View source for Antithrombin

{{Infobox_gene}}

'''Antithrombin''' (AT) is a small protein molecule that inactivates several enzymes of the [[coagulation]] system. Antithrombin is a [[glycoprotein]] produced by the [[liver]] and consists of 432 amino acids. It contains three [[disulfide bond]]s and a total of four possible [[glycosylation]] sites. α-Antithrombin is the dominant form of antithrombin found in [[blood plasma]] and has an oligosaccharide occupying each of its four glycosylation sites. A single glycosylation site remains consistently un-occupied in the minor form of antithrombin, β-antithrombin.<ref name=bjork>{{cite book |last = Bjork | first = I |author2=Olson, JE | title = Antithrombin, A bloody important serpin (in Chemistry and Biology of Serpins) | publisher = Plenum Press | year = 1997 | pages=17–33 | isbn=0-306-45698-2}}</ref> Its activity is increased manyfold by the [[anticoagulant]] drug [[heparin]], which enhances the binding of antithrombin to [[thrombin|factor IIa]] (Thrombin) and [[factor X]]a.<ref>{{Cite journal|last=Finley|first=Alan|last2=Greenberg|first2=Charles|date=2013-06-01|title=Review article: heparin sensitivity and resistance: management during cardiopulmonary bypass|journal=Anesthesia and Analgesia|volume=116|issue=6|pages=1210–1222|doi=10.1213/ANE.0b013e31827e4e62|issn=1526-7598|pmid=23408671}}</ref>

== Nomenclature ==
Antithrombin is also termed '''Antithrombin III''' (AT III). The designations Antithrombin I through to Antithrombin IV originate in early studies carried out in the 1950s by Seegers, Johnson and Fell.<ref>{{cite journal |vauthors=Seegers WH, Johnson JF, Fell C | title = An antithrombin reaction to prothrombin activation | journal = Am. J. Physiol. | volume = 176 | issue = 1 | pages = 97–103 | year = 1954 | pmid = 13124503 }}</ref>

Antithrombin I (AT I) refers to the absorption of [[thrombin]] onto [[fibrin]] after thrombin has activated [[fibrinogen]]. [[Heparin cofactor II|Antithrombin II]] (AT II) refers to a cofactor in plasma, which together with [[heparin]] interferes with the interaction of [[thrombin]] and [[fibrinogen]]. Antithrombin III (AT III) refers to a substance in [[blood plasma|plasma]] that inactivates thrombin. Antithrombin IV (AT IV) refers to an antithrombin that becomes activated during and shortly after [[blood coagulation]].<ref>{{cite journal |vauthors=Yin ET, Wessler S, Stoll PJ | title = Identity of plasma-activated factor X inhibitor with antithrombin 3 and heparin cofactor | journal = J. Biol. Chem. | volume = 246 | issue = 11 | pages = 3712–3719 | year = 1971 | pmid = 4102937 }}</ref> Only AT III and possibly AT I are medically significant. AT III is generally referred to solely as "Antithrombin" and it is Antithrombin III that is discussed in this article.

==Structure==
[[File:antithrombin 1.jpeg|thumb|<span style="font-size:100%;">'''Figure 1.'''</span> The location of the four potential glycosylation sites within the [[tertiary structure]] of an antithrombin [[monomer]] are shown, as taken from the [[protein data bank]] file [http://www.pdb.org/pdb/explore/explore.do?structureId=2ANT 2ANT]. In this structure only Asn 155 is glycosylated by the addition of a single [[N-Acetylglucosamine|''N''-acetylglucosamine]] residue.|left|500px ]]
Antithrombin has a [[half-life]] in [[blood plasma]] of around 3 days.<ref>{{cite journal |vauthors=Collen D, Schetz J, de Cock F, Holmer E, Verstraete M | title = Metabolism of antithrombin III (heparin cofactor) in man: Effects of venous thrombosis of heparin administration | journal = Eur. J. Clin. Invest. | volume = 7 | issue = 1 | pages = 27–35 | year = 1977 | pmid = 65284 | doi = 10.1111/j.1365-2362.1977.tb01566.x }}</ref> 
The normal antithrombin concentration in human [[blood plasma]] is high at approximately 0.12&nbsp;mg/ml, which is equivalent to a [[Concentration#Molarity|molar]] concentration of 2.3 μM.<ref>{{cite journal |vauthors=Conard J, Brosstad F, Lie Larsen M, Samama M, Abildgaard U | title = Molar antithrombin concentration in normal human plasma | journal = Haemostasis | volume = 13 | issue = 6 | pages = 363–368 | year = 1983 | pmid = 6667903 | doi = 10.1159/000214823 }}</ref>
Antithrombin has been isolated from the plasma of a large number of species additional to humans.<ref>{{cite journal | author = Jordan RE | title = Antithrombin in vertebrate species: Conservation of the heparin-dependent anticoagulant mechanism | journal = Arch. Biochem. Biophys. | volume = 227 | issue = 2 | pages = 587–595 | year = 1983 | pmid = 6607710 | doi = 10.1016/0003-9861(83)90488-5 }}</ref> As deduced from protein and [[cDNA]] sequencing, cow, sheep, rabbit and mouse antithrombins are all 433 amino acids in length, which is one amino acid longer than human antithrombin. The extra amino acid is thought to occur at amino acid position 6. Cow, sheep, rabbit, mouse, and human antithrombins share between 84 and 89% amino acid sequence identity.<ref name=olson>{{cite journal |vauthors=Olson ST, Björk I | title = Regulation of thrombin activity by antithrombin and heparin | journal = Sem. Thromb. Hemost. | volume = 20 | issue = 4 | pages = 373–409 | year = 1994 | pmid = 7899869 | doi = 10.1055/s-2007-1001928 }}</ref> Six of the amino acids form three intramolecular [[disulfide bond]]s, [[cysteine|Cys]]8-Cys128, Cys21-Cys95, and Cys248-Cys430.
They all have four potential [[N-glycosylation]] sites. These occur at [[asparagine]] (Asn) amino acid numbers 96, 135, 155, and 192 in humans and at similar amino acid numbers in other species. All these sites are occupied by covalently attached oligosaccharide side-chains in the predominant form of human antithrombin, α-antithrombin, resulting in a molecular weight for this form of antithrombin of 58,200.<ref name=bjork/> The potential glycosylation site at asparagine 135 is not occupied in a minor form (around 10%) of antithrombin, β-antithrombin (see '''Figure 1''').<ref>{{cite journal |vauthors=Brennan SO, George PM, Jordan RE | title = Physiological variant of antithrombin-III lacks carbohydrate side-chain at Asn 135 | journal = FEBS Lett | volume = 219 | issue = 2 | pages = 431–436 | year = 1987 | pmid = 3609301 | doi = 10.1016/0014-5793(87)80266-1 }}</ref>

