Allosteric Disulfide Bonds in Thrombosis and Thrombolysis

Journal of Thrombosis and Haemostasis, 4: 2533–2541

REVIEW ARTICLE Allosteric disulfide bonds in thrombosis and thrombolysis

V . M . C H E N * and P . J . H O G G * *Centre for Vascular Research, University of New South Wales, Sydney; and Children’s Cancer Institute Australia for Medical Research, Sydney, Australia

To cite this article: Chen VM, Hogg PJ. Allosteric disulfide bonds in thrombosis and thrombolysis. J Thromb Haemost 2006; 4: 2533–41.

most frequently acquired amino acid in eight of the 15 taxa Summary. Allosteric disulfide bonds control protein function studied. Other amino acids that have accrued are Met, His, Ser by mediating conformational change when they undergo and Phe, whereas Pro, Ala, Glu and Gly have been lost. reduction or oxidation. The known allosteric disulfide bonds Considering that disulfide bonds follow addition of Cys, this are characterized by a particular bond geometry, the )RHSta- analysis indicates that acquisition of these bonds is a relatively ple. A number of thrombosis and thrombolysis proteins contain recent evolutionary event. one or more disulfide bonds of this type. Tissue factor (TF) was the first hemostasis protein shown to be controlled by an Types and classification of disulfide bonds allosteric disulfide bond, the Cys186–Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in There are two general types of disulfide bond; structural and three forms on the cell surface: a cryptic form that is inert, a functional. The structural bonds, which are the majority, are coagulant form that rapidly binds factor VIIa to initiate involved in the folding of a protein and stabilize the tertiary coagulation, and a signaling form that binds FVIIa and cleaves structure. It is thought that they assist folding by decreasing the protease-activated receptor 2, which functions in inflamma- entropy of the unfolded form [4]. The functional disulfide tion, tumor progression and angiogenesis. Reduction and bonds are the catalytic and allosteric bonds. These disulfide oxidation of the Cys186–Cys209 disulfide bond is central to the bonds regulate protein function. transition between the three forms of TF. The redox state of the The catalytic bonds are the better characterized. Examples of bond appears to be controlled by protein disulfide isomerase these are the disulfide bonds in the active sites of oxidoreduc- and NO. Plasmin(ogen), vitronectin, glycoprotein 1ba,integrin tases, such as the thioredoxin family of proteins [5,6]. These b3 and thrombomodulin also contain )RHStaple disulfides, disulfide bonds cycle between reduced and oxidized configu- and there is circumstantial evidence that the function of these rations in coordination with a dithiol or disulfide in a protein proteins may involve cleavage/formation of these disulfide substrate, resulting in reduction, formation or isomerization of bonds. a disulfide in the substrate. The allosteric bonds have only recently been identified [7]. These bonds control protein

Keywords: glycoprotein 1ba,integrinb3, plasminogen, thrombo- function by triggering a conformational change when they modulin, tissue factor, vitronectin. break and/or form and are the subject of this review. A disulfide bond can be described in terms of the five Introduction dihedral or v angles that make up the bond. A dihedral angle is the angle of rotation about a certain bond, and is defined using Protein disulfide bonds are covalent links between two Cys four atoms [8]. v1 is the dihedral angle about the Ca–Cb bond, residues in the polypeptide chain. It appears that all life-forms v2 about the Cb–Sc bond, v3 about the Sc–Sc¢ bond, v2¢ about make this bond. Mammalian cells, for instance, make about the Sc¢–Cb¢ bond and v1¢ about the Cb¢–Ca¢ bond. These five 3000 different proteins that contain disulfide bonds. angles can be used to estimate the potential energy of a disulfide Addition of disulfide bonds to proteins is a major mechan- bond, which is called the dihedral strain energy (DSE) [8–10]. ism by which proteins have evolved and are still evolving [1–3]. The DSE is a semiquantitative measure, as it does not factor in Analysis of the trend in amino gain and loss in protein bond lengths and van der Waals contacts, for example, but has evolution has shown that Cys residues have accrued in 15 taxa been shown experimentally to reflect the amount of strain in a representing all three domains of life [3]. Moreover, Cys was the disulfide bond [11–14]. There are three basic disulfide bond configurations, based on Correspondence: Philip J. Hogg, Centre for Vascular Research, the sign of the v2, v3 and v2¢ angles; the spirals, hooks or staples University of New South Wales, Sydney NSW 2052, Australia. [15]. If the v3 angle is positive, then the bond is right-handed; if Tel.: +61 2 9385 1004; fax: +61 2 9385 1389; e-mail: it is negative, the bond is left-handed. When the v1 and v1¢ [email protected] angles are considered, there are 20 possible disulfide bond

