Factor XIa

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Schematic representation of the coagulation response

Coagulation Factor XIaCoagulation Factor XIa

IntroductionIntroduction

Factor XIa is unique protease derived from the activation of the coagulation zymogen, factor XI. Factor XIa partcipates in the procoagulant response via contact activation pathway. Synthesized by the liver similar to most vitamin K-dependent coagulation proteins, the zymogen, factor XI circulates in plasma as a 160 kDa disulfide-linked homodimer in complex with high molecular weight kininogen (HK)[1]. Studies show that factor XI is a substrate for various plasma proteins such as factor XIIa, thrombin, meizothrombin and factor XIa (via autoactivation). Proteolysis of the bond generates the active enzyme factor XIa which in turn cleaves its substrate factor IX to produce the serine protease factor IXa.

Crystal structure of factor XI zymogen 2f83

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Protein StructureProtein Structure

Factor XIa is a linked-dimer of similar amino acid composition of approximately 625 residues. The first 18 amino acid residues constitute the signal peptide whereas residues 19-387 and 388-625 represents the heavy- and light- chains of the factor XIa molecule respectively. The protein forms five main distinct domains. Beginning from the N-terminus,each dimeric subunit contains 4 apple domains (, , and ) which are characterized by approximately 90 or 91 amino acid residues. Protein-protein interactions are thought to be the primary role of the apple domains. The is reported to mediate binding to platelet glycoprotein Ib (GPIb)[2] as well as interactions with exosite I of thrombin, and kringle 2 domain of prothrombin. The is the main site of factor XI protein-protein interaction when in complex with high molecular weight kininogen[3]. The C-terminus (heavy chain) of factor XIa contain a trypsin-like catalytic domain [4]. Together with Prekallikrein (PK) a monomeric homolog of factor XIa, they belong to the PAN (plasminogen, apple, nematode) module family which all have a conserved N-terminal apple domain found in hepatocyte growth factor and plasminogen [5].

Secondary structureSecondary structure

About 36 β-strands have been observed in the crystal structure of factor XI with twice as much found in the heavy chain (25 β-strands) compared to the light chain (11 β-strands). The topology of the apple domain reveals 7 antiparallel β-sheets and an α-helix which fold into a compact structure as oppose to an extended structure found in the vitamin K-dependent serine proteases. This core PAN topology is also found in leech antiplatelet protein and hepatocyte growth factor[6]. A single disulfide linkage connects the C- and N-terminals of the dimer whereas two disulfide bond join the helix to the 4β- and 5β-sheets. The apple domains of factor XIa are tightly linked to each other forming a disk-like structure close to the base of the C-terminal catalytic domain. This observation is consistent with the high surface area measurements for the side interfaces between apple domains and (441ÅxÅ) and between and (444ÅxÅ) in contrast to smaller end interfaces between and (380ÅxÅ) and between and (284ÅxÅ).

β-barrel

The FXIa serine protease-like domain found in the light chain region of the enzyme contains 2 connected by a central loop. Factor XI could be classified as an all β protein since β-sheets predominate in its structure with a few helices. It is therefore not suprising that the observed were entirely connected by loops. Although the β-strands which form the β-barrel in factor XIa form an up and down pattern charateristic of a Greek key and Up and Down β-barrels, the interconnect loops are very different. Most serine preteases however contain 2 Greek key β-barrels as a secondary structural element.

β-turn

β-turn in factor XIa

The globular and compact nature of factor XIa as opposed to an elongated form (prevalent in vitamin K-dependent serine proteases) could in part be attributed to the abundance of β-turns in the protein. β-turns are characterized by a hydrogen bond involving carbonyl oxygen (C=O) of residue (i) and amide hydrogen (NH) of residue (i+3). The heavy chain has ~4 β-turns and one such β-turn (residues 566-568) is found in the light chain of factor XIa (see figure on the right). This β-turn based on distance between Cαi-Cαi+3 (5.4Å) and the measured dihedral angles: φ(i+1)=50.5°, ψ(i+1)=47.2° and φ(i+2)=90° and ψ(i+2)=15.8°could be classified as Type I′ according to Hutchinson and Thornton (1994)[7].

α-Helix

N-terminal capping motif in factor XIa

In addition to β-sheets, factor XIa folds into a number of α-helices. The heavy chain region has ~8 helix repeats while about 5 helix repeats are present in the light chain region of the protein. Helices that are capped at either the N or C-terminals forming a capping motif. Helix Capping Motif display a unique hydrogen bonding pattern in addition to hydrophobic interactions. Factor XIa has an N-terminal capping motif in the light chain: residues 523-531 form an α-helix (see the figure on the right). Thr-532 is the Ncap and Glu-526 is found at the N3 position of the helix. The N-terminal capping motif shown in the figure (right hand side of page) appears to belong to the capping box classification. Thus the side chain of Thr-523 forms a H-bond with the backbone of the N3 however, H-bonding between the side chain of N3 and backbone of Ncap is absent. Next to the C-terminal Cys356 of the domain,factor XI assumes an interesting helical element the . Originally classified as a type III turn, the is tight and contains 3 residues (357-360) per turn.