[[Recombinant DNA|Recombinant]] antithrombins with properties similar to those of normal human antithrombin have been produced using [[baculovirus]]-infected insect cells and mammalian cell lines grown in [[cell culture]].<ref>{{cite journal |vauthors=Stephens AW, Siddiqui A, Hirs CH | title = Expression of functionally active human antithrombin III | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 84 | issue = 11 | pages = 3886–3890 | year = 1987 | pmid = 3473488 | pmc = 304981 | doi = 10.1073/pnas.84.11.3886 }}</ref><ref>{{cite journal |vauthors=Zettlmeissl G, Conradt HS, Nimtz M, Karges HE | title = Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells | journal = J. Biol. Chem. | volume = 264 | issue = 35 | pages = 21153–21159 | year = 1989 | pmid = 2592368 }}</ref><ref>{{cite journal |vauthors=Gillespie LS, Hillesland KK, Knauer DJ | title = Expression of biologically active human antithrombin III by recombinant baculovirus in Spodoptera frugiperda cells | journal = J. Biol. Chem. | volume = 266 | issue = 6 | pages = 3995–4001 | year = 1991 | pmid = 1995647 }}</ref><ref>{{cite journal |vauthors=Ersdal-Badju E, Lu A, Peng X, Picard V, Zendehrouh P, Turk B, Björk I, Olson ST, Bock SC | title = Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells | journal = Biochem. J. | volume = 310 | issue = Pt 1 | pages = 323–330 | year = 1995 | pmid = 7646463 | pmc = 1135891 }}</ref>  These recombinant antithrombins generally have different glycosylation patterns to normal antithrombin and are typically used in antithrombin structural studies. For this reason many of the antithrombin structures stored in the [[protein data bank]] and presented in this article show variable glycosylation patterns.

Antithrombin begins in its native state, which has a higher free energy compared to the latent state, which it decays to on average after 3 days. The latent state has the same form as the activated state - that is, when it is inhibiting thrombin. As such it is a classic example of the utility of kinetic vs thermodynamic control of protein folding.

== Function ==
[[File:antithrombin 2.jpeg|thumb|<span style="font-size:100%;">'''Figure 2.'''</span> The reactive arg 393 - ser 394 bond is located on an exposed loop at the surface of the molecule. This loop is termed the reactive site loop (RSL) or reactive centre loop (RCL). |left|460px]]
[[File:antithrombin reactive site loop sequence.jpeg|thumb|<span style="font-size:100%;">'''Figure 3.'''</span> The amino acid sequence of the reactive site loop of human antithrombin is shown.<ref name= Whisstock>{{cite journal |vauthors=Whisstock JC, Pike RN | title = Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparin | journal = J. Mol. Biol. | year=2000 | volume=301 | issue=5 | pages=1287–1305 | pmid = 10966821 | doi = 10.1006/jmbi.2000.3982|display-authors=etal}}</ref> The reactive site loop comprises amino acid sequence numbers 377 to 400 (numbers shown below the above sequence) or amino acids P1 to P17 and P1' to P7' using the Schechter and Berger convention (number shown above the above sequence).<ref name= Schechter>{{cite journal |vauthors=Schechter I, Berger A | title = On the size of the active site in proteases. I. Papain | journal = Biochem. Biophys. Res. Commun.| year=1967 | volume=27 | issue=2 | pages=157–162 | pmid = 6035483 | doi = 10.1016/S0006-291X(67)80055-X}}</ref> The reactive bond is indicated by an arrow.|left|460px]]
Antithrombin is a [[serine protease inhibitor|serpin]] (serine protease inhibitor) and is thus similar in structure to most other [[blood plasma|plasma]] [[protease]] [[enzyme inhibitor|inhibitor]]s, such as [[alpha 1-antichymotrypsin]], [[alpha 2-antiplasmin]] and [[Heparin cofactor II]].