2006 International Society on Thrombosis and Haemostasis 2534 V. M. Chen and P. J. Hogg configurations. All 20 disulfide bond geometries were identified likely mediator [20], although protein disulfide isomerase (PDI) in a dataset of 6874 unique disulfide bonds in X-ray structures has also been implicated [24]. [8]. The main structural disulfide bond is the )LHSpiral. A In this review, we will discuss the presence of allosteric quarter of the disulfide bonds in the dataset were )LHSpirals, disulfide bonds in proteins involved in thrombosis and and this group also had the lowest DSE. A striking finding thrombolysis, and evidence for control of some of these from this analysis was that nearly all the catalytic disulfide proteins by these bonds (Table 1). We will focus initially on bonds were of the +/)RHHook configuration, whereas all the tissue factor (TF), which is the best characterized example to known allosteric disulfide bonds fell into the )RHStaple group. date. Other proteins that will be discussed are plasmin(ogen),

Both of these bond groups have a high and narrow DSE vitronectin, glycoprotein 1ba,integrinb3 and thrombo- distribution, which is consistent with their functional role. modulin. A defining feature of the )RHStaple group is the short distance between the a carbon atoms of the disulfide bond. Tissue factor )RHStaple bonds have a mean Ca–Ca¢ distance of 4.3 A˚ , compared to a mean of 5.6 A˚ for all disulfide bonds [8]. This The blood coagulation process is initiated when the circulating short Ca–Ca¢ distance results from the fact that )RHStaples serine protease, factor (F) VIIa, forms a complex with its mostly link adjacent strands in the same antiparallel b-sheet cofactor, membrane-bound TF, to activate FVII, FX and FIX [7,16]. The strands need to pucker to accommodate the by limited proteolysis. Ternary TF–FVIIa–FXa or binary disulfide bond, and the associated deformation of the b-sheet TF–FVIIa complexes also signal through cleavage of protease- imparts a high torsional energy on the bond. A number of activated receptor 2 (PAR2) to control inflammation, tumor )RHStaple disulfide bonds have been shown to be involved in progression and angiogenesis. TF is a transmembrane glyco- the function of the protein in which they reside. The best protein with structural homology to the class II cytokine characterized are the )RHStaple disulfide bonds in the superfamily. It consists of two fibronectin type III domains, bacterial proteins botulinum neurotoxins [16,17], PapD [18] each with a disulfide bond. and DsbD [19], and the mammalian immune and human Analysis of 16 X-ray TF structures indicated that the immunodeficiency virus (HIV) receptor CD4 [20,21]. The viral N-terminal domain Cys49–Cys57 disulfide bond exists in one envelope glycoprotein, HIV gp120, also appears to be regulated of two configurations: a )RHSpiral or a +/)RHSpiral by cleavage of one or more of its four )RHStaple disulfide (Table 2, Fig. 1). Both configurations are subgroups of the bonds [16,22,23]. Analysis of the )RHStaple disulfide bonds in main structural disulfide bond, the )LHSpiral [8]. This type of nuclear magnetic resonance (NMR) structures indicates that disulfide bond is typical of immunoglobulin-like domains, of theyoftenswitchtothe)LHStaple configuration, which has an which the fibronectin type III domain is an example [25]. The even higher mean torsional energy and shorter mean Ca–Ca¢ disulfide bond links across the two sheets of the b-sandwich distance than the )RHStaple bonds (B. Schmidt and P. J. structure. The C-terminal domain Cys186–Cys209 disulfide Hogg, unpubl. obs.). The )LHStaple bonds, therefore, should bond exists exclusively as a )RHStaple in the 16 structures [26]. also be considered as potential allosteric disulfide bonds. In This bond has a higher DSE and a considerably shorter Ca– fact, it is an open question whether the )RHStaple or Ca¢ distance than the N-terminal RHSpiral (Table 2), which is )LHStaple configuration is the functional form in some in accordance with the typical features of these bonds [8]. The proteins. Cys186–Cys209 disulfide bond straddles the F and G strands of CD4 is a good example of how protein function can be the antiparallel b-sheet at a non-H-bonded site. The disulfide controlled by an allosteric )RHStaple disulfide bond. CD4 is a bond constrains a b hairpin turn adjacent to the stalk region class II cytokine receptor that is expressed on T cells. It is a coreceptor for binding of MHCII to the T-cell receptor and is the primary receptor for HIV. The extracellular part of CD4 Table 1 RHStaple disulfide bonds in selected thrombosis and thrombo- consists of a concatenation of four immunoglobulin-like lysis proteins domains, and the first, second and fourth domains contain a Protein Region Disulfide bond(s)* single disulfide bond. CD4 exists in three forms on the T-cell Tissue factor C-terminal domain 186–209 surface, an ÔoxidizedÕ form in which the Cys130–Cys159 Plasmin(ogen) Catalytic domain 680–747, 737–765 )RHStaple disulfide bond is intact, a ÔreducedÕ form defined Vitronectin Somatomedin B domain 5–9, 21–32 by a cleaved Cys130–Cys159 bond [20], and a disulfide-linked Glycoprotein 1ba N-terminal b finger 4–17 ÔdimerÕ in which the reduced second domains of adjacent Integrin b3 b Tail domain 663–687 molecules swap and the unpaired Cys residues reform across Thrombomodulin EGF-5 399–407 Fibrinogen Interchain 161a–135c, 165a–193b the domains of the two molecules to stabilize the swap [21]. The tPA Catalytic domain 421–496 dimer is the main coreceptor for MHCII [21], whereas the uPA Catalytic domain 197–268, 293–362 monomeric forms of CD4 appear to mediate entry of HIV into uPA Receptor Domains 1 and 2 6–12, 153–170 the cell (L. J. Matthias and P. J. Hogg, unpubl. obs.). Cleavage *Numbering for the mature protein. of the )RHStaple disulfide bond is mediated by an oxidore- EGF, epidermal growth factor; tPA, tissue plasminogen activator; ductase. Thioredoxin, which is secreted by T cells, is the most uPA, urokinase plasminogen activator.

