Update on Antithrombin I (Fibrin)
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©2007 Schattauer GmbH,Stuttgart AnniversaryIssueContribution Update on antithrombinI(fibrin) Michael W. Mosesson 1957–2007) The Blood Research Institute,BloodCenter of Wisconsin, Milwaukee,Wisconsin, USA y( Summary AntithrombinI(fibrin) is an important inhibitor of thrombin exosite 2.Thelatterreaction results in allostericchanges that generation that functions by sequestering thrombin in the form- down-regulate thrombin catalytic activity. AntithrombinIdefi- Anniversar ingfibrin clot,and also by reducing the catalytic activity of fibrin- ciency (afibrinogenemia), defectivethrombin binding to fibrin th boundthrombin.Thrombin binding to fibrin takesplace at two (antithrombin Idefect) found in certain dysfibrinogenemias (e.g. 50 classesofnon-substrate sites: 1) in thefibrin Edomain (two per fibrinogen Naples 1), or areduced plasma γ ’ chain content (re- molecule) throughinteractionwith thrombin exosite 1; 2) at a ducedantithrombin Iactivity),predispose to intravascular singlesite on each γ ’ chain through interaction with thrombin thrombosis. Keywords Fibrinogen,fibrin, thrombin, antithrombin I ThrombHaemost 2007; 98: 105–108 Introduction meric with respecttoits γ chains,and accounts for ~85% of human plasma fibrinogen. Thrombinbinds to its substrate, fibrinogen, through an anion- Low-affinity thrombin binding activity reflects thrombin ex- binding sitecommonlyreferred to as ‘exosite 1’ (1,2). Howell osite1bindinginEdomain of fibrin, as recentlydetailedbyana- recognized nearly acenturyago that the fibrin clot itself exhibits lysesofthrombin-fibrin fragment Ecrystals by Pechiketal. (13) significant thrombin-binding potential (3). Subsequently, (Fig. 2).Incontrast, ‘high affinity’thrombin binding to γ ’ chains thrombin binding associatedwith fibrin formation in plasma was takesplace throughexosite2(14–16) (Fig. 3). The γ ’ chain termed ‘antithrombin I’ by Seegers morethan sixtyyears ago thrombin binding site is situatedbetween residues414 and 427, (4–6), and Icontinue to employthat termfor this activity of fi- and tyrosine sulfation at γ ' 418 and γ ' 422 increases thrombin brin. In recent years,recognition of the functional importanceof binding potential (17).The binding affinity of thrombin for antithrombin Ifor down-regulation of thrombin generation in γ '-containing fibrin moleculesisincreased by concomitant fibrin plasma has brought anew perspective on its physiological role binding to thrombin exosite1(18) (Fig. 1). (7, 8). This present article providesanupdate on the constituents The effect of γ ’ chainbindingtothrombin exosite2ismore in fibrin that comprise antithrombin I, their mechanisms of ac- complexthan binding to thrombin exosite1atthe fibrin Edo- tion, and their physiological role. main. The γ ’ chain-thrombin interaction induces non-competi- tive allostericdown-regulation of amidolytic activity at the Antithrombin Iactivity thrombin catalytic siteand consequentlyslowedreleaseoffibri- nopeptide Aamong othereffects(19). This effect is independent AntithrombinIactivity is defined by twoclassesofnon-sub- of the slow-fast transition induced by Na+ binding (20) or the ef- strate thrombin-binding sites in fibrin (9, 10), one of relatively fect thatisreflected in the slowcleavage of fibrinogen induced This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. lowaffinity in the Edomain (~2sitesper molecule), and the by thrombin binding to thrombomodulin (21). Becauseofde- otherof‘high’ affinity in Ddomains of fibrin(ogen) molecules layedfibrinopeptide cleavage in γ ’ chain-containing fibrinogen containing a γ chainvariant termed γ ' ( γ ' 427L)(10) (Fig. 1). Al- 2, the fibrin that is produced has finer network fibersand con- together γ ' chains comprise ~8%ofthe total γ chainpopulation tains more branchesthan does fibrin 1(19, 22). This structural (11, 12). Virtuallyall γ ’ chains are foundinachromatographic modification of matrix structurealso results in delayedfibri- subfraction termed ‘fibrinogen2’, each moleculeofwhich also nolysis (19). Thedown-regulating effect of the γ ’ peptide se- contains aplatelet-binding γ chain. ‘Fibrinogen1’ishomodi- quenceoncatalytic site activity is similartothat inducedin Correspondence to: Received April 13, 2007 Michael W. Mosesson Accepted May11, 2007 The Blood Research Institute BloodCenterofWisconsin Prepublished onlineJune 12, 2007 PO Box2178, Milwaukee, WI 53201–2178, USA doi:10.1160/TH07–04–0274 Te l.: +1 414 937 3811, Fax: +1 414 937 6784 E-mail: [email protected] 105 Mosesson: Update on antithrombinI(fibrin) Figure1:Diagram of fibrinfeatures that arerelevant to antithrombinI,including details on the thrombin-binding γ ' 427L 1957–2007) chains and non-thrombin-binding γ ' 423P