The physiological target [[protease]]s of antithrombin are those of the [[Coagulation#Contact activation pathway|''contact activation pathway'']] (formerly known as the intrinsic pathway), namely the activated forms of [[Factor X]] (Xa), [[Factor IX]] (IXa), [[Factor XI]] (XIa), [[Factor XII]] (XIIa) and, to a greater extent, [[Factor II]] (thrombin) (IIa), and also the activated form of [[Factor VII]] (VIIa) from the [[Coagulation#Tissue factor pathway|''tissue factor pathway'']] (formerly known as the extrinsic pathway).<ref>{{cite journal |vauthors=Persson E, Bak H, Olsen OH | title = Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity | journal = J. Biol. Chem. | volume = 276 | issue = 31 | pages = 29195–29199 | year = 2001 | pmid = 11389142 | doi = 10.1074/jbc.M102187200 }}</ref> The inhibitor also inactivates [[kallikrein]] and [[plasmin]] {{Citation needed|date=November 2017}}, also involved in blood coagulation. However it inactivates certain other serine proteases that are not involved in coagulation such as [[trypsin]] and the C1s subunit of the enzyme C1 involved in the [[classical complement pathway]].<ref name= olson/><ref>{{cite journal |vauthors=Ogston D, Murray J, Crawford GP | title = Inhibition of the activated Cls subunit of the first component of complement by antithrombin III in the presence of heparin | journal = Thromb. Res. | volume = 9 | issue = 3 | pages = 217–222 | year = 1976 | pmid = 982345 | doi = 10.1016/0049-3848(76)90210-3 }}</ref>

Protease inactivation results as a consequence of trapping the protease in an equimolar complex with antithrombin in which the active site of the protease enzyme is inaccessible to its usual [[Substrate (biochemistry)|substrate]].<ref name=olson/> The formation of an antithrombin-protease complex involves an interaction between the protease and a specific reactive [[peptide bond]] within antithrombin. In human antithrombin this bond is between [[arginine]] (arg) 393 and [[serine]] (ser) 394 (see '''Figure 2''' and '''Figure 3''').<ref name=olson/>
 
It is thought that protease enzymes become trapped in inactive antithrombin-protease complexes as a consequence of their attack on the reactive bond. Although attacking a similar bond within the normal protease substrate results in rapid [[proteolytic]] cleavage of the substrate, initiating an attack on the antithrombin reactive bond causes antithrombin to become activated and trap the enzyme at an intermediate stage of the proteolytic process. Given time, thrombin is able to cleave the reactive bond within antithrombin and an inactive antithrombin-thrombin complex will dissociate, however the time it takes for this to occur may be greater than 3 days.<ref>{{cite journal |vauthors=Danielsson A, Björk I | title = Slow, spontaneous dissociation of the antithrombin-thrombin complex produces a proteolytically modified form of the inhibitor | journal = FEBS Lett | volume = 119 | issue = 2 | pages = 241–244 | year = 1980 | pmid = 7428936 | doi = 10.1016/0014-5793(80)80262-6 }}</ref> However bonds P3-P4 and P1'-P2' can be rapidly cleaved by [[neutrophil elastase]] and the bacterial enzyme [[thermolysin]] respectively, resulting in inactive antithrombins no longer able to inhibit thrombin activity.<ref name =chang>{{cite journal |vauthors=Chang WS, Wardell MR, Lomas DA, Carrell RW | title = Probing serpin reactive-loop conformations by proteolytic cleavage | journal = Biochem. J. | volume = 314 | issue = 2 | pages = 647–653 | year = 1996 | pmid = 8670081 | pmc = 1217096 }}</ref>

The rate of antithrombin's inhibition of protease activity is greatly enhanced by its additional binding to [[heparin]] as is its inactivation by [[neutrophil elastase]].<ref name=chang/>

==Antithrombin and heparin==
Antithrombin inactivates its physiological target enzymes, Thrombin, Factor Xa and Factor IXa with [[rate constant]]s of 7–11 x 10<sup>3</sup>, 2.5 x 10<sup>3</sup> M<sup>−1</sup> s<sup>−1</sup> and 1 x 10 M<sup>−1</sup> s<sup>−1</sup> respectively.<ref name=bjork/><ref name = bedsted>{{cite journal |vauthors=Bedsted T, Swanson R, Chuang YJ, Bock PE, Björk I, Olson ST | title = Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites | journal = Biochemistry | volume = 42 | issue = 27 | pages = 8143–8152 | year = 2003 | pmid = 12846563 | doi = 10.1021/bi034363y }}</ref> The rate of antithrombin-thrombin inactivation increases to 1.5 - 4 x 10<sup>7</sup> M<sup>−1</sup> s<sup>−1</sup> in the presence of heparin, i.e. the reaction is accelerated 2000-4000 fold.<ref name= jordan>{{cite journal |vauthors=Jordan RE, Oosta GM, Gardner WT, Rosenberg RD | title = The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin | journal = J. Biol. Chem. | volume = 255 | issue = 21 | pages = 10081–10090 | year = 1980 | pmid = 6448846 }}</ref><ref>{{cite journal | author = Griffith MJ | title = Kinetics of the heparin-enhanced antithrombin III/thrombin reaction. Evidence for a template model for the mechanism of action of heparin | journal = J. Biol. Chem. | volume = 257 | issue = 13 | pages = 7360–7365 | year = 1982 | pmid = 7085630 }}</ref><ref>{{cite journal |vauthors=Olson ST, Björk I | title = Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects | journal = J. Biol. Chem. | volume = 266 | issue = 10 | pages = 6353–6354 | year = 1991 | pmid = 2007588 }}</ref><ref name=olson2>{{cite journal |vauthors=Olson ST, Björk I, Sheffer R, Craig PA, Shore JD, Choay J | title = Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement | journal = J. Biol. Chem. | volume = 267 | issue = 18 | pages = 12528–12538 | year = 1992 | pmid = 1618758 }}</ref> Factor Xa inhibition is accelerated by only 500 to 1000 fold in the presence of heparin and the maximal rate constant is 10 fold lower than that of thrombin inhibition.<ref name=jordan/><ref name=olson2/> The rate enhancement of antithrombin-Factor IXa inhibition shows an approximate 1 million fold enhancement in the presence of heparin and physiological levels of [[calcium]].<ref name=bedsted/>

