Molecular basis of inherited human antithrombin deficiency

BLOOD VOL 80, NO 9

The Journal of The American Socieg of Hemutology

NOVEMBER 1, 1992 ~

REVIEW ARTICLE

Molecular Basis of Inherited Human Antithrombin Deficiency

By Morris A. Blajchman, Richard C. Austin, FranGoise Fernandez-Rachubinski, and William P. Sheffield

NTITHROMBIN (AT) deficiency is a heterogeneous A group of disorders that is inherited in an autosomal- dominant fashion. Were it not for the presence of inhibitors of coagulation, like AT, the unopposed presence of activated clotting factors in the circulation would result in thrombus formation within intact blood vessels, often with life-threatening consequences. The most abundant of these inhibitors is a plasma glycoprotein generally known as AT III.1*2 The existence of such an anticoagulant was described initially in 1905 by M~rawi t z ,~ who introduced the term progressive antithrombin activity to describe the ability of human plasma to neutralize thrombin slowly. With the discovery of heparin, and its subsequent clinical use as an anticoagulant, it was soon realized that heparin was effec- tive only in the presence of a plasma component. This activity was referred to as heparin cofactor ac t i~ i ty .~ ,~ A number of other AT activities were described subsequently. In 1954, Seegars et aI6 proposed a numerical classification to bring some order to the description of the various AT activities. AT I represented the ability of a fibrin clot to absorb thrombin and thus to neutralize its activity.6 AT I1 represented the previously described heparin cofactor activ- it^.^,^ AT I11 became the term used to describe the progressive antithrombin activity initially described by Morawitz?f6 AT IV represented the thrombin inhibitory activity seen in association with prothrombin activation.6 AT V was designated the AT activity found in the plasma of a patient with hypergammaglobulinemia in which the pathologic globulin inhibited thrombh6s7 AT VI represented the inhibition of thrombin produced by fibrin split products.6,8

With the isolation of the plasma protein that was associated with progressive AT activity, it was realized that this protein (AT 111) was the major plasma anticoagulant and that it also had heparin cofactor activity (AT II).* The availability of large quantities of this protein subsequently provided convincing evidence that the plasma component involved in progressive AT activity and that producing heparin cofactor activity were one and the same.1J A confusion point in the nomenclature was the fact that AT I11 also has been shown to inhibit other activated serine proteases, including factor Xa. Nonetheless, for mainly historical reasons, the term AT I11 continues to be used. Attempts to change the name have been partially success- ful, and in recent years the term antithrombin has come to be used interchangeably with AT IIL9Jo To further confuse

the issue, another protein with both progressive AT and heparin cofactor activities has been described.11J2 This latter moiety has been called heparin cofactor 11, a term used by Tollefsen et all2 to differentiate it from heparin cofactor I (ie, AT 111). In this report, rather than perpetu- ate the continued use of the atavistic term AT 111, we have chosen to use the simpler term antithrombin to describe this plasma glycoprotein exhibiting both progressive AT and heparin cofactor activities.

The existence of a deficiency state involving AT was recognized first by Egebergl3 who described a family, some of whose members suffered from episodes of recurrent venous thromboembolism. The plasma of affected members of this kindred was characterized by the reduction in both progressive AT activity and heparin cofactor activity.13 In fact, this kindred represents the first description ever of an inherited prothrombotic state! Subsequent to this first description of AT deficiency, many other kindreds with classical AT deficiency have been described from diverse geographic locations worldwide, with a reported prevalence of 1:2,000 to 1:5,000.'0J4-16

A decade after the initial description of classical AT deficiency, Sas et all7 described what was believed to be the first report of a kindred with a qualitative AT deficiency caused by the presence in the circulation of a mutant AT protein; AT Budapest. Another decade passed before the specific molecular pathology of an individual with an AT deficiency state was elucidated.18 This was the description of AT-Toyama, by Koide et al,18 who reported the substitution of Arg47 by Cys in this AT molecule. Since this first

From the Canadian Red Cross Society Blood Transfusion Service and the Departments of Pathology and Medicine, McMaster Univer- sity, Hamilton, Ontario, Canada.

Submitted February 12, 1992; accepted June 26,1992. Supported by a Canadian Red Cross Society (CRCS) Blood Services

R&D Fund Grant No. HA-01-92, RC.A. was a recipient of a research fellowship award from the Heart and Stroke Foundation of Canada; F.F.-R. is a recipient of a postdoctoral fellowship from the Medical Research Council (MRC) of Canada; and W.P.S. is a recipient of a scholar award from the CRCSIMileslMRC Scholar Program.

Address reprint requests to M.A. Blajchman, MD, FRCP(C), Room 2N31, McMaster University Health Sciences Centre, 1200 Main St Hamilton, Ontario LSN 325 Canada.

0 1992 by The American Society of Hematology. 0006-4971 /92/80O9-0010$3.00/0

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2160 BLAJCHMAN ET AL

elucidation of a mutant AT molecule, the identification of the molecular defects of many kindreds with AT deficiency has proceeded very rapidly.19-z2 As of March 1992, the specific characterization of AT mutations has been reported for more than 100 kindreds. Because some mutations have been reported more than once, approximately 40 different specific mutations, resulting in an absent or a pathologic AT gene product, causing clinical AT deficiency, have been reported.