Factor FXIa dimerFactor FXIa dimer

The β-sheets form a tight packing against each other with two A4 domains forming a large interface between the dimer subunits. Found in the central position is the main interchain disulfide bond contributed by Cys-321 located on the finger-like loop of the A4 domain. The apple domains form a V-shape in which two A2 domains are distanced ~50Å apart whiles the A1 and A3 domains from adjacent monomers are in close proximity of 5Å apart [8]. The interface made up of Leu-284, Ile-290 and Tyr-329 and the between Lys-331 of one monomer and Glu-287 of the other monomer are absolutely required for dimer formation[9]. Most of the complex protein-protein interactions involving factor XIa are mediated by the apple domains of the dimeric subunits.

Posttranslational ModificationPosttranslational Modification

Unlike most serine proteases which contain a γ-carboxyglutamic acid (Gla) domain, which facilitates the binding of vitamin-K dependent coagulation proteases to phospholipid vesicles, plasma factor XIa lacks the Gla domain. Meanwhile the protease undergoes considerable posttranslational retailing following it synthesis. Approximately : 15 of which are confirmed and 4 potential disulfide linkages are reported to be present in factor XIa molecule. The homodimers are linked by a single disulfide bond at Cys-321 connecting the A4 domains of each subunit [10]. Ser-17 and Thr-22 are phosphorylated [11] whereas 5 N-linked glycosylations (GlcNAc) sites were also reported following glycoproteome analysis [12].

Formation of Factor XIaFormation of Factor XIa

Factor XIa light chain complex with clavatidine and sulfates 3bg8

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Factor XI is partially proteolyzed in vitro by thrombin and factor XIIa generating the active serine-protease, factor XIa. Similar to other chymotrypsin-like proteases, its topology consist of two β-barrels linked through a central loop. Next to the C-terminal Cys-356 of the factor XI heavy chain, the polypeptide forms a 3-10 helix conformation and again turn sharply 90 degrees at Cys-362 forming a disulfide bond with Cys-482 within the active site region.

Thrombin-catalyzed proteolysis of factor XI involves crucial interations with Glu-66, Lys-83 and Gln-84 of the A1 domain (this ensures maximum proximity to the of factor XI) of the factor XI molecule through its exosites I and II regions [13]. Thus binding of thrombin to one subunit of the zymogen dimer promotes cleavage of the bond between contained in the of factor XI. The (residues 370-376) consequently undergoes the greatest conformational change as Ile-370 is displaced ~20Å from its position in factor XI and inserts into the activation pocket of factor XIa producing the oxyanion hole in the active site of the protease [14].

Active Site ResiduesActive Site Residues

Similar to other serine proteases, the catalytic triad residues Ser-557, Asp-462 and His-413 constitute the of factor XIa. A low barrier hydrogen bond (LBHB) formed between the carboxyl group of Asp-462 and imidazole nitrogen of His 413 causes the deprotonation Ser-557 (enhacing its nucleophilicity). Thus catalysis involves a nucleophilic attack by Ser-557 on the carbonyl carbon of the target amino acid at the C-terminal of the substrate producing an intermediate which is stablized by the oxyanion hole. Rearrangement of the resulting tetrahedral intermediate and a second nucleophilic attack by water yields a cleaved peptide with a free carboxyl end [15].

Substrate Recognition and CleavageSubstrate Recognition and Cleavage

The primary substrate of factor XIa is another zymogen, factor IX which is cleavage sequentially at the peptides bonds between Arg145-Ala146 and Arg180-Val181 of factor IX to release an activation peptide [16]. Recognition of the substrate (factor IX) involves residues different from the residues. In the inactive zymogen (factor XI), the highly conserved is buried in the interface between the apple domains and the catalytic domain where it interacts with : Ser-268 from the A3 domain and Asp-488 and Asn-566 in the catalytic domain. Thus following activation, is believed to constitute a switch which undegoes a conformational change breaking its interaction with Ser-268, Asp-488 and Asn 566 facilitating the protease interaction with factor IX [17].

