Disulfide Bonds as Regulators of Integrin Function in Thrombosis and Hemostasis.

ANTIOXIDANTS & REDOX SIGNALING Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2014.6149

FORUM REVIEW ARTICLE

Disulfide Bonds as Regulators of Integrin Function in Thrombosis and Hemostasis Ronit Mor-Cohen 1,2

Abstract

Significance: Disulfide bonds are generally viewed as structure-stabilizing elements in proteins, but some display an alternative functional role as redox switches. Functional disulfide bonds have recently emerged as important regulators of integrin function in thrombosis and hemostasis. Recent Advances: Functional disulfide bonds were identified in the b subunit of the major platelet integrin aIIbb3 and in other integrins involved in thrombus formation that is, avb3 and a2b1. Most of these functional bonds are located in the four epidermal growth factor-like domains of the integrins. Redox agents such as glutathione and nitric oxide and enzymatic thiol isomerase activity were shown to regulate the function of these integrins by disulfide bond reduction and thiol/disulfide exchange. Critical Issues: Increasing evidence suggests that thiol isomerases such as protein disulfide isomerase (PDI) and Erp57 directly bind to the b3 subunit of aIIbb3 and avb3 and regulate their function during thrombus formation. aIIbb3 also exhibits an endogenous thiol isomerase activity. The specific functional disulfide bonds identified in the b3 subunit might be the targets for both exogenous and endogenous thiol isomerase activity. Future Directions: Targeting redox sites of integrins or redox agents and enzymes that regulate their function can provide a useful tool for development of anti-thrombotic therapy. Hence, inhibitors of PDI are currently studied for this purpose. Antioxid. Redox Signal. 00, 000–000.

and constitute targets for therapy of thrombosis and inflammation. Some of the disulfide bonds in integrins have been shown to have a functional role in regulating integrin function (88, 89, 150). Integrin aIIbb3 is the most abundant integrin in platelets and exerts an essential role in thrombus formation by mediating platelet aggregation (105, 115). aIIbb3 and other integrins involved in thrombus formation are subject to redox regulation involving functional disulfide bonds located mainly in their b subunit (37, 147). This review will focus on the current information on the regulation of integrin function by disulfide bonds in thrombosis and hemostasis.

Introduction

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isulfide bonds between cysteine residues have been generally considered to play a structural role in assisting protein folding and stability (123). Other disulfide bonds have been shown to play a functional role as redox switches that can switch between reduced and oxidized states, thereby regulating the function of the proteins they reside in and/or other proteins (48, 53). This redox regulation has emerged over the last decades as an important contributor to many physiological processes, including embryonic development, aging, diabetes, cancer, neurodegenerative, and cardiovascular diseases. Integrins are heterodimeric cell surface adhesion receptors consisting of a and b subunits that contain large numbers of disulfide bonds, especially in their b subunits. Integrins and their ligands play key roles in various processes, including vascular development, immune and inflammatory responses, hematopoiesis, hemostasis, thrombosis, and cancer (55, 56). Abnormalities in integrins stand at the basis of many diseases 1 2

Disulfide Bonds As Regulators of Protein Function

Disulfide bonds are formed by linking the sulfur atoms of two cysteine residues (the cystine residue). In mammalian cells, disulfide bonds are formed in proteins as they mature in the endoplasmic reticulum (ER), Golgi complex, postGolgi complex vesicles, and mitochondrial intermembrane

The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer, Israel. Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

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space (29). Usually, disulfide bonds cannot be identified by the location of the cysteines in the primary protein sequence. However, they can be detected by analysis of threedimensional X-ray and NMR protein structures or by mass spectrometry. Functional disulfide bonds