THE SERINE PROTEASE INHIBITOR SUPERFAMILY: THE SERPINS

AT shares structural and functional homology with other members of a superfamily of proteins known as the serine protease inhibitors or s e r p i n ~ . ~ ~ - ~ ~ The degree of homology amongst the serpins suggests that they evolved from a common ancestral molecule about 300 to 600 million years ago.23 The serpins consist of single-chain glycoprotein molecules, of approximately 400 amino acids in length. More than 40 members of the serpin family have been identified in viruses, plants, and in higher animals, with approximately 30% homology at the amino acid level, with each other.10,26 Although most serpins function as inhibitors of serine proteases, not all have retained such function. Some have evolved other roles, such as carriers of lipophilic molecules (thyroxin- and cortisol-binding globulins), or as peptide hormone precursors (angiotensinogen), or have no recognizable function ( o v a l b ~ m i n ) . ~ ~ ~ ~ ~ The noninhibitory serpins are included in the serpin superfamily on the basis of amino acid homology with the inhibitory serpins.

Amino acid sequence alignment, based on the crystal structure of the cleaved form of the archetypal serpin a-1-antitrypsin, indicates that the serpins share a common, highly ordered structure.27 Each of the inhibitory serpins has a similar reactive center (designated Pl-Pl’), which is believed to act as a pseudosubstrate for the target protease. Protease inactivation by inhibitory serpins is by the formation of a 1:l stoichiometric covalent complex between the active site of the serine protease and the reactive center of the serpin. This inactivation process involves at least two separate but interrelated events-hydrolysis of the serpin’s reactive center by its cognate protease followed by covalent complex f o r m a t i ~ n . ~ ~ - ~ ~

The reactive center’s P1 residue, situated toward the amino terminal of the molecule, has been shown to partially confer inhibitor specificity for the inhibitory serpins. For example, AT, which inhibits proteases that cleave next to an arginyl residue, has an arginine at position P1, while a-1-antitrypsin, the primary inhibitor of elastase, has a methionine at this position. The mutant a-l-antitrypsin- Pittsburgh molecule, in which there is a single Met358Arg substitution at the P1 site, converts the usual elastase specificity of a-1-antitrypsin from elastase to that of thrombin. Thus, a-1-antitrypsin-Pittsburgh was converted from an inhibitor of elastase into a potent inhibitor of thrombin. This mutation manifested clinically as a lethal bleeding d i ~ o r d e r . ~ ~ , ~ ~

Crystallographic studies of serpin structure indicate that the inhibitory serpins exist in a conformation that facilitates

attack by their cognate serine proteases, and that the transition from an intact to a cleaved form is a thermodynam- ically favored event.lO~~~ Recent analysis of the crystal structure of the noninhibitory serpin, ovalbumin, showed that it has a peptide loop analogous to that of the reactive center of the inhibitory serpins. This takes the form of a protruding a-helix, consisting of the reactive center and 10 to 15 amino acid residues amino-terminal to the flanking sequence.34

While the presence of this mobile, exposed reactive loop appears to be common to all members of the serpin family, there is considerable structural diversity elsewhere in the molecule. This diversity likely reflects the various physiologic roles that these molecules assume, either as an allosteric binding site for heparin in AT or heparin cofactor- 11, or as a peptide donor in angiotensinogen.

PRIMARY PROTEIN AND GENE STRUCTURE OF HUMAN ANTITHROMBIN

Plasma-derived human AT consists of a single-chain glycoprotein of molecular mass of approximately 60 Kd, comprising 432 amino acid r e s i d ~ e s . ~ ~ - ~ ~ There are three disulphide bonds linking cysteine residues 8 and 128,21 and 95, and 247 and 430.35,39 The molecule also contains four oligosaccharide side chains, which comprise approximately 15% of the molecular ~ n a s s . ~ , ~ ~ These are coupled to Asn residues 96, 135, 155, and 192, r e ~ p e c t i v e l y . ~ ~ , ~ ~ ~ ~ ~ An underglycosylated form of plasma-derived AT, which represents approximately 10% of the total plasma concentration of AT, is glycosylated at only three of the four Asn residues, not including Asn 135.44,45

Molecular cloning has allowed the determination of the complete nucleotide sequence of human AT cDNA. Al- most simultaneously, three independent research groups reported the isolation of the cDNA of human AT and the identification of the gene.36-38 The work of these three groups of investigators showed that the mRNA contains an open reading frame of 1,392 nucleotides, of which 96 nucleotides encode the 32-amino acid signal peptide, and 1,296 nucleotides the 432-amino acid form of circulating plasma AT. Subsequent analysis showed that the transcrip- tional start site lies 70 nucleotides upstream from the initiator m e t h i ~ n i n e . ~ ~ , ~ ~ At the 3’ end of the gene, the termination codon and the polyA tail are separated by 87 nucleotides containing a typical polyadenylation signal 54 nucleotides downstream from the termination ~ o d o n . ~ ~ - ~ ~

The human gene consists of seven exons and six intervening sequences distributed over approximately 19 kb of the long arm of chromosome 1 (Fig 1).37,48,49 The margins of each of the seven exons comprising the human AT gene are shown in Table 1. In this report, we have chosen to designate exons 3a and 3b as exons 3 and 4, respectively. We believe that numbering the exons 1 to 7 more accurately reflects the structure of this gene than does the nomenclature of Bock et al.49

