, Elsevier Science Publishing Co ., 1050-1738191/$2 .00

TCM Vol . 1, No . 4, 1991



mutants (lacking a region predicted to interact with PAI-1) have considerable resistance to inhibition by this serpin (Madison et al . 1989) . Further support for this notion is presented in a study examining the inhibition of t-PA and urokinase by a series of chimeric PAI-1 molecules . In these mutants, the protein loop between F 17 and P2 ' ( which contains the reactive site region) was replaced by the corresponding regions of antithrombin III, PAI-2, or by a serpin consensus sequence . These chimeric PAL 1 mutants inhibited t-PA and urokinase almost as well as wild-type PAI-1 (Lawrence et al . 1990) . • Serpin Biosynthesis and Regulation Hepatocytes appear to be a major site of synthesis for several serpins ((x 1 -antinypsin, antithrombin III, and C1-inhibitor), but synthesis is also observed in other tissues . For example, a 1 -antitrypsin is detectable by immunocytochemistry in several human tissues, including renal tubules, pancreatic islet cells, small intestine goblet cells, and in certain CNS neurons (Carlson et al . 1988) . Some aspects of the regulation of serpin biosynthesis have been studied in detail . For Cl-inhibitor, synthesis by monocytes and umbilical vein endothelial cells is stimulated by y-interferon (Lotz and Zuraw 1987; Schmaier et al . 1989) . Studies employing several types of endothelial cells, lung or kidney epithelial cells, and hepatoma or other tumor cells have shown that expression of the gene encoding PALI is modulated by various agents, including dexamethasone and other steroid hormones, several cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor p), and thrombin (Loskutoff et al . 1989) . The transfected a 1 -antitrypsin gene is expressed in a cell-specific fashion, with transcription being observed in Hep3B hepatoma, but not in HeLa carcinoma cells (Ciliberto et al . 1985) . In macrophages and hepatocytes, the antitrypsin gene is transcribed from two different promoters (Perlino et al . 1987) . With murine a 1 -antitrypsin, gene activation is not only tissue specific, but also species specific and developmentally regulated (Rheaume et al. 1988) . Mouse and rat hepatocytes rapidly remove from the bloodstream the complex formed betweeen thrombin and TCM hot. 1 , No . 4. 1991

Regulation of serpin biosynthesis : model for a,-antitrypsin . Native a,-antitrypsin is represented by red circles, complexes between a,-antitrpsin and neutrophil elastase by red and green circles, and free elastase by a green circle, (A) Liver: After entering the liver through the portal circulation, a,-antitrpsin-neutrophil-elastase complexes are taken up by hepatocytes, a reaction providing stimulus for increased synthesis of native a,-antitrypsin. (B) Lung: a,-Antitrpsin is produced by monocytes and macrophages, the stimulus for increased biosynthesis being cytokines (transforming growth factor (3 and interleukin 6) released by activated T cells, B cells, and macrophages . In the lung, a 1 -antitrypsin has a major role in inhibiting neutrophil elastase (a,-antitrypsin deficiency is often accompanied by severe emphysema) . (C) Brain : Some neurons contain a,-antitrypsin, but the role of this serpin is not known . Figure 2.

antithrombin HI, by a receptor-mediated pathway. Such a mechanism does not operate for native antithrombin III . The fractional metabolic rate of the complex is over ten times faster than the rate observed with native antithrombin III, Competition experiments indicate that the serpin domain of the complex (and not the serine protease moiety) is responsible for this observation . The pathway employed by the thrombin-antithrombin-III complex is also involved in the rapid metabolic clearance of complexes between other serpins (a,antitrypsin, a 1 -antichymotrypsin, and heparin cofactor II) and their target enzymes (Pizzo 1989) . The idea that conformational changes (taking place when antithrombin III and other serpins react with their target enzymes) create sites responsible for their recognition by cellular receptors and for increased biosynthesis is supported by the following experiments . Human monocytes synthesize a 1 -antitrypsin at an increased rate when they are cultivated in the presence of neutrophil elastase . Augmented biosynthesis is associated with