Most disulfide bonds have an impact on the structure of proteins; they assist in protein folding and stabilize the mature protein. This type of disulfide bond does not change throughout the life of the protein (123). However, some disulfide bonds have a functional role, as they can regulate protein function when they are cleaved (reduced) or form (oxidized) (48, 53). These disulfide bonds can be either allosteric or catalytic. Allosteric control is the change in one site that influences another site by utilizing the flexibility of the protein. Thus, reduction or oxidation of an allosteric bond results in a functional change at another site of the protein (86). Several types of change in protein function can be triggered by an allosteric bond reduction or oxidation, including changes in ligand binding, in substrate hydrolysis, in protein proteolysis, or in oligomer formation. The allosteric disulfide bonds can be modified by several means (26). They can be reduced or oxidized by oxidoreductases that contain catalytic disulfides at their active sites. These catalytic disulfides can be either reduced or oxidized and can reduce or oxidize an allosteric disulfide of a substrate protein. Allosteric disulfide bonds can be also modified by thiol/disulfide exchange when a conformational change in the protein brings the thiol of a free cysteine close to an allosteric disulfide bond where it cleaves the disulfide bond and creates a new bond. This process, also known as disulfide bond isomerization, can be catalyzed by oxidoreductases that have the ability to isomerize disulfide bonds, also known as thiol isomerases. In some cases, multiple thiol/disulfide exchanges occur in a cascade within the protein, and this rearrangement or ‘‘shuffling’’ of disulfide bonds can cause large-scale changes in the protein conformation as was shown for integrins and will be discussed next (38, 60). Functional disulfides can be also modified by low-molecular-weight (LMW) thiols, such as glutathione, that can form a mixed disulfide with the protein cysteine, that is, S-gluthanylation, and by reactive oxygen and nitric oxide (NO) species such as NO, which can react with a free cysteine, that is, S-nitrosylation (38).

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bonds at this time is their configuration. This is defined by the five chi (v) angles, which are the rotation around the bonds linking the six atoms of the two cysteine residues (Ca - Cb Sc - Sc¢ - Cb¢ - Ca¢) (Fig. 1) (112). There are 20 possible disulfide bond configurations, and all of them were identified in protein crystal structures. The functional disulfides have been associated with specific configurations. The most frequent functional configuration is the minus right-handed (RH) - RHStaple. The minus left-handed (LH) - LHHook, and - / + RHHook configurations are also associated with functional disulfides. About one quarter of disulfide bonds are - LHSpirals, so this bond configuration is representative of the structural disulfides (11, 26, 50, 112, 113). The configuration of disulfide bonds in proteins in the database, for which known structures are available, can be analyzed using the Disulfide Bond Analysis tool (49). Experimental identification of functional disulfides is based on differential cysteine labeling and mass spectrometry (11, 27, 42, 84). A recent study identified multiple redox-regulated disulfide bonds on the surface of cultured mouse T-cells in vitro and on the surface of mouse splenocytes during inflammation in vivo. The in vitro method was based on alkylating free thiols present on the cell surface with untagged maleimide derivatives before treating the cells with different reducing agents or thiol isomerases, followed by labeling the newly reduced thiols with biotinylated maleimide. The biotinylated cell surface proteins were purified and mass spectrometry was used for identification of the proteins and, when applicable, the specific disulfide bonds that were reduced in this process. In the in vivo study,

Identification of functional disulfide bonds

Given the essential role of functional disulfide bonds and redox regulation in many physiological processes, many efforts have been made to identify those functional disulfides and characterize their function. Bioinformatics and experimental data have been useful for identification of functional disulfide bonds. Functional disulfide bonds have common structural figures that can be extracted from the NMR and crystal structures in the Protein Data Bank (136). Some functional disulfide bonds are not stable and form in ‘‘forbidden’’ regions of primary and secondary structures, in which they exert strain on the protein structure (137–139). In addition, some functional disulfide bonds are surface exposed, which enables them to react with modifying agents and oxidoreductases (139). However, the most informative aspect of functional disulfide

FIG. 1. Disulfide bond configuration. (A) The configuration of a disulfide bond is defined by the five bond angles (v angles) linking the six atoms of the cystine residue. Ca is the main chain carbon atom, and Cb is the side chain carbon atom of each cysteine residue. The v angles are recorded as being either positive or negative. The three basic types of bond configurations (Spirals, Hooks, and Staples) are based on the signs of the central three angles, and they can be either RH or LH depending on whether the sign of the v3 angle is positive or negative, respectively. These six bond types expand to 20 when the v1 and v1¢ angles are taken into account. (B) Examples of the structures of the emerging allosteric configurations: - RHStaple, - LHHook, and - / + RHHook. Adapted with permission from Cook and Hogg (26). LH, left handed; RH, right handed.