Several restriction fragment length polymorphisms (RFLP) have been described within the human AT gene as follows: in the 5’ untranslated region; in exon 2, the signal peptide; in exon 4, codon 305; in exon 4, codon 395; in




INHERITED ANTITHROMBIN DEFICIENCY 2161

H I k b

V n n i i f i r 1 1

+I : I .I I : I+ exon 1 2 3 4 5 6 7

W (38) (4) (5) 0

e- l frameshift mutation P single base substiion resulting in a stop codon

portion of the gene deleted

Fig 1. Schematic diagram indicating the site of reported AT gene mutations associated with type 1 AT deficiency. The numbers in parentheses refer to the exon numbers used by Bock et aLQ

intervening sequence 5, position 160; and in exon 6, codon 359.22,373-52 Recently, we identified two previously unre- ported repeat sequences in intervening sequence 5 of the gene. These repeat sequences are likely to be similar to the previously described variable number tandem repeat (VNTR) sequences that have been discovered in the human g e n ~ m e . ~ ~ ” ~ Using the polymerase chain reaction (PCR) method, we hope to detect various length polymorphisms related to these VNTRs to perform linkage analyses in the various kindreds that have been reported to have identical mutations. Such data will likely provide important information about whether the so-called hot spots noted in the human AT gene are real or related to a founder effect.56

The 5’ upstream region of the human AT gene is relatively unexplored. To date, only two studies have addressed the question of genetic control of AT gene expression. The upstream region of the AT gene lacks a discernible TATA However, it has been reported to contain an enhancer region, identified on the basis of homology to the Jk-Ck enhancer of the Ig K chain gene.47 These studies indicate that this putative enhancer region is sensitive to liver-specific cellular elements in regulating AT gene expre~s ion .~~ This putative enhancer region lies between nucleotides -173 and -129 relative to the start codon. The second reported study examined the binding of nuclear proteins to upstream DNA sequences.57 Such investigations provided evidence that an as-yet-unidentified

Table 1. Margins of the Seven Exons Comprising the Human AT Gene

No. of Amino Exon 5’-Margin 3‘-Margin Acid Residues

1

2 codon -18 3 (3A)* codon 105 4 (38) codon 177 5 (4) codon 223 6 (5) codon 354 7 (6) codon 375

70 bp 5’ to the start codon

codon -19 14

codon 104 122 codon 176 72 codon 222 46 codon 353 131 codon 374 21 84 bp 3’ to the 58

stop codon ~ ~~

*The numbers in parentheses refer to the exon numbers used by Bock et al.49

protein binds to a region between nucleotides -89 and -68.57 If this region of the AT gene were indeed critical to the control of human AT gene expression, then its deletion from reporter constructs should decrease or abolish pro- moter activity. However, such experiments have yet to be reported.

A number of independent studies have shown that the human AT gene is localized to the long arm of chromosome 1.58-61 Linkage analysis had previously shown that the human AT gene was linked to the red blood cell D u Q gene.58 More recent studies have clearly established the localization of the human AT gene to chromosome 1. This was accomplished both by observing AT deficiency in individuals in a kindred with a finite chromosomal deletion in the regions lq22 to lq25, and by precise mapping of partial AT cDNA clones to the region lq23 to 1q25.58-61

MECHANISM OF ACTION OF ANTITHROMBIN

AT has been shown to be the primary inhibitor of the various activated serine protease clotting factors, including thrombin, IXa, Xa, XIa, XIIa, kallekrein, plasmin, urokinase, and t ryp~in .1 ,2 ,~~-~~ It is likely that the most important of these physiologically is the inhibition of thrombin, which is relatively slow under physiologic conditions, but greatly accelerated in the presence of h e ~ a r i n . ~ ~ - ~ ~ As indicated above, protease inactivation by AT involves

the formation of a 1:l molar complex between the active site serine of the protease and the reactive center of AT. The reactive center Arg393-Ser394 of AT is situated toward the carboxy-terminal domain of the molecule.10.30,76.77 When thrombin interacts with AT, the two molecules form a covalent, stable, stoichiometric complex that is rapidly removed from the c i r c ~ l a t i o n . ~ ~ , ~ ~ The formation of this stable covalent complex initially involves the cleavage of the Arg393-Ser394 peptide bond by the serine active site of thrombin and then the association between the serine at the active site of thrombin and the arginine (Pl) of the cleaved AT by the formation of a covalent ester linkage.30~78.80,81 The margins of the thrombin-interactive domain of AT have yet to be defined precisely. However, some naturally occurring AT mutants that show impaired inhibitory activity have mutations adjacent to the reactive center. These natural mutations suggest that the margins of the reactive center of the human AT molecule spans, at least, from residues 382

The inhibition of thrombin, as well as other serine proteases of the coagulation pathway, by AT is relatively slow in the absence of heparin but can be enhanced more than a thousandfold in its p r e ~ e n c e . ~ ~ , ~ ~ This enhancement is mediated by the binding of heparin to AT, resulting in a conformational change in the inhibitor, thought to promote attack by the protease.83 Thus, heparin binding to AT has been shown to activate Lys 236 such that it enhances its ability to undergo chemical modification. Heparin binding has also been shown to alter the overall fluorescence spectrum of AT, suggesting a conformational change as the result of such i n t e r a ~ t i o n . ~ ~