AT-III binds to a specific pentasaccharide sulfation sequence contained within the heparin polymer

GlcNAc/NS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S)

Upon binding to this pentasaccharide sequence, inhibition of protease activity is increased by heparin as a result of two distinct mechanisms.<ref>{{cite journal |vauthors=Johnson DJ, Langdown J, Li W, Luis SA, Baglin TP, Huntington JA | title = Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation | journal = J. Biol. Chem. | volume = 281 | issue = 46 | pages = 35478–35486 | year = 2006 | pmid = 16973611 | pmc = 2679979 | doi = 10.1074/jbc.M607204200 }}</ref> In one mechanism heparin stimulation of Factor IXa and Xa inhibition depends on a conformational change within antithrombin involving the reactive site loop and is thus [[allosteric]].<ref name=Langdown>{{cite journal |vauthors=Langdown J, Johnson DJ, Baglin TP, Huntington JA | title = Allosteric activation of antithrombin critically depends upon hinge region extension | journal = J. Biol. Chem. | volume = 279 | issue = 45 | pages = 47288–47297 | year = 2004 | pmid = 15326167 | doi = 10.1074/jbc.M408961200 }}</ref> In another mechanism stimulation of thrombin inhibition depends on the formation of a [[ternary complex]] between AT-III, thrombin, and heparin.<ref name=Langdown/>

===Allosteric activation===
[[File:Antithrom+heparin.jpeg|thumb|<span style="font-size:100%;">'''Figure 4.'''</span> Two [[X-ray crystallography|crystal structure]]s for antithrombin. Model '''A''' is taken from the [[Protein Data Bank|pdb]] file [http://www.pdb.org/pdb/explore/explore.do?structureId=2ANT 2ANT] and model '''B''' from pdb file [http://www.pdb.org/pdb/explore/explore.do?structureId=1AZX 1AZX]. Model '''B''' is complexed with a pentasaccharide while model '''A''' is uncomplexed.|left|400px]]
Increased Factor IXa and Xa inhibition requires the minimal heparin pentasaccharide sequence. The conformational changes that occur within antithrombin in response to pentasaccharide binding are well documented.<ref name=Whisstock/><ref name=Schreuder>{{cite journal |vauthors=Schreuder HA, de Boer B, Dijkema R, Mulders J, Theunissen HJ, Grootenhuis PD, Hol WG | title = The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions | journal = Nature Structural & Molecular Biology | volume = 1 | issue = 1 | pages = 48–54 | year = 1994 | pmid = 7656006 | doi = 10.1038/nsb0194-48 }}</ref><ref name=Carrell>{{cite journal |vauthors=Carrell RW, Stein PE, Fermi G, Wardell MR | title = Biological implications of a 3 A structure of dimeric antithrombin | journal = Structure | volume = 2 | issue = 4 | pages = 257–270 | year = 1994 | pmid = 8087553 | doi = 10.1016/S0969-2126(00)00028-9 }}</ref>

In the absence of heparin, amino acids P14 and P15 (see '''Figure 3''') from the reactive site loop are embedded within the main body of the protein (specifically the top of [[beta sheet]] A). This feature is in common with other [[serpin]]s such as [[heparin cofactor II]], [[alpha 1-antichymotrypsin]] and [[myeloid and erythroid nuclear termination stage specific protein|MENT]].

The conformational change most relevant for Factor IXa and Xa inhibition involves the P14 and P15 amino acids within the [[N-terminus|N-terminal]] region of the reactive site loop (circled in '''Figure 4''' model '''B'''). This region has been termed the hinge region. The conformational change within the hinge region in response to heparin binding results in the expulsion of P14 and P15 from the main body of the protein and it has been shown that by preventing this conformational change, increased Factor IXa and Xa inhibition does not occur.<ref name=Langdown/> It is thought that the increased flexibility given to the reactive site loop as a result of the hinge region conformational change is a key factor in influencing increased Factor IXa and Xa inhibition. It has been calculated that in the absence of the pentasaccharide only one in every 400 Antithrombin molecules (0.25%) is in an active conformation with the P14 and P15 amino acids expelled.<ref name=Langdown/>

===Non-allosteric activation===
[[File:Throm+anti+hep complex.jpeg|thumb|<span style="font-size:100%;">'''Figure 5.'''</span> The structure of an antithrombin-thrombin-heparin [[ternary complex]] taken from pdb [http://www.pdb.org/pdb/explore/explore.do?structureId=1TB6 1TB6].|left|400px]]
Increased thrombin inhibition requires the minimal heparin pentasaccharide plus at least an additional 13 monomeric units.<ref>{{cite journal |vauthors=Petitou M, Hérault JP, Bernat A, Driguez PA, Duchaussoy P, Lormeau JC, Herbert JM | title = Synthesis of Thrombin inhibiting Heparin mimetics without side effects | journal = Nature | volume = 398 | issue = 6726 | pages = 417–422 | year = 1999 | pmid = 10201371 | doi = 10.1038/18877 }}</ref> This is thought to be due to a requirement that antithrombin and thrombin must bind to the same heparin chain adjacent to each other. This can be seen in the series of models shown in '''Figure 5'''.