INTEGRIN DISULFIDE BONDS IN THROMBOSIS AND HEMOSTASIS

mice were treated with lipopolysaccharide (LPS), a strong inflammatory inducer, and splenocytes were purified from LPStreated and control mice. The splenocytes were labeled with biotin-maleimide, and cell surface proteins were purified and analyzed as in the in vitro assay. Identified proteins include activating and inhibitory receptors, antigen presenting, intracellular transport, cell adhesion receptors, and integrins. Thus, this approach proved useful for identifying proteins regulated by functional disulfide bonds and the functional disulfide bonds themselves (84). Disulfide Bonds in Integrin Structure and Function The integrin family

Integrins are a central class of cell surface adhesion receptors that mediate cell–matrix and cell–cell interactions. Integrin family members are heterodimeric receptors formed by noncovalent association of a and b subunits. The family of mammalian integrins comprises 8 b and 18 a subunits that assemble into 24 distinct integrins which can be divided according to their ligand binding specificity and expression in specific cells. One set of integrins recognizes the tripeptide sequence RGD in molecules such as fibrinogen, fibronectin, and vitronectin. Another set mediates adhesion to basement membrane laminin. There is also a set of collagen receptors, a pair of related integrins (a4b1, a9b1) that recognize both extracellular matrix proteins and Ig-superfamily counter receptors such as VCAM and a set of leukocyte-specific receptors which also recognize Ig-superfamily counter receptors (56) (Fig. 2). Integrin structure and function

Integrins have been intensively studied over the last three decades since the recognition of the integrin receptor family (55). The understanding of integrin structure and function has particularly advanced since the elucidation of the crystal structure of integrin avb3 followed by the elucidation of aIIbb3 and axb2 crystal structures (142–144, 146, 152). The a and b subunits of integrins consist of a single transmembrane (TM) domain, a large extracellular domain, and a small cytoplasmic tail (CT) of *20–70 residues. The extracellular domain of integrins consists of a large globular ‘‘head’’ region formed by a and b N-terminal domains that contain the ligand binding sites, followed by two a and b ‘‘legs’’. The

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cytoplasmic domains anchor to the cytoskeleton and signaling proteins, enabling the integrins to link the exterior and the interior of the cells, thereby mediating cell adhesion, spreading, and migration (56). Integrins are signaling receptors that can signal bidirectionally across the plasma membrane. Intracellular signals can change the conformation of integrins and increase their affinity for ligands, a process known as ‘‘inside out signaling.’’ Ligand binding to the integrin head transmits signals into the cell, which induces cascades of intracellular events, including clustering of integrin receptors, phosphorylation of proteins, and cytoskeleton rearrangement, a process known as ‘‘outside in signaling’’ (56, 115). Integrins involved in thrombosis and hemostasis

Thrombus formation occurs after vascular injury, when the exposed subendothelial matrix activates platelets and initiates, along with coagulation, the production of fibrin from fibrinogen. Platelets adhere to the site of vascular damage, recruit and activate additional platelets to form platelet aggregates, and, finally, retract along with the fibrin polymers that stabilize the platelet plug until wound healing occurs (57). Integrins and other receptors on the surface of platelets play an essential role in this process. Platelets express six integrins on their surface: aIIbb3, avb3, a2b1, a5b1, a6b1, and aLb2. Most of these integrins contribute, along with other adhesion receptors, to platelet adhesion to the exposed subendothelial matrix, which is the initial step in platelet thrombus formation. Some integrins were also shown to play an important role in the sequential steps of thrombus formation, including downstream signaling and activation of platelets, platelet aggregation, and fibrin clot retraction. The most important and abundant platelet integrin is aIIbb3, also known as GPIIb-IIIa complex. This is the only integrin that is uniquely expressed in platelets and is the major platelet receptor with 80,000 to 100,000 copies per platelet (94, 118, 129). The aIIb and b3 subunits of the receptor are the products of the ITGA2B and ITGB3 genes, respectively, both of which are located on chromosome 17 (117). aIIbb3 is activated after platelet activation, enabling it to bind its ligands fibrinogen, fibrin, and von Willebrand factor (vWF) and to carry out its main role in thrombus formation: platelet aggregation. aIIbb3 engagement by its ligands is also important for platelet adhesion and spreading on

FIG. 2. The mammalian integrin family. The mammalian 8 b and 18 a subunits can form 24 distinct integrins that can be separated by their ligand specificity and expression in specific cells. There are integrins that recognize the tripeptide sequence RGD (blue), integrins which bind collagen (yellow), integrins that bind laminin (green), and leukocyte-specific integrins (pink). a4b1 and a9b1 are related integrins that recognize both extracellular matrix proteins and Ig-superfamily counter receptors such as VCAM. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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damaged extracellular matrix, and for fibrin clot retraction (57, 104, 105, 115). The crucial role of aIIbb3 in platelets’ thrombus formation is well demonstrated by its absence in the hereditary disorder Glanzmann thrombasthenia (GT), which is associated with a severe bleeding tendency. Importantly, aIIbb3 became a major target for antithrombotic therapy in patients with coronary arterial thrombosis. A remarkable progress in revealing aIIbb3 structure and function was achieved after the elucidation of the ectodomain crystal structure of its homologous integrin avb3, both sharing the same b3 subunit, and the complete ectodomain crystal structure of aIIbb3 (143, 144, 152). Integrin aIIb and b3 subunits can be divided into several structurally defined domains (Fig. 3A). The aIIb ectodomain consists of the bpropeller domain, which along with the b A-domain in the b3