The heparin-binding domain of AT appears to consist of two regions, encompassing amino acids 41 to 49 and 107 to

to 407.19,49,82





156, respe~tively. '~.~ Both regions consist of clusters of basic amino acids localized toward the amino-terminus of the molecule. Two lines of evidence indicate that the region encompassing amino acid residues 41 to 49 is involved in heparin binding. Biochemical experiments have shown that the chemical modification of Trp49 results in impaired heparin binding to AT.85 This modification also abolishes the heparin-catalyzed inhibition of thrombin by modified AT.85 The second line of evidence implicating the 41-49 region involves experiments of nature. The study of affected individuals has shown that mutations at Pro41 and Arg47 interfere directly or indirectly with heparin binding to AT.1s,86-97 The second heparin-binding region has been proposed to consist of the positively charged amino acid residues Lys 107, Lys 114, Lys 125, Arg129, Arg 132, and Lys 133 at the surface of the AT m o l e ~ u l e . ~ ~ ~ ~ ~ Evidence in support of this hypothesis was provided when the chemical modification of the Lys and Arg residues encompassing amino acids 107 to 156 resulted in an AT moiety with impaired heparin cofactor a ~ t i v i t y . ~ ~ , ~ ~ Additional evidence that this region might be important for heparin binding consists of data that indicate that heparin binds to a peptide comprising amino acid residues 114 to 156.98 Moreover, polyclonal antibodies with specificity toward the synthetic peptide comprising residues 124 to 145 block the binding of heparin to AT.99 Another piece of evidence implicating this region, as involved in heparin binding, is the recent report of a naturally occurring mutation located within this second heparin-binding region, at Arg 129. In AT-Geneva, there is an Argl29Gln substitution.lOOJO1

Interestingly, the analysis of a three-dimensional structure model of AT (based on the crystal structure of a-1 antitrypsin) puts the two heparin-binding regions adjacent to each 0 t h e r . 2 ~ J ~ ~ Thus, these two putative heparin- binding regions are adjacent to each other when the molecule assumes its three-dimensional format. Moreover, together these two regions provide a clearly defined positively charged domain suitable for heparin binding to AT. However, the relative contribution of each of these two heparin-binding regions to the presumed heparin-binding domain of the intact molecule has not been established. In this context, it has been shown recently that the disulphide bond between Cys8 and Cys128 (located within the second heparin-binding domain) is required for the integrity of heparin binding to intact AT.'" It has been proposed that the integrity of this disulphide linkage is required for Arg47 to be in a position to facilitate favorable cooperativity for heparin binding with Arg129.1°1 Thus, it is likely that the two heparin-binding regions 41-49 and 107-156 together cooperatively constitute one heparin-binding domain when plasma AT assumes its tertiary structure.

CLASSIFICATION OF ANTITHROMBIN DEFICIENCY

In attempting a classification of AT deficiency, a number of difficulties arise. Some of these are listed in Table 2. First is the absence of a decision by an appropriate international committee concerning the name of the thrombin inhibitor under discussion. Various opinions range from antithrombin, antithrombin 111, heparin cofactor I, serpin I, etc. As

Table 2. Problems With the Classification of AT Deficiency

1. The name of the progressive thrombin inhibitor (AT 111 v AT). 2. Multiple classification schemes. 3. Nomenclature to describe specific defects(top0nyms v mo-

4. Homozygosity v heterozygosity. 5. Repeat reporting of the same molecular defect. 6. Numbering of the exons of the human AT gene.

lecular defect).

indicated earlier, we would recommend the term antithrombin.

A second difficulty occurs because different classification schemes have been proposed to describe the various types of AT deficiency state. Some investigators prefer to classify AT deficiency into only two types,21,22 whereas others have classified AT deficiency into up to five different types.2O In this review, we propose a classification based on the known, or presumed, site of the molecular pathology of the AT gene and its resultant effect on the circulating AT molecule. In our proposed classification, AT deficiency is divided into four types, based on the site of the mutation (see Table 3).

Another problem with the classification of AT deficiency is the nomenclature used for the past 2 decades to describe the various types of AT deficiency. In general, the various investigators have used the name of the city where the propositus resided (ie, a toponym). The use of toponyms may have been necessary before the capability of elucidat- ing the specific molecular pathology existed. Presently, it is relatively easy to characterize a specific mutation. There- fore, the use of toponyms should be discouraged. We would suggest that all newly characterized mutations be designated by the specific mutation. For example, AT-Toyama, with a substitution of the Arg47 residue by Cys, should be referred to as AT-Arg47Cy~.'~ Thus, the repetitive reporting of the same molecular defect might be discouraged. In this regard, the AT-Toyama defect, first described in 1984, has been reported nine different times from different geographic locations. Thus, the Arg47Cys mutation has been reported as AT-Toyama, -Tours, -Alger, -Amiens, -Barcelona-2, -Paris-1, -Paris-2, -Padua-2, and -Kumamo- to.18,s6-92 While it is of considerable interest that the same mutation occurs in different parts of the world, suggesting a mutational hot it is possible that many of these kindreds, particularly the European ones, derive from the same founder.