©1991,

Elsevier Science Publishing

increased levels of a,-antitrypsin mRNA (Perlmutter ct al . 1988) . In other words, a 1 -antitrypsin that has reacted with its principal target enzyme (but not the native inhibitor molecule) conveys a signal that, upon interaction with cellular receptors, increases its own production. Further support for this idea is provided by the observation that synthetic peptides mimicking the carboxy terminus of a,-antitrypsin bind to HepG2 hepatoma cells and induce them (as well as monocytes) to produce increased quantities of this serpin (Perlmutter et al . 1990) . The results described in the preceding paragraphs show cell- and organ-specific expression of a,-antitrpsin and other serpin genes . Under physiologic conditions, the a 1 -antitrypsin concentration is probably maintained at a level sufficient for controlling basal proteolytic activity by constitutive protein secretion . When increased quantities of elastase are released, the production of a 1 -antitrypsin can be regulated by a feedback mechanism, the stimulus for increased production being uptake of

Co ., 1050-17381911$2 .00

149



a t -antitrypsin-protease complexes by

antitrypsin Va1 358,

hepatocytes (Figure 2A) . Local concentrations (in the lung and at sites of

inhibitory spectrum of wild-type a,antitrypsin Met 358 , but resists oxidation,

injury) can be increased in response to a

would be efficacious in circumstances where neutrophil or macrophage activa-

series of inflammatory stimuli (for example, macrophage activation with cytokine production) that induce release of preformed serpin as well as de novo biosynthesis (Figure 2B) . In the acute phase reaction that accompanies many pathologic conditions, the production of inflammatory mediators can be sufficient to cause a substantial increase of hepatic biosynthesis; in these circumstances, the plasma

which retains the

tion generates sufficient reactive oxygen species to inactivate the natural serpin (Courtney et al. 1985) . Also, the double mutant a,-antitrypsin Ala 357 Arg358 is efficient in vivo in preventing the circulatory collapse induced by activated factorXII fragment, a well-documented kininmediated reaction (Schapira et al . 1987) . Furthermore, a,-antitrypsin Arg 358 seems to attenuate the course of Pseudomonas

level of a,-antitrypsin increases two- to fourfold . The role of local production of

aeruginosa hypotensive septicemia in pigs (Colman et al . 1988) . Third, inhib-

a,-antitrypsin in other tissues (brain, adrenal cortex, and so forth) is intrigu-

itors of serpin function will be identified (that is, agents protecting serine pro-

ing, since the target enzymes at these

teases from inhibition by serpins) . An

sites have not been identified (Figure 2C) . Interest in the role of protease

agent that could prevent inhibition of t-PA or urokinase by PAI-1 and other

inhibitors in the brain has been stimulated by the observation that the pre-

serpins has the potential of being a

cursor of amyloid

0 protein (which accumulates in patients with Alzheimer's disease and Down syndrome) contains a

useful adjunct in thrombolytic therapy . Also, the availability of such a compound would enable the testing of whether decreased fibrinolytic activity (because

Kunitz-type inhibitor domain that can inhibit trypsin and chymotrypsin

of increased levels of PAI-I) is participating in the hypercoagulable state ob-

(Selkoe 1990) . The extent to which the scenario illustrated in Figure 2 for a,-

served after myocardial infarction and

antitrypsin is applicable to other serpins is not known .

other inflammatory processes . Fourth, gene therapy may become a viable therapeutic approach for patients with inherited serpin deficiencies . This approach is currently under investigation for a,-

• Therapeutic Potential of Serpins

antitrypsin deficiency (Crystal 1990) .