Evolutionary conservationEvolutionary conservation

Template:STRUCTURE 2f83 Analysis of vertebrate genomes has shown that human Factor XI and Prekallikrein are both products of gene duplication events during evolution[18].Prekallikrein, a monomeric homolog of factor XI is the zymogen form of the protease α-kallikrein and both zymogens(factor XI and prekallikrein) are 58% identical in their primary structure[19]. Interestingly, studies show that the ancestral predecessor of both zymogens is a protease bearing the highly conserved 4 apple domains[20]. The active site serine protease-like domain is however the most conserved amongst coagulation proteins

Factor XIa DeficiencyFactor XIa Deficiency

In contrast to a dysfunctional protein often reported in patients with defects in the vitamin K-dependent proteases, most cases of factor XIa deficiency are associated with low circulating amounts of the protein in the plasma [21]. Factor XI deficiency is a rare autosomal recessive [1] disorder with a prevalence rate of about 1% in human populations. Individuals with the disease experience slight to mild bleeding diathesis which moderately increase during a surgical challenge. Studies of the structural features of factor XI/FXIa has hightened in recent times due its implication both venous[22] and arterial[23] thrombosis, pathology of sepsis and ischemia-reperfusion damage in the central nervous system. Mutations in the A4 domain of factor XIa often interfere with the ability of the protein to dimerize.

Amino acid substitutions such as Phe283Leu[24] and Gly350Glu[25] in the heavy chain results in an increased dimer dissociation and absence of dimer formation respectively. Some mutations in the factor XI A4 domain and catalytic domains are inherited as autosomal recessive bleeding diathesis however, other amino acid substitutions are exert a dominant negative effect on the normal monomer subunit affecting protein secretion. Studies suggest that dimerization is not affected under dominant negative mutations but the mutant subunit traps the normal subunit in the cell preventing its secretion. Majority of these missense mutations:Ser225Phe, Cys398Tyr, Gly400Val and Trp569Ser which produce a dominant negative effect involves residues found in the catalytic domain[26].

3D structures of Factor XI3D structures of Factor XI

Factor XIFactor XI

2j8j, 2j8l – hFXIa A4 domain – human – NMR
2f83 - hFXI zymogen

Factor XI inhibitor complexFactor XI inhibitor complex

3bg8 – hFXIa + clavatadine
1zom, 1zsj, 1zsk, 1zlr, 1zmj, 1zml, 1zmn, 1zpz, 1zrk, 1ztj, 1ztk, 1ztl, 1zpb, 1zpc, 2fda, 1zsl, 1zhm, 1zhp, 1zhr - hFXI catalytic domain (mutant) + inhibitor
1zjd - hFXI catalytic domain (mutant) + Kunitz protease inhibitory domain
1xx9 - hFXI catalytic domain + ecotin (mutant)
1xxd, 1xxf - hFXI catalytic domain (mutant) + ecotin (mutant)