In the structures shown in Figure 5 the [[C-terminus|C-terminal]] portion (P' side) of the reactive site loop is in an extended conformation when compared with other un-activated or heparin activated antithrombin structures.<ref name=Li/> The P' region of antithrombin is unusually long relative to the P' region of other serpins and in un-activated or heparin activated antithrombin structures forms a tightly [[hydrogen bond]]ed [[Turn (biochemistry)#Types of turns|β-turn]]. P' elongation occurs through the breaking of all hydrogen bonds involved in the [[Turn (biochemistry)#Types of turns|β-turn]].<ref name=Li>{{cite journal |vauthors=Li W, Johnson DJ, Esmon CT, Huntington JA | title = Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin | journal = Nature Structural & Molecular Biology | volume = 11 | issue = 9 | pages = 857–862 | year = 2004 | pmid = 15311269 | doi = 10.1038/nsmb811 }}</ref>

The hinge region of antithrombin in the Figure 5 complex could not be modelled due to its conformational flexibility, and amino acids P9-P14 are not seen in this structure. This conformational flexibility indicates an [[chemical equilibrium|equilibrium]] may exist within the complex between a P14 P15 reactive site loop inserted antithrombin conformation and a P14 P15 reactive site loop expelled conformation. In support of this, analysis of the positioning of P15 Gly in the Figure 5 complex (labelled in model B) shows it to be inserted into [[beta sheet]] A (see model C).<ref name=Li/>

==Effect of glycosylation on activity==
α-Antithrombin and β-antithrombin differ in their affinity for heparin.<ref name=mccoy>{{cite journal |vauthors=McCoy AJ, Pei XY, Skinner R, Abrahams JP, Carrell RW | title = Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity | journal = J. Mol. Biol. | volume = 326 | issue = 3 | pages = 823–833 | year = 2003 | pmid = 12581643 | doi = 10.1016/S0022-2836(02)01382-7 }}</ref> The difference in [[dissociation constant]] between the two is threefold for the pentasaccharide shown in '''Figure 3''' and greater than tenfold for full length heparin, with β-antithrombin having a higher affinity.<ref>{{cite journal |vauthors=Turk B, Brieditis I, Bock SC, Olson ST, Björk I | title = The oligosaccharide side chain on Asn-135 of alpha-antithrombin, absent in beta-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change | journal = Biochemistry | volume = 36 | issue = 22 | pages = 6682–6691 | year = 1997 | pmid = 9184148 | doi = 10.1021/bi9702492 }}</ref> The higher affinity of β-antithrombin is thought to be due to the increased rate at which subsequent conformational changes occur within the protein upon initial heparin binding. For α-antithrombin, the additional glycosylation at Asn-135 is not thought to interfere with initial heparin binding, but rather to inhibit any resulting conformational changes.<ref name=mccoy/>

Even though it is present at only 5–10% the levels of α-antithrombin, due to its increased heparin affinity, it is thought that β-antithrombin is more important than α-antithrombin in controlling thrombogenic events resulting from tissue injury. Indeed, thrombin inhibition after injury to the [[aorta]] has been attributed solely to β-antithrombin.<ref>{{cite journal |vauthors=Frebelius S, Isaksson S, Swedenborg J | title = Thrombin inhibition by antithrombin III on the subendothelium is explained by the isoform AT beta | journal = Arterioscler. Thromb. Vasc. Biol. | volume = 16 | issue = 10 | pages = 1292–1297 | year = 1996 | pmid = 8857927 | doi = 10.1161/01.ATV.16.10.1292 }}</ref>

== Role in disease ==
{{details|Antithrombin III deficiency}}
Evidence for the important role antithrombin plays in regulating normal blood coagulation is demonstrated by the correlation between [[inherited disorder|inherited]] or [[acquired disorder|acquired]] antithrombin deficiencies and an increased risk of any affected individual developing thrombotic disease.<ref>{{cite journal |vauthors=van Boven HH, Lane DA | title = Antithrombin and its inherited deficiency states | journal = Semin. Hematol. | volume = 34 | issue = 3 | pages = 188–204 | year = 1997 | pmid = 9241705 }}</ref> [[Antithrombin deficiency]] generally comes to light when a patient suffers recurrent venous [[thrombosis]] and [[pulmonary embolism]].

===Acquired antithrombin deficiency===
Acquired antithrombin deficiency occurs as a result of three distinctly different mechanisms.  The first mechanism is increased excretion which may occur with renal failure associated with proteinuria [[nephrotic syndrome]].  The second mechanism results from decreased production as seen in [[Acute liver failure#Coagulopathy|liver failure]] or cirrhosis or an immature liver secondary to [[premature birth]].  The third mechanism results from accelerated consumption which is most pronounced as consequence of severe injury [[Trauma (medicine)|trauma]] but also may be seen on a lesser scale as a result of interventions such as [[surgery|major surgery]] or [[cardiopulmonary bypass]].<ref>{{cite journal |vauthors=Maclean PS, Tait RC | title = Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options | journal = Drugs | volume = 67 | issue = 10 | pages = 1429–1440 | year = 2007 | pmid = 17600391 | doi = 10.2165/00003495-200767100-00005 }}</ref>