FIG. 3. aIIbb3 domain structure. (A) A model of the ectodomain of integrin aIIbb3 in the open conformation manually extended at the a and b knees. The domains comprising the aIIb and b3 subunits are shown. (B) Integrin aIIbb3 X-ray structure (PDB 4O02) of the closed conformation. The figure was prepared using UCSF Chimera (100). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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subunit provides the ligand binding sites. The remaining extracellular portion of aIIb consists of the thigh, calf-1, and calf-2 domains. The b3 subunit consists of the N-terminal b A-domain, hybrid domain, plexins, semaphorins, and integrins (PSI) domain; four tandem epidermal growth factor (EGF)-like domains (EGF-1, 2, 3, and 4) that are rich in cysteines; and a unique b-tail domain (bTD). The structures of the TM and the CT domains of aIIb and b3 were determined by NMR and were shown to interact with each other through their TM and membrane proximal CT regions. Unclasping of these interactions by intracellular signals that cause binding of cytoplasmic proteins to aIIb and b3 CTs, followed by rearrangement of the extracellular region of the integrin, is the basis for physiological agonistinduced aIIbb3 activation (83, 124, 126–128). Activation of aIIbb3 can be also induced by other pathways that do not involve intracellular signaling and CTs. These pathways include activation by small agonists containing the ligand mimetic RGD motif (4, 122), activation by Mn2 + (122), activation by antibodies to ligand-induced binding sites (LIBS) epitopes in the extracellular domain of the receptor (31, 41), activation by small peptides (24, 30, 95, 98), and activation by reducing agents such as dithiothreitol (DTT). The latter pathway will be discussed in the next chapter. Surprisingly, both aIIbb3 and avb3 integrins were crystallized in a bent conformation, in which the headpiece is folded over the legs, with each leg bent at a ‘‘genu’’ or knee. The a genu lies at the junction between the thigh and calf-1 domains and the b genu at the EGF-2 domain (Fig. 3B) (144, 149, 152). Many studies indicated that this extremely bent conformation represents the low affinity state of the integrins and that integrin activation involves a switchblade-like opening of the ectodomain at the knee fulcrum, resulting in its extension along with the opening of the integrin head region which enables ligand binding (6, 122, 151, 152). Other studies, however, showed that such large-scale conformational change is not required for ligand binding (14, 143, 145). Moreover, several intermediate conformations have been proposed to be in equilibrium between the extreme bent and extended conformational states, which can have different affinities for the ligand (122, 141, 144). Thus, there is no simple known relationship between the conformation and affinity of integrins at this time. avb3, known as the vitronectin receptor, is present on platelets in small amounts (several hundred copies per platelet) and its function in platelets is unclear. GT patients with a defect in the b3 subunit do not express aIIbb3 and avb3, but do not appear to be more seriously affected than GT patients who have a defect in the aIIb subunit and do not express only aIIbb3 (25). In contrast to platelets, endothelial cells express large amounts of avb3 and on vascular injury, its surface expression increases (109). avb3 was shown to mediate fibrin clot retraction when expressed in mammalian cell lines (20, 87, 111), suggesting that during vascular injury, avb3 in endothelial cells can contribute to clot retraction mediated by platelets aIIbb3, especially at the edges of the injury, and promote closure of the wound corners. Endothelial avb3 was also suggested to play a role in fibrin formation during vascular injury by the binding of protein disulfide isomerase (PDI) as will be discussed later (23). The second most important platelet integrin after aIIbb3 is a2b1, with 2000–4000 copies per platelet. a2b1 and GPVI are