Another problem with present classifications is that there is no indication of the zygosity of the mutation in a given kindred. For example, the clinical or phenotypic expression of a particular defect is, in part, dependent on the zygosity; ie, whether the individual is homozygous or heterozygous for the disorder. While most individuals with AT deficiency

Table 3. Proposed Classification of Inherited AT Deficiency

Type 1 : AT gene product absent from the plasma. Type 2: AT gene defect affecting the thrombin-binding domain of the

Type 3: AT gene defect affecting the heparin-binding domain of the

Type 4: None of the above (miscellaneous group).

molecule.

molecule.





have been reported to have a heterozygous defect, there have been reports of homozygous individuals and even some with doubly heterozygous defects.ts~s7J04~to5 Reports of AT deficiency should have the zygosity clearly indicated as this has relevance to the clinical management (see below).

INHERITED ANTITHROMBIN DEFICIENCY

The prevalence of AT deficiency in individuals with symptomatic thrombotic disease is approximately 5%.'06 In the normal population, AT deficiency has been estimated to affect from 1 to 2,000 to 1 to 5,000 individual^.'^-'^ However, it has been reported recently that the incidence of AT deficiency may be higher than previously estimated.'" The prevalence of AT deficiency in normal individuals has been estimated, in a Scottish study, to be 0.4% or 1 in 25O.lo7 Sixteen of over 4,000 blood donors, ages 18 to 65 (2,611 male and 1,578 female), had persistent AT deficiency.Io7 One had type 1 deficiency, 2 had type 2 deficiency, and 13 had a type 3 deficiency (as defined in Table 3). These findings suggest a prevalence of 1 in 4,200 for type 1 AT deficiency; a 1 in 2,100 prevalence for a type 2 deficiency; and a prevalence of 1 in 350 for type 3 deficiency. Further studies are required to define more precisely the prevalence of AT deficiency, which may vary from one population to another.

Type 1 AT deficiency is characterized by a decrease of both antigenic and functional levels to approximately 50% of those observed in normal individuals. For the purposes of this review, we define type 1 AT deficiency as being a quantitative defect only. We specify that no gene product of the affected AT allele should be detectable in the plasma of an affected type 1 individual. Table 4 lists some of the possible causes of a type 1 AT deficiency. Table 5 summarizes the elucidated mutations that have been associated with type 1 AT deficiency. There have been two reports where a complete AT gene deletion was associated with type 1 AT deficiency.60J08 In one of these kindreds, the AT deficiency was associated with a visible deletion of a portion of the long arm of chromosome 1.60 In our laboratory, we have recently characterized a kindred with a partial, but extensive, gene deletion of one AT allele.84 All genetic information upstream of a point between exons 2 and 3 is deleted. We have recently established the exact location of the breakpoint to be 480 nucleotides upstream to the 5' boundary of exon 3.1a9 There have been reports of 11 frameshift mutations associated with type 1 AT deficiency, resulting from either an insertion of one nucleotide or a deletion of one to four nucleo-

Type 1 ATdeficiency.

Table 4. Putative Causes for Inherited Type 1 AT Deficiency, Defined as a Quantitative Deficiency Without Evidence in the Plasma of a

Gene Product From the Affected Allele

1. A complete gene deletion. 2. A partial gene deletion. 3. A mutation causing a transcription defect. 4. A mutation causing a translation defect. 5. A mutation causing a protein secretion defect. 6. Combinations of the above.

t i d e ~ . ~ l ~ - ' l ~ In addition, there have been several reports of kindreds with a type 1 deficiency resulting from a single C to T substitution in the first base of codon 129, mutating an Arg (CGA) codon to a stop (TGA) ~ o d o n . " ~ , ~ ~ ~ The details of these mutations are summarized in Fig 1.

The molecular pathology of most kindreds with type 1 AT deficiency has not yet been elucidated. Of 16 kindreds examined by Bock and Pro~hownik,"~ only one was reported to be completely lacking in one AT allele. All others had RFLP patterns indistinguishable from normal individuals. Such analyses suggests that the molecular defects in most kindreds with AT deficiency are caused by point mutations at key positions within the AT gene. The potential causes of such mutations are listed in Table 4. The advent of direct sequencing of PCR-amplified portions of genomic DNA from affected individuals should make it possible to characterize various other mutations leading to type 1 AT deficiency.

Type 2 AT deficiency is characterized by a mutation within the AT gene resulting in a circulating protein with impaired serine protease inhibitory activity. All type 2 mutations characterized to date have been single nucleotide substitutions altering codons between amino acid residues 382 and 407, the region adjacent to the AT reactive center Arg393-Ser394. The reported type 2 AT variants are summarized in Fig 2. While there have been reports of at least 24 kindreds from diverse geographic locations having type 2 AT deficiency, there have been only 14 specific mutations identified.49is2,90~"8-'35 Patients with type 2 AT deficiency have normal plasma AT antigen 1e~els. l~ However, they have impaired serine protease inhibitory activity both in the presence and absence of heparin. The first reported elucidation of a type 2 deficiency was that of A T - D e n ~ e r . ~ ~ ~ . ' ~ ~ This mutation was shown to be a Ser394Leu substitution, which rendered the AT molecule incapable of complexing with its cognate protease, t h r ~ m b i n . ~ ~ ~ J ~ ~