===Inherited antithrombin deficiency===
The incidence of inherited antithrombin deficiency has been estimated at between 1:2000 and 1:5000 in the normal population, with the first family suffering from inherited antithrombin deficiency being described in 1965.<ref>{{cite journal |vauthors=Lane DA, Kunz G, Olds RJ, Thein SL | title = Molecular genetics of antithrombin deficiency | journal = Blood Rev. | volume = 10 | issue = 2 | pages = 59–74 | year = 1996 | pmid = 8813337 | doi = 10.1016/S0268-960X(96)90034-X }}</ref><ref>{{cite journal | author = Egeberg O | title = Inherited antithrombin deficiency causing thrombophilia | journal = Thromb. Diath. Haemorrh. | volume = 13 | pages = 516–530 | year = 1965 | pmid = 14347873 }}</ref> Subsequently, it was proposed that the classification of inherited antithrombin deficiency be designated as either type I or type II, based upon functional and [[Immunoelectrophoresis|immunochemical]] antithrombin analyses.<ref>{{cite journal |vauthors=Sas G, Petö I, Bánhegyi D, Blaskó G, Domján G | title = Heterogeneity of the "classical" antithrombin III deficiency | journal = Thromb. Haemost. | volume = 43 | issue = 2 | pages = 133–136 | year = 1980 | pmid = 7455972 }}</ref> Maintenance of an adequate level of antithrombin activity, which is at least 70% that of a normal functional level is essential to ensure effective inhibition of blood coagulation proteases.<ref>{{cite journal |vauthors=Lane DA, Olds RJ, Conard J, Boisclair M, Bock SC, Hultin M, Abildgaard U, Ireland H, Thompson E, Sas G | title = Pleiotropic effects of antithrombin strand 1C substitution mutations | journal = J. Clin. Invest. | volume = 90 | issue = 6 | pages = 2422–2433 | year = 1992 | pmid = 1469094 | pmc = 443398 | doi = 10.1172/JCI116133 }}</ref> Typically as a result of type I or type II antithrombin deficiency, functional antithrombin levels are reduced to below 50% of normal.<ref name=Lane>{{cite journal |vauthors=Lane DA, Olds RJ, Thein SL | title = Antithrombin III: summary of first database update | journal = Nucleic Acids Res. | volume = 22 | issue = 17 | pages = 3556–3559 | year = 1994 | pmid = 7937056 | pmc = 308318 }}</ref>

====Type I antithrombin deficiency====
Type I antithrombin deficiency is characterised by a decrease in both antithrombin activity and antithrombin concentration in the blood of affected individuals. Type I deficiency was originally further divided into two subgroups, Ia and Ib, based upon heparin affinity. The antithrombin of subgroup Ia individuals showed a normal affinity for heparin while the antithrombin of subgroup Ib individuals showed a reduced affinity for heparin.<ref name=Sas>{{cite journal | author = Sas G | title = Hereditary antithrombin III deficiency: biochemical aspects | journal = Haematologica | volume = 17 | issue = 1 | pages = 81–86 | year = 1984 | pmid = 6724355 }}</ref> Subsequent functional analysis of a group of 1b cases found them not only to have reduced heparin affinity but multiple or 'pleiotrophic' abnormalities affecting the reactive site, the heparin binding site and antithrombin blood concentration. In a revised system of classification adopted by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, type Ib cases are now designated as type II PE, Pleiotrophic effect.<ref name=database1>{{cite journal |vauthors=Lane DA, Olds RJ, Boisclair M, Chowdhury V, Thein SL, Cooper DN, Blajchman M, Perry D, Emmerich J, Aiach M | title = Antithrombin III mutation database: first update. For the Thrombin and its Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis | journal = Thromb. Haemost. | volume = 70 | issue = 2 | pages = 361–369 | year = 1993 | pmid = 8236149 }}</ref>

Most cases of type I deficiency are due to [[point mutation]]s, deletions or minor insertions within the antithrombin gene. These genetic mutations result in type I deficiency through a variety of mechanisms:
*Mutations may produce unstable antithrombins that either may be not exported into the blood correctly upon completion biosynthesis or exist in the blood for a shortened period of time, e.g., the deletion of 6 [[base pair]]s in [[codon]]s 106-108.<ref>{{cite journal |vauthors=Olds RJ, Lane DA, Beresford CH, Abildgaard U, Hughes PM, Thein SL | title = A recurrent deletion in the antithrombin gene, AT106-108(-6 bp), identified by DNA heteroduplex detection | journal = Genomics | volume = 16 | issue = 1 | pages = 298–299 | year = 1993 | pmid = 8486379 | doi = 10.1006/geno.1993.1184 }}</ref>
*Mutations may affect [[mRNA]] processing of the antithrombin gene.
*Minor insertions or deletions may lead to [[frame shift]] mutations and premature termination of the antithrombin gene.
*Point mutations may also result in the premature generation of a termination or [[stop codon]] e.g. the mutation of codon 129, [[Genetic code#RNA codon table|CGA]]→[[Genetic code#RNA codon table|TGA]] ([[Genetic code#RNA codon table|UGA]] after transcription), replaces a normal codon for arginine with a termination codon.<ref>{{cite journal |vauthors=Olds RJ, Lane DA, Ireland H, Finazzi G, Barbui T, Abildgaard U, Girolami A, Thein SL | title = A common point mutation producing type 1A antithrombin III deficiency: AT129 CGA to TGA (Arg to Stop) | journal = Thromb. Res. | volume = 64 | issue = 5 | pages = 621–625 | year = 1991 | pmid = 1808766 | doi = 10.1016/S0049-3848(05)80011-8 }}</ref>

====Type II antithrombin deficiency====
Type II antithrombin deficiency is characterised by normal antithrombin levels but reduced antithrombin activity in the blood of affected individuals. Originally it was proposed that type II deficiency be further divided into three subgroups IIa, IIb and IIc depending upon which antithrombin functional activity is reduced or retained.<ref name=Sas/>

* Subgroup IIa - Decreased thrombin inactivation, decreased factor Xa inactivation and decreased heparin affinity.
* Subgroup IIb - Decreased thrombin inactivation and normal heparin affinity.
* Subgroup IIc - Normal thrombin inactivation, normal factor Xa inactivation and decreased heparin affinity.