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the major collagen receptors in platelets. Similar to aIIbb3, a2b1 needs to be activated to bind collagen with a high affinity. It was suggested that platelets first bind collagen by GPVI and then when the platelets become activated, a2b1 binds collagen and strengthens the adhesion. Adhesion of a2b1 to collagen also induces outside-in signaling in platelets that supports platelet responses (62–64). b1 and a2 null mice show a relatively normal phenotype with normal adhesion to collagen but defective platelet aggregation in response to soluble collagen (15, 68, 92). Humans with defective or lack of a2b1 exhibit defective aggregation and adhesion to collagen associated with prolonged bleeding time and thrombocytosis (45). a5b1 and a6b1 are thought to be the fibronectin and the laminin receptors on platelets, respectively, and to have a supplementary role in platelet adhesion at the injury site (103, 116). aLb2 was shown to be expressed on the surface of activated, but not resting platelets and to play a role in platelet localization to local inflammation sites (101, 102). The effect of disulfide bonds on integrin structure and function

All integrins contain a large number of cysteine residues located in their ectodomains that are highly conserved. These cysteines were generally believed to be engaged in disulfide bonds that are structurally important for protein folding and stabilization of the integrins. The disulfide bonds in integrins are not uniformly distributed but are located mainly in the b subunit where they are concentrated in the four integrin EGFlike domains. Elucidation of the aIIbb3 crystal structure revealed that this integrin contains 18 cysteines in its aIIb subunit and 56 cysteines in its b3 subunit, of which 30 are in the EGF region (Fig. 4). Each of the four b3 EGF domains contain eight cysteines that form four disulfide bonds in a distinctive pattern of 1–5, 2–4, 3–6, and 7–8, except for EGF-1, which lacks the 2–4 disulfide bond (143, 152). The cysteines engaged in the 2–4, 3–6, and 7–8 bonds, and the disulfide bonds themselves are highly conserved not only in the EGF domains of integrins but also in other EGF domains, suggesting that they are important for the EGF structure. In contrast, the 1–5 bonds are absent in other proteins’ EGF domains, and so are unique for b integrins (6, 121) (Fig. 5). It has become recently evident that disulfide bonds and unpaired cysteines in b subunits of integrins, and particularly in the EGF domains, play a role in regulating integrin function. Most studies were performed with aIIbb3 integrin, but other integrins were also shown to be regulated by functional disulfides. Evidence supporting these observations is presented next. Labeling thiols in aIIbb3 and avb3. Earlier, the general concept has been that all cysteines in aIIbb3 are engaged in disulfide bonds (5, 13, 93). More recently, studies revealed that both aIIb and b3 contain thiols that can be labeled by membrane-impermeable thiol-modifying agents (37, 39, 78, 147), implying that some cysteines in aIIbb3 might be presented as free thiols. Using the maleimide reagent 1biotinamido-4-[4¢-(maleimidomethyl)-cyclohexanecarbox amido]butane (biotin-BMCC), both subunits of purified aIIbb3 were labeled but only the b3 subunit was labeled in intact platelets. While there were 2–3 free thiols in the purified resting conformer of the integrin, there were 4–5 free thiols in the purified active conformer, implying that

FIG. 4. Distribution of cysteines in the b3 subunit. A model of the b3 open structure based on its crystal structure (PDB 3IJE, residues 1–695). The structure was extended by torsion at the b-knee, between EGF-1 and EGF-2 domains. The structure is presented in ribbons, and the cysteine residues are exhibited as spheres. The figure was generated by UCSF Chimera (100).

reduction of disulfide bonds is associated with aIIbb3 activation. The labeled thiols in the b3 subunit were localized to a 30 kDa region containing residues 450–650 that contain the cysteine-rich EGF domains (147). Disruption of aIIbb3 integrin complex by ethylenediaminetetraaceticacid (EDTA) was required to obtain optimal labeling of thiols in aIIb and b3 by 3-(N-maleimido-propionyl)biocytin (MPB) (37). Since EDTA does not, by itself, generate new thiols, the increased labeling suggests that cryptic thiols which are sterically hindered from the labeling reagent were exposed after disruption of the integrin. Using 4-(chloromercuri) benzenesulfonic acid (pCMBS), labeling of aIIb and b3 was obtained even without disruption of the aIIbb3 complex (78). The b3 subunit of avb3 was also labeled by MPB after stimulation of endothelial cells and activation of avb3 with Mn2 + , suggesting that disulfide bond reduction and thiol formation in the b3 subunit is a general mechanism for both aIIbb3 and avb3 activation (120). Taken together, these studies imply that some disulfide bonds in aIIbb3 and avb3, and specifically in the b3EGF domains, are not always bonded and appear as thiols that serve as redox regulators of integrin function. The effect of reducing and thiol-blocking agents on integrin function. Earlier studies showed that reducing agents in-

fluence aIIbb3 integrin function. The reducing agent DTT in concentrations above 1 mM caused slow progressive platelet