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Table 2 Features of the tissue factor disulfide bonds* It has been recognized for some time that TF activity on the Disulfide unperturbed cell surface is regulated by a post-translational ) Domain bond Configuration DSE (kJ mol 1)Ca–Ca¢ (A˚ ) event [28]. TF can be de-encrypted in vitro by membrane disruption via freeze–thaw [29,30], exposing cells to calcium ) à N-terminal 49–57 RHSpiral 12.3 ± 0.9 6.11 ± 0.06 ionophores [26,31,32], or exposure to thiol alkylators such as +/)RHSpiral 11.5 ± 0.4§ 6.05 ± 0.04 C-terminal 186–209 )RHStaple 15.2 ± 0.7– 3.89 ± 0.05 HgCl2 and N-ethylmaleimide [26,33]. It does not appear to require proteolysis of TF. A number of studies have indicated *The 2HFT X-ray structure has been omitted from this analysis be- that there are phosphatidylserine-independent as well as cause of the unusual state of the 49–57 disulfide bond, which was found in bound and cleaved forms in the crystal structure. phosphatidylserine-dependent elements to de-encryption Numbering for the mature protein. [26,33–36]. The phosphatidylserine-independent step is a func- àThe DSE and distance between the a carbon atoms (± SE) of the tion of the extracellular domain of TF. Ionophore-mediated disulfide bond were calculated from eight X-ray structures from PDB de-encryption of TF procoagulant activity was equivalent in IDs 1BOY, 1DAN, 1FAK, 1TFH, 1W2K, 2A2Q and 2AER. Both BHK, mouse fibroblast and human glioblastoma cell lines molecules of the crystal dimer of 1TFH are included in this analysis. §The values were calculated from eight X-ray structures from PDB IDs expressing full-length or cytoplasmic domain-deleted TF in the 1J9C, 1JPS, 1W0Y, 1WQV, 1WSS, 1WUN, 1WV7 and 1Z6J. presence or absence of phosphatidylserine blockade with –The values were calculated from 16 X-ray structures from PDB IDs annexin V [36,37]. 1BOY, 1DAN, 1FAK, 1J9C, 1JPS, 1TFH, 1W0Y, 1W2K, 1WQV, Several lines of evidence suggest that the post-translational 1WSS, 1WUN, 1WV7, 1Z6J, 2A2Q and 2AER. Both molecules of the event associated with de-encryption takes place in the vicinity crystal dimer of 1TFH are included in this analysis. DSE, dihedral strain energy. of the Cys186–Cys209 disulfide bond. Encrypted TF is characterized by slow binding of FVIIa and inefficient cleavage of FX to FXa, but efficient cleavage of a tripeptidyl substrate which connects the second fibronectin type III domain with that interacts only with the active site of FVIIa [34]. This the transmembrane domain [27] (Fig. 1). The allosteric bond observation suggests that de-encryption is not a result of a is exposed to solvent and cleavable by dithiothreitol in the change in the interaction of FVIIa with the N-terminal domain crystal structure [27]. Both TF disulfide bonds are conserved in of TF [38]. Scanning alanine mutagenesis studies have identi- species from trout to mammals (V. M. Chen and P. J. Hogg, fied several residues (Tyr157, Lys159, Ser163, Gly164, Lys165, unpubl. obs.). Lys166, Tyr185) near the Cys186–Cys209 bond that are important for procoagulant activity [39,40]. In addition, the )RHStaple disulfide bond constrains a WKS motif at residues 158–160 that is important for procoagulant function [41]. Furthermore, a monoclonal antibody that binds in the vicinity N-terminal of the allosteric disulfide bond recognizes cryptic but not domain coagulant TF [42]. An intact Cys186–Cys209 bond is important for TF procoagulant activity. Elimination of the Cys186–Cys209 bond of cellular TF by mutating both Cys residues to Ser or Ala Cys49-Cys57 considerably reduces activation of the macromolecular sub- –RHSpiral strates FX and FIX [42,43], but does not impair FVIIa amidolytic activity [42]. Elimination of the N-terminal disulfide bond, in contrast, has no effect on either activity [42,43]. Using a biotin-linked thiol alkylator, it was shown that unpaired Cys thiols exist in cryptic TF and are reduced or lost upon de-encryption with a calcium ionophore or brief exposure