In the revised system of classification again adopted by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, type II antithrombin deficiency remains subdivided into three subgroups: the already mentioned type II PE, along with type II RS, where mutations effect the reactive site and type II HBS, where mutations effect the antithrombin heparin binding site.<ref name=database1/> For the purposes of an antithrombin mutational database compiled by members of the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, type IIa cases are now classified as type II PE, type IIb cases as type II RS and type IIc cases as type II HBS.<ref name=database2>Imperial College London, Faculty of Medicine, [http://www1.imperial.ac.uk/medicine/about/divisions/is/haemo/coag/antithrombin/ Antithrombin Mutation Database] {{webarchive |url=https://web.archive.org/web/20090615172423/http://www1.imperial.ac.uk/medicine/about/divisions/is/haemo/coag/antithrombin/ |date=June 15, 2009 }}. Retrieved on 2008-08-16.</ref>

===Toponyms===
Presently it is relatively easy to characterise a specific antithrombin genetic mutation. However prior to the use of modern characterisation techniques investigators named mutations for the town or city where the individual suffering from the deficiency resided i.e. the antithrombin mutation was designated a [[toponym]].<ref name=Blajchman>{{cite journal |vauthors=Blajchman MA, Austin RC, Fernandez-Rachubinski F, Sheffield WP | title = Molecular basis of inherited human antithrombin deficiency | journal = Blood | volume = 80 | issue = 9 | pages = 2159–2171 | year = 1992 | pmid = 1421387 }}</ref> Modern mutational characterisation has since shown that many individual antithrombin toponyms are actually the result of the same genetic mutation, for example Antithrombin-Toyama, is equivalent to Antihrombin-Kumamoto, -Amien, -Tours, -Paris-1, -Paris-2, -Alger, -Padua-2 and -Barcelona.<ref name=database2/>

<center>
<gallery widths="300px" heights="300px">
Image:Pleio_mutations.jpeg|
Image:Active_site_mutations.jpeg|
Image:Hep_mutations.jpeg|
</gallery>
</center>

==Medical uses==
Antithrombin is used as a [[Biologic medical product|protein therapeutic]] that can be purified from human plasma<ref name=ThrombateLabel>[http://www.talecris-pi.info/inserts/thrombate.pdf Thrombate III label]</ref> or produced recombinantly (for example, Atryn, which is produced in the milk of [[Genetic engineering|genetically modified]] goats.<ref>[http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/FractionatedPlasmaProducts/ucm134042.htm FDA website for ATryn (BL 125284)]</ref><ref name=ATrynLabel>[http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/FractionatedPlasmaProducts/UCM134045.pdf Antithrombin (Recombinant) US Package Insert  ATryn for Injection February 3, 2009]</ref>)

Antithrombin is approved by the FDA as an [[anticoagulant]] for the prevention of clots before, during, or after surgery or birthing in patients with hereditary antithrombin deficiency.<ref name=ThrombateLabel/><ref name=ATrynLabel/>

Antithrombin has been studied in [[sepsis]] to reduce [[diffuse intravascular coagulation]] and other outcomes. It has not been found to confer any benefit in critically ill people with sepsis.<ref>{{cite journal|last1=Allingstrup|first1=Mikkel|last2=Wetterslev|first2=Jørn|last3=Ravn|first3=Frederikke B.|last4=Møller|first4=Ann Merete|last5=Afshari|first5=Arash|title=Antithrombin III for critically ill patients: a systematic review with meta-analysis and trial sequential analysis|journal=Intensive Care Medicine|date=9 February 2016|volume=42|issue=4|pages=505–520|doi=10.1007/s00134-016-4225-7|pmid=26862016}}</ref>

==Cleaved and latent antithrombin==
[[File:Latent antithrombin.jpeg|thumb|<span style="font-size:100%;">'''Figure 6.'''</span> Latent antithrombin|left|300px]]
Cleavage at the reactive site results in entrapment of the thrombin protease, with movement of the cleaved reactive site loop together with the bound protease, such that the
loop forms an extra sixth strand in the middle of [[beta sheet]] A. This movement of the reactive site loop can also be induced without cleavage, with the resulting crystallographic structure being identical to that of the physiologically latent conformation of [[plasminogen activator inhibitor-1]] (PAI-1).<ref>{{cite journal |vauthors=Mottonen J, Strand A, Symersky J, Sweet RM, Danley DE, Geoghegan KF, Gerard RD, Goldsmith EJ | title = Structural basis of latency in plasminogen activator inhibitor-1 | journal = Nature | volume = 355 | issue = 6357 | pages = 270–273 | year = 1992 | pmid = 1731226 | doi = 10.1038/355270a0 }}</ref> For this reason the conformation of antithrombin in which the reactive site loop is incorporated uncleaved into the main body of the protein is referred to as latent antithrombin. In contrast to PAI-1 the transition for antithrombin from a normal or native conformation to a latent conformation is irreversible.