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FIG. 5. Sequence alignment of EGF domains of integrins and of other proteins. Sequence alignment of the four EGF domains of human b3 and human b2 and three representative EGF domains of other proteins are shown. Eight conserved cysteines are labeled in gray. The pairs of cysteines that form disulfide bonds are shown by black lines above the alignment. The disulfide bond 1–5 is unique among integrins and is labeled in a black box. The protein structures were aligned using Multiprot (114). The sequence alignment, based on the structural alignment, was generated using JalView (134). EGF, epidermal growth factor. aggregation in the presence of fibrinogen (77). DTT and 2mercaptoethanol induced fibrinogen binding and platelet aggregation in normal platelets but not in platelets from GT patients that lacked aIIbb3 (153). Yan and Smith (147, 148) showed that DTT can activate purified aIIbb3 and also cause slow progressive platelet aggregation by reducing disulfide bonds in b3. DTT treatment increased the amount of labeled thiols from a mean of 2.5 in the inactive integrin to a mean of 6.6 in the DTT activated integrin. These thiols were localized to the cysteine-rich EGF region of b3, suggesting that reduction of two disulfide bonds in this region is associated with aIIbb3 activation. Reduction of disulfide bonds in b1 and b2 integrins by DTT activated those integrins (21, 28, 32, 91), suggesting a role for disulfide bond reduction in the activation of these integrins as well. Disulfide bond reduction by ultraviolet light C irradiation was also shown to induce aIIbb3 activation, as well as a4b1 and a5b1 activation (125). On the other hand, blockade of free thiols by various thiol-modifying agents, including biotin-BMCC (147), Nethylmaleimide (81), dithiobisnitrobenzoic acid (70, 89), and pCMBS (70, 78), inhibited aIIbb3 activation and platelet aggregation induced by physiological agonists, Mn2 + , activating antibodies and peptides, or DTT, showing that free thiols are necessary for aIIbb3 activation by various signalingdependent and -independent pathways. The observation that these reagents can block DTT activation of aIIbb3 implies that the reduction of disulfide bonds is not, by itself, sufficient to induce aIIbb3 activation, but rather requires an additional thiol/disulfide exchange step. Furthermore, a set of comprehensive studies revealed that enzymatically catalyzed disulfide bond exchange is also necessary for integrin outside-in signaling events following by ligand binding, including sustained fibrinogen binding to aIIbb3, adhesion of aIIbb3 to fibrinogen, and adhesion of a2b1 to collagen (69–71). Disulfide bond exchanges are also necessary for aIIbb3- and avb3-mediated fibrin clot retraction, a process that requires both integrin activation and post-ligation outside-in signaling (87). Taken together, those studies suggest that disulfide bond reduction and thiol/disulfide bond exchanges are involved in the activation and signaling of integrins. Cysteine substitutions in the EGF domains and bTD of b3 integrins. Substantial evidence pointing at the involvement

of disulfide bonds in integrin function has been provided by studies of mutations in cysteines engaged in disulfide bonds. Several naturally occurring mutations that disrupt disulfide

bonds in the EGF domains of b3 (Cys549Arg in EGF-3 and Cys560Arg, Cys560Phe, and Cys598Tyr in EGF-4) were identified in GT patients. The patients’ aIIbb3 was constitutively active, suggesting that these bonds are important for aIIbb3 activation (1, 17, 90, 108). Several artificial cysteine substitutions in the EGF region were also shown to result in constitutively active aIIbb3 when the mutated integrins were expressed in mammalian cell lines. A systemic mutagenesis of 29 cysteine residues in the b3 subunit revealed that all cysteine mutations within the EGF domains of b3 activated aIIbb3, while cysteine mutations in other regions did not (65). These results led the authors to the conclusion that all disulfide bonds in the EGF domains of b3 are important for keeping aIIbb3 in an inactive state with no unique cysteine that is critical for aIIbb3 activation. However, the authors mutated only one of the cysteines from each cysteine pair forming the disulfide bonds, assuming that mutating the second cysteine or both cysteines in the pair would have the same effect. In two later studies by our group, disulfide bonds in the EGF domains and the subsequent bTD of b3 were disrupted by mutating each of the cysteine of the pair, as well as both cysteines to serine. These studies revealed that in some disulfide bonds, mutating one cysteine resulted in integrin activation, while mutating the partner cysteine or both cysteines in the pair did not. These results show that it is not the opening of these bonds, but rather the existence of a thiol in one of the cysteines in the bond which induces the activation process, suggesting that these bonds have a regulatory role in the activation of the integrin (88, 89). Altogether, the mutational studies show that disulfide bonds in the b3 EGF domains and bTD have different roles in integrin structure and function: some disulfide bonds play a fundamental structural role. They stabilize the protein folding, and their disruption strongly impairs aIIbb3 expression. Other disulfide bonds are important for the stabilization of the integrin inactive conformer, and their disruption results in constitutive activation of aIIbb3. There are also specific disulfide bonds that have a regulatory role in integrin activation involving disulfide bond exchange. The suggested role of the disulfide bonds in the b3 EGF domains and bTD is summarized in Figure 6. Notably, several disulfide bonds have more than one role; the Cys549– Cys558 bond, for example, which is the 7–8 bond in EGF-3, has two roles: (i) It is structurally important for stabilizing the EGF-3-fold, and its disruption, either by the natural mutation Cys549Arg or by an artificial mutation Cys549Ser,