ReferencesReferences

  1. ↑ Thompson RE, Mandle R Jr, Kaplan AP. Association of factor XI and high molecular weight kininogen in human plasma. J Clin Invest. 1977 Dec;60(6):1376-80. PMID:915004 doi:http://dx.doi.org/10.1172/JCI108898
  2. ↑ Baglia FA, Gailani D, Lopez JA, Walsh PN. Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI. J Biol Chem. 2004 Oct 29;279(44):45470-6. Epub 2004 Aug 17. PMID:15317813 doi:10.1074/jbc.M406727200
  3. ↑ Herwald H, Jahnen-Dechent W, Alla SA, Hock J, Bouma BN, Muller-Esterl W. Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain. J Biol Chem. 1993 Jul 5;268(19):14527-35. PMID:7686159
  4. ↑ Bouma BN, Griffin JH. Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII. J Biol Chem. 1977 Sep 25;252(18):6432-7. PMID:893417
  5. ↑ Tordai H, Banyai L, Patthy L. The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS Lett. 1999 Nov 12;461(1-2):63-7. PMID:10561497
  6. ↑ Tordai H, Banyai L, Patthy L. The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS Lett. 1999 Nov 12;461(1-2):63-7. PMID:10561497
  7. ↑ Hutchinson EG, Thornton JM. A revised set of potentials for beta-turn formation in proteins. Protein Sci. 1994 Dec;3(12):2207-16. PMID:7756980 doi:http://dx.doi.org/10.1002/pro.5560031206
  8. ↑ Papagrigoriou E, McEwan PA, Walsh PN, Emsley J. Crystal structure of the factor XI zymogen reveals a pathway for transactivation. Nat Struct Mol Biol. 2006 Jun;13(6):557-8. Epub 2006 May 14. PMID:16699514 doi:10.1038/nsmb1095
  9. ↑ Dorfman R, Walsh PN. Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization. J Biol Chem. 2001 Mar 2;276(9):6429-38. Epub 2000 Nov 22. PMID:11092900 doi:10.1074/jbc.M010340200
  10. ↑ McMullen BA, Fujikawa K, Davie EW. Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains. Biochemistry. 1991 Feb 26;30(8):2056-60. PMID:1998667
  11. ↑ Imami K, Sugiyama N, Kyono Y, Tomita M, Ishihama Y. Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column. Anal Sci. 2008 Jan;24(1):161-6. PMID:18187866
  12. ↑ Chen R, Jiang X, Sun D, Han G, Wang F, Ye M, Wang L, Zou H. Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. J Proteome Res. 2009 Feb;8(2):651-61. PMID:19159218 doi:10.1021/pr8008012
  13. ↑ Papagrigoriou E, McEwan PA, Walsh PN, Emsley J. Crystal structure of the factor XI zymogen reveals a pathway for transactivation. Nat Struct Mol Biol. 2006 Jun;13(6):557-8. Epub 2006 May 14. PMID:16699514 doi:10.1038/nsmb1095
  14. ↑ Friedrich R, Panizzi P, Fuentes-Prior P, Richter K, Verhamme I, Anderson PJ, Kawabata S, Huber R, Bode W, Bock PE. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature. 2003 Oct 2;425(6957):535-9. PMID:14523451 doi:10.1038/nature01962
  15. ↑ Friedrich R, Panizzi P, Fuentes-Prior P, Richter K, Verhamme I, Anderson PJ, Kawabata S, Huber R, Bode W, Bock PE. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature. 2003 Oct 2;425(6957):535-9. PMID:14523451 doi:10.1038/nature01962
  16. ↑ Sinha D, Marcinkiewicz M, Navaneetham D, Walsh PN. Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation. Biochemistry. 2007 Aug 28;46(34):9830-9. Epub 2007 Aug 4. PMID:17676929 doi:10.1021/bi062296c
  17. ↑ Papagrigoriou E, McEwan PA, Walsh PN, Emsley J. Crystal structure of the factor XI zymogen reveals a pathway for transactivation. Nat Struct Mol Biol. 2006 Jun;13(6):557-8. Epub 2006 May 14. PMID:16699514 doi:10.1038/nsmb1095
  18. ↑ Ponczek MB, Gailani D, Doolittle RF. Evolution of the contact phase of vertebrate blood coagulation. J Thromb Haemost. 2008 Nov;6(11):1876-83. Epub 2008 Aug 28. PMID:18761718 doi:10.1111/j.1538-7836.2008.03143.x
  19. ↑ McMullen BA, Fujikawa K, Davie EW. Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule. Biochemistry. 1991 Feb 26;30(8):2050-6. PMID:1998666
  20. ↑ Ponczek MB, Gailani D, Doolittle RF. Evolution of the contact phase of vertebrate blood coagulation. J Thromb Haemost. 2008 Nov;6(11):1876-83. Epub 2008 Aug 28. PMID:18761718 doi:10.1111/j.1538-7836.2008.03143.x
  21. ↑ Saito H, Ratnoff OD, Bouma BN, Seligsohn U. Failure to detect variant (CRM+) plasma thromboplastin antecedent (factor XI) molecules in hereditary plasma thromboplastin antecedent deficiency: a study of 125 patients of several ethnic backgrounds. J Lab Clin Med. 1985 Dec;106(6):718-22. PMID:4067382
  22. ↑ 10706899
  23. ↑ Wang X, Cheng Q, Xu L, Feuerstein GZ, Hsu MY, Smith PL, Seiffert DA, Schumacher WA, Ogletree ML, Gailani D. Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice. J Thromb Haemost. 2005 Apr;3(4):695-702. Epub 2005 Feb 23. PMID:15733058 doi:10.1111/j.1538-7836.2005.01236.x
  24. ↑ Riley PW, Cheng H, Samuel D, Roder H, Walsh PN. Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis. J Mol Biol. 2007 Mar 23;367(2):558-73. Epub 2006 Dec 29. PMID:17257616 doi:10.1016/j.jmb.2006.12.066
  25. ↑ Kravtsov DV, Wu W, Meijers JC, Sun MF, Blinder MA, Dang TP, Wang H, Gailani D. Dominant factor XI deficiency caused by mutations in the factor XI catalytic domain. Blood. 2004 Jul 1;104(1):128-34. Epub 2004 Mar 16. PMID:15026311 doi:10.1182/blood-2003-10-3530
  26. ↑ Kravtsov DV, Wu W, Meijers JC, Sun MF, Blinder MA, Dang TP, Wang H, Gailani D. Dominant factor XI deficiency caused by mutations in the factor XI catalytic domain. Blood. 2004 Jul 1;104(1):128-34. Epub 2004 Mar 16. PMID:15026311 doi:10.1182/blood-2003-10-3530

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