to thiol-oxidizing doses of HgCl2 [26]. Moreover, the de- Cys186-Cys209 encryption could not be explained by exposure of phosphat- –RHStaple C-terminal idylserine on the outer leaflet of the plasma membrane [26]. domain Evidence of a role for the unpaired Cys thiols in TF de- encryption is provided by the observation that pretreatment of cells with the thiol-alkylating compounds N-ethylmaleimide or methyl methanethiosulfonate blocks TF activation by calcium

ionophore or HgCl2 [26]. The finding that dithiol crosslinkers can activate TF implies that at least two Cys thiols are involved Fig. 1. Structure of the extracellular part of tissue factor (TF) and in de-encryption [26]. features of the two disulfide bonds. The TF structure is that from the complex with p-aminobenzamidine and FVIIa [78], which has a resolution The allosteric disulfide bond is also involved in TF signaling. of 1.8 A˚ . The two disulfide bonds are shown in yellow. The figure was An intact Cys186–Cys209 disulfide bond is required for high- generated using PyMol (40). affinity binding of FVIIa and full coagulant as well as ternary

2006 International Society on Thrombosis and Haemostasis 2536 V. M. Chen and P. J. Hogg

TF–FVIIa–FXa complex signaling through PAR2 activation Plasmin(ogen) [42]. Binary TF–FVIIa complex signaling, on the other hand, is sensitive to thiol blockade [42]. Further insight into the role of Plasminogen is a serine protease zymogen that is activated by the C-terminal disulfide bond in TF signaling was obtained urokinase or tissue plasminogen activator by proteolysis at a when the Cys residues were ablated individually. Mutating discrete site in the protease domain. Plasmin functions in clot Cys209 to Ala resulted in TF with lower affinity for FVIIa, lysis and cell migration, and is the precursor for an inhibitor of although the complex still activated PAR2. Mutating Cys186 angiogenesis known as angiostatin [46]. Plasmin contains five to Ala, in contrast, disabled both the coagulant and signaling kringle domains followed by a serine protease module. Tumor functions. These findings indicated that an available thiol at cells process plasmin to release angiostatin fragments consisting 1 Cys186 is required for binary TF–FVIIa complex signaling and of kringle domains 1–42, 1–4 and 1–3 [47–49]. The first event in implies that cleavage of the allosteric disulfide bond also release of angiostatin from plasmin is cleavage of two disulfide controls TF signaling. bonds, Cys462–Cys541 and Cys512–Cys536, in the fifth kringle The Cys186–Cys209 disulfide bond appears to be cleaved by domain. Cleavage of these bonds is facilitated by phosphogly- the oxidoreductase PDI [42]. This is analogous to cleavage of cerate kinase, which is secreted by tumor cells [50]. the CD4 )RHStaple by thioredoxin [20]. PDI contains two The mechanism by which phosphoglycerate kinase facilitates thioredoxin-like domains, each with a +/)RHHook catalytic cleavage of the kringle 5 disulfides is not known. Phosphogly- disulfide bond in a Cys-Gly-His-Cys motif. It is expressed on cerate kinase contains seven Cys residues, and none is involved the cell surface in proportion to cellular levels of the protein, in cleavage of the plasmin disulfide bonds [51]. We have and influences the redox state of surface protein thiols/ observed that plasmin(ogen) contains one or more redox-active disulfides [44]. The oxidoreductase has also been implicated disulfide bonds, and alkylation of the unpaired Cys with N- in the transfer of NO across the plasma membrane [45]. PDI ethylmaleimide blocks release of angiostatin from plasmin by coprecipitates with cryptic TF in HaCAT cells but not with hydroxide ion (T. Ganderton and P. J. Hogg, unpubl. obs.). coagulant TF. Moreover, small interfering RNA knockdown We hypothesize that reduction of the kringle five disulfides is of PDI expression increases coagulant activity [42], whereas mediated by the redox-active disulfide bond in plasmin, and stable overexpression of PDI suppresses coagulant activity (V. that phosphoglycerate kinase positively influences the catalytic M. Chen and P. J. Hogg, unpubl. obs.). PDI blockade with activity of this disulfide bond by binding to the enzyme. bacitracin inhibits binary TF–FVIIa complex signaling, and Plasmin contains two )RHStaple disulfide bonds in the washout of bactricin results in reassociation of PDI with TF protease domain, Cys680–Cys747 and Cys737–Cys765. The and full recovery of TF–FVIIa signaling [42]. These findings Cys680–Cys747 bond links the A2 and D2 b-strands, and imply that PDI cleavage of the TF allosteric disulfide bond the Cys737–Cys765 bond links the oxyanion-stabilizing loop to renders the cofactor cryptic while enabling binary TF–FVIIa the S1-entrance frame loop [52]. This disulfide bond, which is complex signaling (Fig. 2). largely conserved in the trypsin family, anchors the ends of the