The region amino-terminal to the reactive center has been characterized by a number of mutations. The first of these reported was AT-Hamilton, consisting of an Ala382Thr substitution.82 Unlike AT-Denver, the AT- Hamilton molecule is capable of being a substrate for thrombin and factor Xa, but is incapable of inhibiting either protease.28 Two molecular defects at position 384 have been described; Ala 384 Pro and Ala 384 Ser.90J20-123 Both of these mutant ATs behave similarly to AT-Hamilton in that they can be substrates for their cognate proteases, but do not form inhibitory complexes with them.z2J22J36

Recently, we described an interesting P2 mutation. A Gly392Asp substitution was found to render the mutant protein, termed AT-Stockholm, devoid of thrombin- inhibiting activity. Evidence provided by this mutation suggests that the P2 residue may importantly influence the reactive center of AT.124

Three AT mutants with substitutions at Arg393 have been described. These include Arg393His, Arg393Cys, and A r g 3 9 3 P r 0 . ' ~ ~ - ~ ~ ~ These mutant proteins are of considerable interest because they are associated with increased heparin affinity.'" The mechanism whereby these substitutions

Type 2ATdeficiency.





t

Table 5. Mutations Associated With Type 1 AT Deficiency

t l t l t t t

Location Mutation Specific Pathology Reference

G;g;!2k2LM Pro Ser

ND ND IVS-2 Codon 48 Codon 81 Codon 119 Codon 129 Codon 208 Codon 228 Codon 245 Codon 245 Codons 290,291 Codons 308,309 Codon 370 Codon 408

Thr Met Leu MI LA NO-2 D:;:A OSLO KYOTO UTAH

Cys Ser

Complete gene deletion Complete gene deletion Partial gene deletion 1-bp insertion (+T) 1-bp deletion (-T) 1-bp deletion (-T) CGA to TGA 1-bp insertion (+A) 1-bp insertion (+A) 1-bp deletion (-A) 2-bp deletion (FAG) 2-bp deletion (-AG) 4-bp deletion (-TGGA) 1-bp deletion (-A) 1-bp insertion (+A)

ND ND 480 bp 5' to exon 3 Frameshift Frameshift Frameshift Mutation to stop codon Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift

60 108 109 110 111 112

113,116 110 114 115 115 114 114 110 113

~~ ~

Abbreviations: ND, not determined (the margins of this deletion have not yet been determined precisely); IVS-2, intervening sequence 2 (intron 2).

enhance heparin affinity is unclear. It may be relevant that the heparin affinity of the mutation Arg393His can be returned to normal by decreasing the pH, suggesting that normally the AT molecule may be held in a constrained conformation because of ionic interactions involving Arg393.137 The Arg393Cys mutation also presents an interesting phenomenon. This mutation is associated, on cross- immunoelectrophoresis, with an additional anodal peak.

THROMBIN BINDING REGION

] COOH NH2 I / \ 432

/ \ / \

/ \ /- \

/ THROMBIN \ \ \ \ * \

/ /

/ /

'302 304 392 393 394 402 404 406 401'

Ala Ala Gly Arg Ser Phe Ala Arg Pro

CAMERIDGE-1 SUDBURY VUJCENZA

His CYS Pro GLASGOW-1 NORTHWICK PARK PESCARA SHE F F I E L D

NRANCHES

MILAN04 CHICAGO FRANKFURT-1

Fig 2. Schematic diagram summarizing, at the amino acid level, the reported AT gene mutations associated with type 2 AT deficiency.

The additional 120-Kd band can be seen on nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and on Western blot analysis has been shown to be a high molecular weight complex between AT and albumin. Thus, the Arg393Cys mutation circulates as a disulphide-linked heterodimer between albumin and

Mutations have also been reported between residues 402-407.49J17J34J35J39 The prototype of this mutation is AT-Utah (Pr0407Leu).~~>l~~ Pro407, like Ala382, the position of the AT-Hamilton mutation, is highly conserved amongst the inhibitor serpins. This AT deficiency is characterized by both a decreased level of AT antigen activity and the presence of a mutant protein with impaired serine protease reactivity. It has been hypothesized that this mutation is associated with an alteration in the polypeptide chain resulting both in an impairment of the function and the stability of the m0lecule.4~ In addition, it is possible that this mutation may result in a relative inability of hepato- cytes to secrete this mutant protein. It is interesting that AT-Utah was classified initially as a type 1 variant.139 Subsequent to the initial report, however, it was realized that small amounts of abnormal molecules were present in the plasma of affected individual^.^^ This is similar to AT-Oslo, the very first described kindred with AT deficiency, in also being classified as a classical, or type 1, deficiency.l3 Subsequent analysis detected trace amounts of altered protein in the plasma and the deficiency was elucidated subsequently to be caused by an Ala404Thr substitution.118

In addition to the characterized type 2 AT variants, there appear to be several AT-deficient kindreds with variants that behave like type 2 variants, but that have not yet been characterized. These include AT-Alberg, AT-Hvidore, and AT-Trent~n ." '@~~~

Type 3 AT deficiency is characterized by the presence of normal plasma progressive AT activity but impaired heparin cofactor a~tivity. '~. '~ The mutations in the type 3 variants have been shown to lie within the first putative heparin-binding region of AT

AT.128,129,138

Type 3 ATdeficiency.