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FIG. 6. Suggested role of disulfide bonds in the b3 EGF domains and bTD. Human integrin b3 EGF domains and bTD are presented as ribbons (PDB 3IJE). Cysteine residues are shown by spheres and colored according to their suggested roles in integrin structure and function. Disulfide bonds for which there are enough data to implicate their role are in solid colors. Uncertain function of bonds is transparent. Bonds for which there are no available data or the data could not point to a specific role are not colored. Yellow: Structural; Blue: Stabilization of the inactive conformer; Red: Regulatory role; Green: Structural and stabilization of the inactive conformer (Yellow + Blue); Violet: Regulatory role and stabilization of the inactive conformer (Red + Blue); Brown: Enables activation. The figure was generated using Pymol (http://pymol.org/). bTD, b-tail domain. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars profoundly reduces its surface expression. (ii). It stabilizes the inactive conformer of the integrin, and its disruption by the same mutations results in constitutively active integrin (90). This bond has the common - LHSpiral configuration (Table 1), and it is highly conserved in other EGF domains,

Table 1. Classification of Disulfide Bonds in the b3 Subunit of aIIbb3a by Their Configuration Domain

Cys1

Cys2

Configuration

Possible role

PSI

5 13 26 177 232 374 406 437 448 462 473 486 495 508 523 528 536 549 560 567 575 588 601 608 614 617 663

23 435 49 184 273 386 433 457 460 471 503 501 506 521 544 542 547 558 583 581 586 598 604 655 635 631 687

- LHSpiral + / - LHSpiral + RHSpiral - / + RHHook - RHHook - LHSpiral + / - RHSpiral - / + RHHook - LHSpiral - LHSpiral - / + RHHook - RHSpiral - LHSpiral - LHSpiral - LHHook - RHSpiral - LHSpiral - LHSpiral - LHHook + / - RHSpiral - LHSpiral - LHSpiral - RHHook + / - LHSpiral - / + RHHook - / + LHHook - RHStaple

Structural Structural Structural Allosteric Structural Structural Structural Allosteric Structural Structural Allosteric Structural Structural Structural Allosteric Structural Structural Structural Allosteric Structural Structural Structural Structural Structural Allosteric Structural Allosteric

bI Hybrid EGF-1 EGF-2

EGF-3

EGF-4

Ankle bTD

a According to aIIbb3 crystal structure, PDB 3FCS (152). bTD, b-tail domain; EGF, epidermal growth factor; LH, left handed; PSI, plexins, semaphorins and integrins; RH, right-handed.