NO is also involved in control of the redox state of the TF two loops during activation. It holds the S1-entrance frame allosteric disulfide bond. Cryptic but not coagulant TF is S- tight while allowing the oxyanion-stabilizing loop to change nitrosylated, and TF can be encrypted with S-nitrosoglutathi- conformation [52]. It is possible that the Cys680–Cys747 or one [42]. S-Nitrosylation of TF, therefore, appears to play a Cys737–Cys765 disulfide bond is the redox-active disulfide role in maintaining the cofactor in an encrypted state (Fig. 2). bond that we have measured.

Coagulant Cryptic Signaling

VIIa VIIa X

TF TF TF PAR2 PDI PDI HS SNO HS SH SS S S S S

Fig. 2. Proposed model for switching between cryptic, coagulant and signaling tissue factor (TF). A protein disulfide isomerase (PDI) active site thiol cleaves the TF allosteric disulfide bond, resulting in a mixed disulfide bond between the two proteins that renders TF coagulation cryptic. S-Nitrosylation of the unpaired TF thiol controls the stability of this complex. Denitrosylation of TF leads to cleavage of the disulfide bond holding the TF–PDI complex together, with formation of the TF allosteric disulfide bond and coagulant TF. Denitrosylation of TF can also lead to cleavage of protein-activated receptor 2 (PAR2) by the TF–PDI–FVIIa complex and signaling.