(residues 41 and 47).18,86392-97,101 As indicated earlier, a very interesting type 3 mutation has been reported at position 129, in the second heparin-binding region of AT. Thus, AT-Geneva has a Glnl29Arg substitution.lo3 A second variant in this region has recently been described with an Leu99Phe substitution.143 The large variety of reported type 3 AT mutants is summarized in Fig 3. Heterozygous individuals with type 3 AT deficiency do not appear to be at increased risk for thromboembolic events.lM,14 In contrast, homozygously affected type 3 individuals have been reported with clear-cut histories of thromboembolic disease.145-147 Such homozygous individuals appear to be at increased risk for thromboembolic events.lo6

In addition to the above characterized type 3 AT variants, there appear to be several type 3 variants that are characterized by reduced serine protease inhibitory activity in the presence of heparin. They have, on crossed immunoelectrophoresis, a population of circulating AT molecules with reduced heparin affinity. However, the molecular pathology of these variants has not yet been elucidated and include AT-Barcelona-1, -Johannesburg, and -Roma.148-150

We propose that all kindreds with AT mutations that cannot be classified readily into one of the above three types be classified as having type 4 AT deficiency. For example, AT-Dublin-1 is a variant that was identified initially during studies of AT activity in children being administered asparaginase c h e m ~ t h e r a p y . ~ ~ , ~ ~ ~ This variant was shown recently to be an amino terminal variant

Type 4ATdeJiciency.

HEPARIN BINDING REGIONS I II

\ I \ \ I \ \ I \ \ I \ \ I \

\ \

\ f

\ \ f \ f

4 7 \ f 99 129 \ Pro Arg Leu Arg I ! -

I V V

FR ANCONVILLE CLICHW CLICHlc2 DUBLK?

Cys His Ser TOYAMA ROUEN-1 ROUEN-2 TOURS PADUA-1 ALGER BLIGNY AMIENS

BARCELONA-? PARIS-1 PARIS-2 PADUA-2

KUMAMOTO

Fig 3. Schematic summary, at the amino acid level, of reported AT gene mutations associated with type 3 AT deficiency.

with an aberrant signal peptidase cleavage site, but with normal function.52 An unnamed type 4 variant has also been characterized by overglycosylation, resulting in decreased heparin affinity.152 In this variant, there is an Ile7Asn substitution resulting in an additional glycosylation site. Thus, this additional glycosidic side chain appears to cause the reduced heparin affinity seen with this molecule. AT-Budapest, the first described AT mutant, appears to have impairment of both heparin binding and serine protease inhibitor activity.17 AT-Budapest is characterized by normal AT antigenic levels. Recently, the specific mutation of AT-Budapest has been characterized as a Pro429Leu substitution, causing the apparent increase in molecular mass, as determined by SDS-PAGE electrophoresis, under nonreducing ~0nd i t ions . l~~ Under reducing conditions, the apparent increased molecular mass is no longer evident. The increase in molecular mass can be explained by the failure of AT-Budapest to form a normal disulphide bond between Cys247 and Cys430. Thus, this mutation results in the formation of partially unfolded molecule, phenotypi- cally giving the appearance of a molecule with increased molecular mass under nonreducing conditions.153 Other AT mutations that we would classify as type 4 include AT- Rouen-4, an Arg24Cys substitution; AT-Truro, a Glu237Lys m u t a t i ~ n ~ ~ , ~ ~ ~ ; and a novel missence Ser349Pro mutation, associated with familial recurrent venous thrombosis.lS5

LABORATORY DIAGNOSIS OF ANTITHROMBIN DEFICIENCY

The diagnosis of AT deficiency is based on the quantitative and qualitative determination of AT antigenic and functional activity levels. The latter is based primarily on the ability of an individual's plasma to inactivate thrombin in the presence (or absence) of heparin. For initial screen- ing purposes, most laboratories use a synthetic chromogenic substrate to measure residual thrombin activity, following thrombin inactivation by AT.lS6 This result is a measure of the functional AT activity that is always reduced in patients with AT deficiency. In general, an activity level lower than 70% of that of normal pooled plasma represents an individual with AT deficiency. In most cases of hereditary AT deficiency, the AT levels are reduced to approximately 50% of normal.

We have found recently that the use of factor Xa as the substrate for AT is preferable to the use of thrombin in such assays.lS7 The use of factor Xa provides for the more reliable detection of an AT deficiency because the use of thrombin simultaneously measures both AT and heparin cofactor I1 activities.

To determine whether there is a quantitative AT deficiency, the AT antigen concentration is obtained, usually using an immunochemical method. The radial immunodiffusion method of Mancini et alls8 and the electroimmuno- diffusion method of L a ~ r e l l ' ~ ~ are both in general use to measure AT antigenic activity. Thus, if AT antigenic and functional activities are both decreased, a quantitative (type 1) AT deficiency can be assumed. Such an observation indicates that one of the AT alleles is not functional, as outlined in Table 4. A qualitative AT deficiency is implied if reduced heparin cofactor activity is accompanied by the





presence of normal antigenic activity. Determination of progressive thrombin and/or factor Xa inactivation, as well as the performance of crossed immunoelectrophoresis with or without heparin, are required to identify the functional domain of the impaired AT molecule.160 For example, a sample with decreased progressive thrombin and/or factor Xa inactivation together with a normal crossed immunoelectrophoresis pattern, both in the presence or absence of heparin, suggests an abnormality at or near the reactive center of the molecule. However, a sample with normal progressive inactivation of thrombin and/or factor Xa but with an abnormal crossed immunoelectrophoresis pattern, in the presence of heparin, is presumed to have an abnormal heparin-binding region.