Native antithrombin can be converted to latent antithrombin (L-antithrombin) by heating alone or heating in the presence of [[citrate]].<ref>{{cite journal |vauthors=Chang WS, Harper PL | title = Commercial antithrombin concentrate contains inactive L-forms of antithrombin | journal = Thromb. Haemost. | volume = 77 | issue = 2 | pages = 323–328 | year = 1997 | pmid = 9157590 }}</ref><ref>{{cite journal |vauthors=Wardell MR, Chang WS, Bruce D, Skinner R, Lesk AM, Carrell RW | title = Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1 | journal = Biochemistry | volume = 36 | issue = 42 | pages = 13133–13142 | year = 1997 | pmid = 9335576 | doi = 10.1021/bi970664u }}</ref> However, without extreme heating and at 37&nbsp;°C (body temperature) 10% of all antithrombin circulating in the blood is converted to the L-antithrombin over a 24-hour period.<ref>{{cite journal |vauthors=Carrell RW, Huntington JA, Mushunje A, Zhou A | title = The conformational basis of thrombosis | journal = Thromb. Haemost. | volume = 86 | issue = 1 | pages = 14–22 | year = 2001 | pmid = 11487000 }}</ref><ref name=Zhou>{{cite journal |vauthors=Zhou A, Huntington JA, Carrell RW | title = Formation of the antithrombin heterodimer in vivo and the onset of thrombosis | journal = Blood | volume = 94 | issue = 10 | pages = 3388–3396 | year = 1999 | pmid = 10552948 }}</ref> The structure of L-antithrombin is shown in '''Figure 6'''.

The 3-dimensional structure of native antithrombin was first determined in 1994.<ref name=Schreuder/><ref name=Carrell/> Unexpectedly the protein crystallized as a [[protein dimer|heterodimer]] composed of one molecule of native antithrombin and one molecule of latent antithrombin. Latent antithrombin on formation immediately links to a molecule of native antithrombin to form the heterodimer, and it is not until the concentration of latent antithrombin exceeds 50% of the total antithrombin that it can be detected analytically.<ref name=Zhou/> Not only is the latent form of antithrombin inactive against its target coagulation proteases, but its dimerisation with an otherwise active native antithrombin molecule also results in the native molecules inactivation. The physiological impact of the loss of antithrombin activity either through latent antithrombin formation or through subsequent dimer formation is exacerbated by the preference for dimerisation to occur between heparin activated β-antithrombin and latent antithrombin as opposed to α-antithrombin.<ref name=Zhou/>

A form of antithrombin that is an intermediate in the conversion between native and latent forms of antithrombin has also been isolated and this has been termed '''prelatent antithrombin'''.<ref>{{cite journal |vauthors=Larsson H, Akerud P, Nordling K, Raub-Segall E, Claesson-Welsh L, Björk I | title = A novel anti-angiogenic form of antithrombin with retained proteinase binding ability and heparin affinity | journal = J. Biol. Chem. | volume = 276 | issue = 15 | pages = 11996–12002 | year = 2001 | pmid = 11278631 | doi = 10.1074/jbc.M010170200 }}</ref>

==Antiangiogenic antithrombin==
[[Angiogenesis]] is a physiological process involving the growth of new [[blood vessel]]s from pre-existing vessels. Under normal physiological conditions angiogenesis is tightly regulated and is controlled by a balance of [[Angiogenesis#Chemical stimulation|angiogenic stimulators]] and [[Angiogenesis inhibitor|angiogenic inhibitors]]. [[Tumor]] growth is dependent upon angiogenesis and during tumor development a sustained production of angiogenic stimulatory factors is required along with a reduction in the quantity of angiogenic inhibitory factors tumor cells produce.<ref>{{cite journal | author = O'Reilly MS | title = Antiangiogenic antithrombin | journal = Semin. Thromb. Hemost. | volume = 33 | issue = 7 | pages = 660–666 | year = 2007 | pmid = 18000792 | doi = 10.1055/s-2007-991533 }}</ref> The cleaved and latent form of antithrombin potently inhibit angiogenesis and tumor growth in animal models.<ref>{{cite journal |vauthors=O'Reilly MS, Pirie-Shepherd S, Lane WS, Folkman J | title = Antiangiogenic activity of the cleaved conformation of the serpin antithrombin | journal = Science | volume = 285 | issue = 5435 | pages = 1926–1928 | year = 1999 | pmid = 10489375 | doi = 10.1126/science.285.5435.1926 }}</ref> The prelatent form of antithrombin has been shown to inhibit angiogenesis [[in-vitro]] but to date has not been tested in experimental animal models.

==References==
{{Reflist|2}}

== Further reading ==
* {{Cite thesis | degree = | chapter = | title = Antithrombin (III) - Establishing Pediatric Reference Values, Relevance for DIC 1992 versus 2007 | url = http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000011534 | author = Panzer-Heinig, Sabine | year = 2009 | publisher = Medizinische Fakultät Charité - Universitätsmedizin Berlin }}

==External links==
* The [[MEROPS]] online database for peptidases and their inhibitors: [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=I04.018 I04.018]
* {{MeshName|Antithrombin+III}}
* {{UCSC gene info|SERPINC1}}

{{Coagulation}}
{{Serpins}}
{{Alpha globulins}}

[[Category:Coagulation system]]
[[Category:Serine protease inhibitors]]