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The disulfide bonding of the eight Cys residues of the Vitronectin somatomedin B domain derived by proteolysis of plasma Vitronectin is found in the circulation, where it functions in blood vitronectin has a 1–2, 3–6, 4–7 and 5–8 pattern [54]. The coagulation and fibrinolysis, and in the extracellular matrix, where bonding pattern for the recombinant somatomedin B it plays a role in cell adhesion and migration [53]. Vitronectin binds domain expressed in Escherichia coli is 1–2, 3–4, 5–6 and the serine protease inhibitor plasminogen activator inhibitor 1 7–8 [55]. Three of the four disulfide bonds, therefore, are (PAI-1) and cellular receptors, including integrins and urokinase linking different Cys residues in the two forms of the plasminogen activator (uPAR). All three ligands bind to the protein. This degree of disulfide flexibility in a protein is N-terminal somatomedin B domain. This domain is about 50 very unusual. We have analyzed the disulfide bond geom- amino acids in length and contains eight Cys residues that etry and energy of the individual NMR models (40 in total) form four disulfide bonds. The disulfide bonding, however, is of the two forms of the protein (Table 3). different in plasma-derived vs. recombinant protein (Table 3). The DSEs of the disulfide bonds of the plasma protein are all of lower energy than the lowest-energy disulfide bond of the Table 3 Features of the vitronectin somatomedin B domain disulfide recombinant protein. In particular, the Cys5–Cys9 and Cys32– bonds Cys39 disulfide bonds of the recombinant protein have very high mean DSEs of 40.6 and 46.2 kJ mol)1, respectively. For Disulfide Overall DSE Overall ) Source bond* Configurations (kJ mol 1) Ca–Ca¢ (A˚ ) reference, the average DSE of 55 005 disulfide bonds in NMR structures is 26.5 kJ mol)1 (95% confidence intervals; 26.3– Plasmaà 5–9 +/)RHStaple (8) 13.1 ± 1.2– 5.13 ± 0.15– 26.6 kJ mol)1) (B. Schmidt and P. J. Hogg, unpubl. obs.). ) RHHook (3) There is a high degree of conformational flexibility in the )/+RHHook (3) +RHSpiral (3) geometry of the disulfide bonds in both forms of the protein. +/)RHHook (1) For instance, the Cys5–Cys9 disulfide bond exists in seven +/)RHSpiral (1) different configurations in the plasma protein and five config- )RHStaple (1) urations in the recombinant protein. A notable feature of the 19–31 +RHSpiral (8) 12.9 ± 1.1 5.92 ± 0.07 disulfide bonds is that three of the four in the plasma protein +/)RHSpiral (5) +/)LHHook (3) (Cys5–Cys9, Cys21–Cys32 and Cys25–Cys39) and two of the +RHHook (2) four in the recombinant protein (Cys5–Cys9 and Cys32–Cys39) )/+RHHook (1) can exist in allosteric )RHStaple or )LHStaple configurations. )/+LHHook (1) This feature is probably an important factor that contributes to ) 21–32 +/ LHStaple (10) 15.4 ± 1.1 4.38 ± 0.10 the disulfide flexibility of this protein. )LHStaple (9) )LHHook (1) The malleability of the disulfide bonds in the somatomedin B 25–39 +/)RHSpiral (11) 12.3 ± 1.0 5.76 ± 0.09 domain may help explain the conflicting results for PAI-1 )RHSpiral (3) effects on vitronectin activity ([54] and references therein). For )RHHook (2) instance, different disulfide bond isomers may bind PAI-1, +RHHook (1) integrins and uPAR with different affinities. Ligand binding +/)RHHook (1) +RHSpiral (1) may also trigger disulfide interchange, which controls further )RHStaple (1) ligand binding. Recombinant§ 5–9 )RHStaple (9) 40.6 ± 1.7 5.35 ± 0.09 )LHSpiral (7) )/+RHHook (2) Glycoprotein 1ba )LHStaple (1) +/)LHStaple (1) GPIb–IX–V is a complex of glycoproteins on the platelet 19–21 +/)LHStaple (14) 25.5 ± 1.2 5.34 ± 0.02 surface that binds von Willebrand factor (VWF) under shear +/)RHHook (5) conditions and mediates platelet adhesion to the subendothe- +/)RHStaple (1) lium of the disrupted vessel wall to initiate platelet clotting [56]. ) 25–31 /+RHHook (13) 18.7 ± 2.0 4.99 ± 0.03 GP1ba and GP1bb are covalently linked by disulfide bonds +/)LHStaple (7) 32–39 +/)RHSpiral (13) 46.2 ± 2.3 7.16 ± 0.02 and non-covalently associated with GPIX and GPV. The N- +/)LHStaple (4) terminal globular domain of GP1ba is the major ligand- )LHStaple (3) binding region, with partially overlapping binding sites for the *Numbering for the mature protein. A1 domain of VWF, a-thrombin, amb2 integrin, and P-selectin. Numbers in parentheses are the number of disulfide bonds with that The N-terminal domain consists of a b hairpin constrained by a configuration. disulfide bridge between Cys4 and Cys17, followed by eight àNMR structure of the somatomedin B domain of plasma vitronectin leucine-rich repeats of parallel b strands, and a C-terminal cap (1S4G, 20 models). containing two disulfide bridges between Cys209 and Cys248 §NMR structure of recombinant somatomedin B domain (1SSU, 20 models). and between Cys211 and Cys264. Residues Val227–Ser241 –The error is ±SE. form a large loop that plays a critical role in binding the VWF DSE, dihedral strain energy. A1 domain [57–59].

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A comparison of three X-ray structures of GP1ba indicated and extracellular domains enables crosstalk between the that whereas the leucine-rich repeats are relatively rigid, there is intracellular and extracellular environments [64]. considerable flexibility in the N-terminal b hairpin, the residue The aIIbb3 integrin is the most abundant integrin on the 227–242 loop, the region encompassing residues 249–254, and platelet surface. It binds fibrinogen, VWF, fibronectin and the C-terminal cap [58,59]. These regions, therefore, are most vitronectin to facilitate platelet adhesion and aggregation. likely to be involved in conformational change upon ligand During normal blood flow, the integrin is maintained in a binding. The Cys4–Cys17 disulfide bond is a potential allosteric resting or inactive state. Platelet stimulation by agonists such as disulfide bond. It has a )RHStaple configuration with a thrombin and ADP trigger inside-out signaling, leading to a characteristically short Ca–Ca¢ distance (Table 4). Comparison conformational change in aIIbb3 and ligand binding. There are of the three unliganded X-ray structures [58,59] with four 58 Cys residues in the b3 integrin subunit. NMR studies suggest structures of GP1ba cocrystallized with ligand (VWF or a- that the switch between the resting and active conformations of thrombin) [57,60–62] shows that the calculated strain energy of b3 and b2 integrin centers around the Cys-rich region of the C- the )RHStaple bond doubles when ligand is bound (Table 4). terminal domain, which forms a fulcrum for integrin rear- Free thiols appear in GP1ba following platelet activation, rangement on activation [65]. Most activating antibodies target and PDI is in close proximity to the receptor on the platelet epitopes in the Cys-rich region. surface [63]. In addition, incubation of resting platelets with Platelet activation results in a marked increase in the number anti-PDI antibodies followed by thrombin activation results in of cell surface protein thiol groups [63,66], and exposure of enhanced binding of three anti-GP1ba antibodies that recog- activated platelets to hydrophilic thiol-alkylating agents inhib- nize epitopes in the N-terminal domain (residues 1–73), but its ligand binding to a number of integrins, including aIIbb3, inhibits binding of a fourth monoclonal antibody that recog- a2b1 and avb3 [67,68]. In particular, platelet activation results in nizes residues 269–282 [63]. It is possible that platelet activation increased free thiols in integrin b3 [69]. The new thiols are in triggers cleavage of the Cys4–Cys17 )RHStaple disulfide bond close proximity, as their labeling is blocked by a trivalent by PDI and that straining of the disulfide bond upon ligand arsenical, which crosslinks closely spaced thiols to form stable binding may facilitate this cleavage. cyclic dithioarsinites [70]. This suggests that the new thiols in