To further characterize AT variants at the molecular level, the determination of the amino acid sequence and/or the DNA sequence is desirable. Development of molecular techniques such as PCR have enabled the rapid elucidation of the molecular defects involved in patients with AT deficiency.l6l

reported in such studies, is overestimated. This is because the clinical diagnosis of venous thromboembolism is usually not confirmed with objective testing. Thus, many subjects with clinically suspected deep venous thrombosis do not actually have deep venous thrombosis.162 Furthermore, in most of the reports of thromboembolism, in AT-deficient individuals, it is not clear whether the diagnosis of venous thrombosis was made without knowledge of the AT status of the study subjects. Such knowledge has the potential to cause diagnostic suspicion bias, which could result in the overestimation of the prevalence of venous thrombosis in such a population.

We have recently performed a cross-sectional study to estimate the prevalence of objectively proven thrombotic events in patients with AT deficiency.163 The large kindred chosen had a type 2 AT deficiency (AT-Hamilton).8Z Sixty-seven subjects, constituting the majority (83%) of the available family members, were evaluated. Six of 31 (19.4%) AT-deficient subjects had evidence of one or more thrombotic events. None of the 36 nondeficient subjects had evidence of a thrombotic event. The initial thrombotic episode, in five of the six subjects who had such an episode, had occurred in association with predisposing f a ~ t 0 r s . l ~ ~ On

TREATMENT OF PATIENTS WITH ANTITHROMBIN DEFICIENCY

Hereditary AT deficiency is a well-recognized cause of thrombophilia,16J06 and individuals with this disorder are at increased risk to develop venous thrombosis and are often treated with anticoagulants indefinitely. Therefore, the management of asymptomatic AT-deficient individuals is problematic, because data concerning the relative risks and benefits of lifelong anticoagulant, or replacement therapy, are lacking. This is largely because reliable estimates of the prevalence of venous thrombosis in AT-deficient individuals are not yet available. Based on the available literature, the reported prevalence of venous thrombosis in AT deficiency subjects is approximately 50%.'06 In our opinion, it is likely that the true prevalence of venous thrombosis,

the basis of this study, we have postulated that the incidence of thrombotic events in people with AT deficiency may be significantly lower than heretofore estimated and that they occur predominantly in association with predisposing factors. Thus, we believe that lifelong prophylaxis with anticoagulants, or replacement, is probably not warranted in asymptomatic AT-deficient individuals, and that use of prophylaxis in such individuals be limited to high-risk p e r i o d ~ . ~ ~ ~ J ~ In contrast, patients with established deep venous thrombosis should be treated with heparin and oral anticoagulants, with the length of treatment dependent on the clinical setting. In some situations, AT replacement is probably indicated; however, at this time there are insuffi-

HEPARIN BINDING REGIONS I II

THROMBIN BINDING REGION

e I COOH NH2 I I \ I \ / 432

I \ I \ I \ I \ / \

/ / \

I \ / \ / THROMBIN \

/ \

/ \

I \ \ \

I \ I

\ I \ I

\ / I \ I

I \ I \

\ /

4I\ I 99 129 237 349 '382 301 392 393 394 402 404 400 407' 429

Atg 1;

A I 9 AI S; P[ i 9 ; ; 9 7 7 9 A m Cys Phr Gln Lyr Pro Thr Asp Leu Thr Met Leu Leu

-- - I 24 1 4 t --

Fig 4. A schematic diagram showing the two putative heparin-binding regions and the thrombin-binding region of AT. The characterized type 2,3, and 4 AT mutants are shown, indicating the amino acid substitution in each case.





cient data available to evaluate the value of AT replacement. Recommendations for the treatment of AT deficiency have recently been published and should be consulted for further details.164

Patients with type 3 AT deficiency are considered not to be at risk for thromboembolic events, except if they are homozygous or double heterozygotes.lwJo6J4 Recently Vidaud et allw reported a kindred with double heterozygous AT deficiency in two brothers. In one allele the mutation appeared to be a type 3 deficiency, while in the other there was a type 1 deficiency. Both brothers had evidence of recurrent thromboembolism. The mother of the two affected brothers had a typical type 3 AT deficiency phenotype and was asymptomatic; the father, who manifested several thromboembolic episodes, appeared to have a type 1 AT deficiency.lo4

There has been only a single report of a kindred with homozygous type 1 AT deficiency.Io5 In this kindred, two homozygously affected offspring died within the first 3

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weeks of life. The data from this consanguineous family indicate that homozygous type 1 and probably type 2 AT deficiency is incompatible with long life, except if lifelong replacement therapy were to be administered, as has been reported with homozygous protein C deficiency.165

SUMMARY

Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.

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1992 80: 2159-2171

MA Blajchman, RC Austin, F Fernandez-Rachubinski and WP Sheffield Molecular basis of inherited human antithrombin deficiency

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Molecular basis of inherited human antithrombin deficiency