Present and Future References Therapeutic options for serpin-deficiency states currently focus on stimulation of biosynthesis or replacement therapy . For example, administration of the anabolic steroid danazol increases the concentration of Cl-inhibitor in patients with hereditary angioedema and protects them against angioedema attacks (Gelfand et al. 1976). Infusion of exogenous antithrombin III prevents the development of thrombotic complications in patients with inherited deficiency of this serpin (Menache et al . 1990) . These options are likely to be expanded in the future in at least four directions. First, compounds that modulate serpin biosynthesis without having the systemic effects of steroid

Banda MJ, Rice AG, Griffin GL, Senior RM : 1988 . a,-Proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase . J Blot Chem 263 :4481-4484. Bock SC: 1991 . Antithrombin HI genetics, structure and function . In Hoyer LW, ed . Recombinant Technology in Hemostasis and Thrombosis : 21st Red Cross Symposium . New York, Plenum (in press) . Bruch M, Weiss V. Engel J : 1988 . Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage of their reactive sites . J Biol Chem 263 :16,626-16,630.

ond, mutated serpins with altered or improved inhibitory characteristics may

Carlson JA, Rogers BB, Sifers RN, Hawkins HK, Finegold MJ, Woo SLC : 1988. Multiple tissues express alpha,-antitrypsin in transgenic mice and man. J Clin Invest 82 :26-36 .

become available . For example, a,-

Ciliberto G, Dente L, Cortese R : 1985 . Cell-

hormones are under development (see preceding section and Figure 2A) . Sec-

150

specific expression of a transfected human a,-antitrypsin gene . Cell 41 :531-540 . Colman RW, Flores DN, de la Cadena RA, et al. : 1988 . Recombinant a,-antitrypsin Pittsburgh attenuates experimental Gramnegative septicemia. Am J Pathol 130:418426 . Courtney M, Jallat S, Tessier L-H, Benavente A, Crystal RG, Lecocq J-P : 1985 . Synthesis in E. coli of a 1 -antitrypsin variants of therapeutic potential for emphysema and thrombosis . Nature 313 :149-151 . Crystal RG : 1990 . a,-Antitrypsin deficiency, emphysema, and liver disease : genetic basis and strategies for therapy. J Clin Invest 85 :1343-1352 . de Agostini A, Patston PA, Marottoli V, Carrel S, Harpel PC, Schapira M : 1988 . A common neoepitope is created when the reactive center of Cl-inhibitor is cleaved by plasma kallikrein, activated factor XII fragment, C 1 esterase, or neutrophil elastase. I Clin Invest 82 :700-705 . Gelfand JA, Sherins RJ, Ailing DW, Frank MM : 1976 . Treatment of hereditary angioedema with danazol: reversal of clinical and biochemical abnormalities . N Engl J Med 295 :1444-1448 . Gettins P : 1989 . Absence of large-scale conformational change upon limited proteolysis of ovalhumin, the prototypic serpin . J Biol Chem 264 :3781-3785 . Harpel PC : 1987 . Blood proteolytic enzyme inhibitors : their role in modulating blood coagulation and fibrinolytic enzyme pathways . In Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis, 2nd ed. Philadelphia, JB Lippincott, pp 219-234 . Huber R, Carrell RW: 1989. Implications of the three-dimensional structure of a,antitrypsin for structure and function of serpins. Biochemistry 28:8951-8966 . Janin J, Chothia C : 1990. The structure of protein-protein recognition sites . J Biol Chem 265 :16,027-16,030 . Lawrence DA, Strandberg L, Ericson J, Ny T : 1990 . Structure-function studies of the SERPIN plasminogen activator inhibitor type 1 . J Biol Chem 265 :20,293-20,301 . Loebermann H, Tokuoka R, Deisenhofer J, Huber R : 1984 . Human a,-proteinase inhibitor. crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function . I Mol Biol 177 :531-556 . Loskutoff DJ, Sawdey M, Mimuro J : 1989 . Type 1 plasminogen activator inhibitor. Prog Hemost Thromb 9:87-115 . Lotz M, Zuraw BL : 1987 . Interferongamma is a major regulator of CI-inhibitor synthesis by human blood monocytes . J Immunol 139 :3382-3387.

01991, Elsevier Science Publishing Co ., 1050-1738/91/$2 .00

TCM Vo1. 1, No . 4, 1991



Madison EL, Goldsmith EJ, Gerard RD, Gething M-J, Sambrook JF : 1989 . Serpinresistant mutants of human tissue-type plasminogen activator . Nature 339 :721724 .