integrin b3 are derived from reduction of a disulfide bond. This observation is concordant with the finding that dithiothreitol Integrin b3 activates aIIbb3 by cleaving a disulfide bond in b3 [71]. The Integrins reside on the cell surface in equilibrium between active unpaired Cys residues were mapped to the Cys-rich domain, and inactive conformations. Bidirectional signaling resulting in and Cys457 and Cys495 were implicated but not proven to be a series of reversible conformational changes in the cytoplasmic involved [71]. Notably, the Cys663–Cys687 disulfide bond in the b tail domain is a )RHStaple bond (Table 1) and ablation of this bond by mutating both Cys to Ala results in a Table 4 Features of the glycoprotein 1ba disulfide bonds constitutively active aIIbb3 [72]. Bound Disulfide DSE Platelet activation also triggers reduction of the active site )1 ˚ to bond* Configuration (kJ mol )Ca–Ca¢ (A) disulfide(s) of surface PDI [63]. Inhibition of PDI with Self 4–17– )RHStaple 7.7 ± 0.9 4.62 ± 0.10 bacitracin or anti-PDI antibodies partly recapitulates the 209–248** )LHHook 5.6 ± 0.5 5.59 ± 0.01 platelet aggregation blockade seen with thiol alkylation, as 211–264** )LHSpiral 5.6 ± 0.5 5.43 ± 0.06 well as blockade of granule secretion and P-selectin expression. IIaà 4–17 )RHStaple 15.2 ± 1.4 4.58 ± 0.06 These observations suggest that PDI is involved in the thiol/ ) 209–248 LHHook 6.2 ± 0.7 5.71 ± 0.04 disulfide regulation of integrin conformation [66,67]. In another 211–264 )LHSpiral 6.1 ± 0.7 5.47 ± 0.02 VWF§ 4–17 )RHStaple 12.8 ± 1.4 4.63 ± 0.03 parallel with control of TF function, exposure of activated 209–248 )LHHook 6.1 ± 0.4 5.54 ± 0.03 aIIbb3 to glutathione and the NO donor nitroprusside converts 211–264 )LHSpiral 4.8 ± 0.8 5.41 ± 0.07 theintegrintoitsrestingstate[73]. *Numbering for the mature protein.

The dihedral strain energy (DSE) and distance between the a carbon Thrombomodulin atoms (± SE) of the disulfide bond were calculated from three X-ray structures from PDB IDs 1P9A and 1QYY. Both molecules of the Thrombomodulin is a transmembrane receptor that binds crystal dimer of 1QYY are included in the analysis. thrombin and switches it from a procoagulant to an antico- àThe values were calculated from two X-ray structures from PDB IDs 1P8V and 1OOK. agulant by activating protein C, which then degrades activated §The values were calculated from three X-ray structures from PDB IDs FV and FVIII [74]. The extracellular part of thrombomodulin 1SQ0 and 1M0Z. Both molecules of the crystal dimer of 1M0Z are consists of a lectin-like N-terminal domain, followed by a included in the analysis. hydrophobic segment, six epidermal growth factor (EGF)-like – The 4–17 disulfide bond constrains the b hairpin in the N-terminal domains, and an O-glycosylated serine/threonine-rich domain. domain. **The 209–248 and 211–264 disulfide bonds are in the C-terminal cap Thrombin binds to EGF domains 4–6 [75]. of the N-terminal domain. EGF domains are about 40 amino acids in length and have VWF, von Willebrand factor. six Cys residues that form three disulfide bonds that are