chains. Thedonor-acceptorglutamine (Q) and y( lysine(K) residues that arecommontoall γ chains, andthat become reciprocally cross- linkedbyfactor XIII areshown in red. Residues that areunique to γ ' chainsare blue.Thrombin molecules arered and exosites 1and 2are green.The arrowindicates athrombin mol- Anniversar ecule that is bound by both of itsexosites to a th γ ' chain-containing fibrin moleculeand to theE 50 domain. Other thrombinmoleculesshown in this diagramare bound only by their exosites 1 to Edomains.The EA and EB polymerization sitesinthe fibrin Edomain that bind to com- plementarysites in neighboring Ddomains are indicated. thrombin by other exosite2-binding proteins likeGP1bα or pro- In addition to the fulllength γ ' chains,ashortenedversion of thrombin fragment 2(23–26), amonoclonalantibody directed this chain, γ ' 423P,ispresentinmost plasmas (33–36).Webelieve againstanepitope in thrombin exosite2(27), and DNAaptamer that γ ' 423P chains arise by post-translational proteolytic process- HD-22 (28).This suggests that thrombin exosite2bindinginter- ing of intact γ ' 427L fibrinogen chains, buttodatewehavenot actions,for examplewith GP1bα or γ ’ chains,playarole in vivo beenabletoidentify the basis for this occurrence.Sincethe ulti- in regulating thrombin generation.Inaddition to the effects of γ ’ mate C-terminal γ ' 424 to 427 sequence is requiredfor thrombin chainbindingonfibrin formation and lysis, fibrin-mediated en- binding at the γ ' site (17), γ ' 423P chains lackthrombin binding po- hancement of factor XIII activation (29–32) wasslowerinthe tential, and their formation would reduce antithrombin Iactivity presenceoffibrinogen2compared with fibrinogen 1(19). Thus, at the expense of the γ ' 427L chains. γ ’ chain-thrombin interactionsplayanimportant roleinregulat- ing factor XIII activation. Antithrombin Iand itsrelationship to thrombotic disease The concept that antithrombinIisanimportant regulator of thrombin activity in clotting blood (8) is basedupon anumber of prior observations and reports: i) Fibrin from certaincongenital dysfibrinogens, e.g. fibrinogen NewYork I(9) and fibrinogen Naples I(18, 37, 38), exhibit reducedthrombin binding capacity and areassociatedwith marked venous or arterial thromboem- bolism. ii) Paradoxically, severe thromboembolic disease, both venous and arterial, occurs in afibrinogenemia and in hypofibri- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. nogenemia (39–47) ofteninassociation with the infusion of fi- brinogen. iii)Increased levels of prothrombin activation frag- ment F 1+2 (48, 49) or thrombin-antithrombin (TAT)complexes (47,48) arefound in afibrinogenemic plasma (i.e.congenital antithrombin Ideficiency),and these abnormal levels can be normalized by fibrinogen infusions (47, 49), further suggesting that an underlying hypercoagulable state exists in this condition. Figure2:Three-dimensional structureoftwo thrombinmol- iv)The reportthat an afibrinogenemic subjectdeveloped occlus- eculesboundtoafibrinEfragmentthat is projected from a iveperipheral arterial thrombosis in the absence of afibrinogen ribbon diagram of afibrin molecule. Thethrombin-fibrin complex infusion(47) seems to be analogous to studies in ferric chloride- is drawn as aribbon diagramalong atwo-fold symmetryaxis perpendi- injured afibrinogenemic mice, whichdeveloped abundantin- cular to theplane of thepage.Aα ,Bβ ,and γ chain fragmentsare blue, green,and red, respectively. Thrombinmoleculesare in beige,and the travascularthrombi at the siteofinjurythat characteristically residues included in exosite 1are in orange.The PPACKinhibitor bound embolized downstream (50).v)The demonstration by Dupuyet to theactivesiteisinmagenta. Adapted from Pechiketal. (13). al.(47) that increased thrombin generation in their patient’splas- 106 Mosesson: Update on antithrombinI(fibrin) 1957–2007) y( Anniversar th 50 Figure3:Crystal structureofthe γ ' pep- tide ( γ ' 408–427) making contacts with basic residues(blue)inexosite2of thrombin. From Pineda et al. (16).The γ ' pep- tide interactions closely reproduce heparin binding at this site(56),thereby explaining why thebindingofthe γ ' peptide and heparin are mutuallyexclusive and whythrombin bound to fibrin is resistanttoinactivationbyantithrom- bin and heparin cofactor II (14, 57). ma wasnormalizedbyaddition of fibrinogen underscored the On theotherhand,Drouet et al. (51)suggestedthat subjects thrombin inhibitoryrole of plasma fibrinogen. Demonstrating with elevated γ ' -fibrinogen/totalfibrinogenratios correlated that fibrinogen 2(γ A/γ ' )had amore profound effect in normaliz- with ahigherincidence of arterial thrombosis and Lovely et al. ing thrombin generation in afibrinogenemic plasma than did fi- (52)reported an association between elevatedlevelsof