Perlmutter DH, Travis J, Punsal PI : 1988 . Elastase regulates the synthesis of its inhibitor, a t -proteinase inhibitor, and exaggerates the defect in homozygous PiZZ a, PI deficiency. J Clin Invest 81 :1774-1780.

Menache D, O'Malley JP, Schorr JB, et al . : 1990- Evaluation of the safety, recovery, half-life, and clinical efficacy of antithrombin III (human) in patients with hereditary antithrombin III deficiency. Blood 75 :3339 .

Perlmutter DH, Glover GI, Rivetna M, Schasteen CS, Fallon RJ : 1990 . Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes . Proc Natl Acad Sci USA 87 :3753-3757 .

Owen MC, Brennan SO, Lewis JH, Carrell RW: 1983, Mutation of a,-antitrypsin to antithrombin : a,-antitrypsin Pittsburgh (Met 358 -*Arg), a fatal bleeding disorder. N Engl J Med 309 :694-698 . Patston PA, Roodi N, Schifferli JA, Bischoff R, Courtney M, Schapira M : 1990 . Reactivity of a r -antitrypsin mutants against proteolytic enzymes of the kallikrein-ldnin, complement, and fibrinolytic systems . I Biol Chem 265 :10,786-10,791 . Patthy L: 1985 . Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules . Cell 41 :657-663 . Perlino E, Cortese R, Ciliberto G : 1987 . The human a,-antitrypsin gene is transcribed form two different promoters in macrophages and hepatocytes . EMBO 1 6:27672771 .

TCM

FOR

Pickup DJ, Ink BS, Hu W, Ray CA, Joklik WK : 1986 . Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases . Proc Natl Acad Sci USA 83 :7698-7702 . Pizzo SV : 1989 . Serpin receptor 1 : a hepatic receptor that mediates the clearance of antithrombin III-proteinase complexes . Am J Med 87(3B) :1OS-14S . Rheaume C, Latimer JJ, Baumann H, Berger FG : 1988 . Tissue- and species-specific regulation of murine a,-antitrypsin gene transcription . J Biol Chem 263 :15,118-15,121 . Schapira M, Ramus M-A, Waeber B, et al . : 1987 . Protection by recombinant a,antitrypsin Ala357 Arg358 against arterial hypotension induced by factor XII fragment . J Clin Invest 80 :582-585. Schmaier AH, Murray SC, Heda GD, et al . :

COLLEAGUES

1989 . Synthesis and expression of Clinhibitor by human umbilical vein endothelial cells . J Biol Chem 264 :18,173-18,179 . Selkoe DJ : 1990 . Deciphering Alzheimer's disease : the amyloid precursor protein yields new clues . Science 248 :1058-1060 . ShiehB-H, Potempa 1, Travis J : 1989 . The use of a2 -antiplasmin as a model for the demonstration of complex reversibility in serpins . J Biol Chem 264 :13,420-13,423 . Stein PE, Tewkesbury DA, Carrell RW: 1989 . Ovalbumin and angiotensinogen lack serpin S-R conformational change . Biochem J 262 :103-107 . Stein PE, Leslie AGW, Finch IT, Turnell WG, McLaughlin PJ, Carrell RW : 1990 . Crystal structure of ovalbumin as a model for the reactive centre of serpins . Nature 347 :99-102 . Tamaki T, Aoki N : 1982- Cross-linking of ay -plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor . J Biol Chem 257 :14,767-14,772 . Travis I, Salvesen GS : 1983 . Human plasma proteinase inhibitors . Anne Rev Biochem 52:655-709 . Wright TH, Oian HX, Huber R : 1990 . Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin : its relationship to the structure of cleaved a-l-proteinase inhibitor . J Mol Biol 213 :513-528 . TCM

ABROAD

Consider giving a subscription to TCM to colleagues abroad where US dollars or other hard currency is not available . Simply complete the subscription order card bound into any issue, giving the recipient's name and address labelled "send to" ; after "signature," give your own name and address and mark this "bill to ." Renewal notices will be sent to your address and the recipient will receive the journal . Please inform the recipient of your action .

TCM Vol. 1, No . 4, 1991

01991, Elsevier Science Publishing Co ., 1050-1738/91/$2 . 00

151