2006 International Society on Thrombosis and Haemostasis Allosteric disulfide bonds in hemostasis 2539 typically bound in a 1–3, 2–4, 5–6 pattern. The EGF-5 domain particular, has a very high DSE of 41.4 kJ mol)1.Asforthe of thrombomodulin, however, has an usual disulfide bonding somatomedin B domain of vitronectin, there is a high degree of pattern [76,77]. Its disulfides are bound in a 1–2, 3–4, 5–6 conformational flexibility in the geometry of the disulfide pattern. The thrombomodulin EGF-4 domain has a conven- bonds. This is more apparent for the EGF-5 disulfide bonds. tional disulfide pattern. The disulfide bond geometry and The average number of disulfide configurations for the EGF-4 energy of individual NMR models (26 in total) of the EGF-4 bonds is four, compared to nine for the EGF-5 bonds. The and EGF-5 domains has been analyzed (Table 5). EGF-4 disulfide bonds are mostly of the spiral configuration, The most striking feature of the analysis is the generally high which is typical of structural bonds [8]. Two of the three EGF-5 energy of two of the three disulfide bonds in EGF-4 (Cys351– disulfide bonds, on the other hand, can exist in allosteric Cys360 and Cys372–Cys386) and all three disulfide bonds in )RHStaple or )LHStaple configurations. EGF-5 (Cys390–Cys395, Cys399–Cys407 and Cys409– The significance of this analysis of the EGF-5 disulfide bonds Cys421). The Cys399–Cys407 disulfide bond in EGF-5, in for the function of thrombomodulin is not known. Interest- ingly, however, EGF-5 can fold into several different disulfide- bonded isomers [78]. The 1–3, 3–4, 5–6 isomer binds to Table 5 Features of the thrombomodulin epidermal growth factor thrombin with higher affinity than the conventional 1–3, 2–4, (EGF)-4 and EGF-5 disulfide bonds 5–6 isomer. It may be, for example, that the allosteric Disulfide Overall DSE Overall configuration of the 3–4 and 5–6 disulfide bonds of EGF-5 ) Domain bond* Configurations (kJ mol 1) Ca–Ca¢ (A˚ ) confers a means of regulating thrombin affinity by allowing for disulfide interchange in the domain. EGF-4 351–360 +/)RHSpiral (4) 30.6 ± 3.0à 5.75 ± 0.13à )RHSpiral (3) +LHHook (2) Conclusions +RHHook (1) +/)RHHook (1) The current evidence indicates that the allosteric disulfide bond +/)LHSpiral (1) in the membrane-proximal domain of TF is the key to switching ) 356–370 LHSpiral (12) 13.3 ± 1.1 4.72 ± 0.05 of TF between cryptic, coagulant and signaling functions. The 372–386 +/)LHSpiral (7) 39.5 ± 4.4 5.78 ± 0.13 )LHSpiral (2) cleavage or formation of this disulfide bond appears to be )/+RHHook (2) controlled by PDI and NO. There are some interesting parallels )/+LHHook (1) between control of TF function and possible control of other EGF-5 390–395 +/)LHSpiral (8) 37.3 ± 2.2 5.53 ± 0.12 thrombosis and thrombolysis proteins by allosteric disulfide +RHHook (3) bonds. There is circumstantial evidence that the allosteric +RHSpiral (2) +/)LHHook (3) disulfide bond in the N-terminal b finger of GP1ba is cleaved by +LHHook (2) PDI upon ligand binding. A disulfide bond in integrin b3 also +/)RHSpiral (2) appears to be regulated by PDI and NO. It is likely that )LHSpiral (2) additional allosteric disulfide bonds will be identified as more ) /+RHHook (1) structures of hemostasis proteins are solved and more and better )LHHook (1) +/)RHStaple (1) tools for probing their function are developed. Their character- +/)LHStaple (1) ization will result in a more detailed understanding of the 399–407 +/)RHStaple (7) 41.4 ± 2.1 5.47 ± 0.09 mechanisms of thrombosis and thrombolysis. )LHHook (5) )RHHook (3) )/+LHHook (3) Acknowledgements +/)RHHook (2) +/)LHHook (1) We thank Bryan Schmidt for assistance with the data mining )RHSpiral (1) and Wolfram Ruf for many helpful discussions. +/)RHSpiral (1) +LHSpiral (1) +/)LHSpiral (1) Disclosure of Conflict of Interests )RHStaple (1) 409–421 )RHHook (15) 39.9 ± 1.9 5.67 ± 0.13 This study was supported by grants from the Australian )LHHook (4) Research Council, the National Health and Medical Research +/)RHSpiral (3) Council of Australia, the Cancer Council NSW and an ) +/ LHHook (2) infrastructure grant from the NSW State Government. )LHSpiral (1) )LHStaple (1)

*Numbering for the mature protein. References

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2006 International Society on Thrombosis and Haemostasis 2540 V. M. Chen and P. J. Hogg

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