consistent with its role in stabilizing the integrin EGF-3 structure and the integrin inactive conformer. Three disulfide bonds have a proposed regulatory role in integrin activation; two of them, Cys523–Cys544 and Cys560–Cys583, are the integrin unique 1–5 bonds in the EGF-3 and the EGF-4 domains, respectively, and one, Cys608–Cys655, is the first disulfide bond in the bTD. The integrin unique 1–5 bonds are present only in integrins and not in EGF domains of other proteins, suggesting that they are not important to the structure of the EGF fold, but rather have an effect on integrin function. This notion is also supported by structural data, showing that the 1–5 integrin unique bonds reduce the flexibility between the EGF domains relative to non-integrin EGF domains, by shortening the linker region between them (Fig. 5) (6, 121, 143, 152). Thus, these bonds control the rigidity of the interface between the EGF domains and can induce allosteric rearrangement of disulfide bonds that cause reorganization of the EGF domains relative to each other, affording transmission of a conformational change along the molecule (19). The fact that two of four integrin unique bonds exhibit properties of a functional bond supports this notion. Interestingly, a regulatory role in integrin activation was shown for the Cys560–Cys583 bond in both aIIbb3 and avb3 integrins, but for the Cys523–Cys544 bond a regulatory role was shown only for avb3; while in aIIbb3, a role in stabilizing the inactive conformer was implied. Substituting Cys523 or Cys544 by serine results in a constitutively active aIIbb3 and avb3, while substituting both Cys523 and Cys544 by serine (the C523S/C544S mutant) results in constitutively active aIIbb3 but not avb3. A molecular dynamics analysis revealed that the aIIbb3 C523S/C544S mutant stabilized at an active conformation with an extended angle between EGF-2 and EGF-3 domains, while the avb3 C523S/C544S mutant stabilized at a wild-type (WT)-like inactive conformation with no pronounced change in the orientation of the EGF-2/EGF-3 interface (88). This is the only disulfide bond currently known to exhibit such a pronounced functional difference between the two integrins when disrupted, showing that although the b3 subunit is the same in these integrins, it can be structurally affected by the different a subunits, resulting in different roles of the same disulfide bond between

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the two integrins. Notably, the configuration of this bond is also distinct for the two integrins: In avb3, it is - RHStaple, which is the most common allosteric configuration. In aIIbb3, it is - LHHook, which was also classified as allosteric, implying that this bond also displays a functional role in aIIbb3. The Cys560–Cys583 bond is in the - LHHook allosteric configuration in both aIIbb3 and avb3, consistent with its functional role in both integrins (Table 1). Conceivably, the other two 1–5 unique bonds, Cys437–Cys457 in EGF-1 and Cys473–Cys503 in EGF-2, stabilize the inactive conformer of aIIbb3 and avb3 (88). Their configuration, - / + RHHook, has also been associated with allosteric disulfides (11), suggesting that they exert a functional effect yet to be discovered. The other non-integrin unique disulfide bonds in the EGF domains do not seem to have a functional role, but are rather structurally important for the EGF folding or for stabilization of the inactive conformer (65, 88, 89). Consistent with that, they have non-allosteric configurations. A unique role was suggested for the 2–4 Cys567–Cys581 in EGF-4 (Fig. 6). Disruption of this bond by mutations that disrupt each one of the cysteines or both cysteines in the pair resulted not only in reduced expression of aIIbb3 but also in prevention of its activation: All mutants were inactive and in contrast to WT integrins, could not be activated by activating antibodies. This implies that the integrity of this bond is essential for aIIbb3 activation (89). An important feature of a functional disulfide bond is its surface accessibility; high surface accessibility makes it accessible for reducing agents and thiol isomerases that can modify it. Notably, in all four integrin-unique disulfide bonds, at least one cysteine is relatively accessible, in agreement with their proposed functional role (Fig. 7). The Cys560–Cys583 bond has the highest surface accessibility with both cysteines in the bond exposed to solvent; reinforcing the notion that it has a regulatory role in integrin function. In contrast, the other non-unique disulfide bonds have a relatively poor surface accessibility, in agreement with their structural role.

FIG. 7. Surface accessibility of cysteines in the b3 EGF domains and bTD. The structure of human b3 integrin is shown by white ribbons and solvent accessible surface (PDB 3IJE). Atoms of the cysteines that form disulfide bonds among the EGF domains and bTD are shown by spheres. They are colored by their relative surface accessibility to water, as calculated by PSAIA (85): The most accessible side chain is in dark red, and the least accessible side chain is in light pink. C1– C5 bonds in each domain are labeled by residue number. The figure was generated using Pymol (http://pymol.org/). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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The Cys608–Cys655 in the bTD is so far the only bond outside the EGF domains for which evidence of a regulatory role in integrin activation was provided. The bTD was shown to be flexibly connected to EGF-4 by two weak hydrophobic bonds. In addition, the EGF-4/bTD interface was suggested to be involved in activation of integrins, as it comprises the binding site for activation-sensitive antibodies against b3 (anti-LIBS2) and b2 (KIM185-activating antibody) (3, 76). The Cys608–Cys655 is relatively close to the EGF-4/bTD interface, and its disruption can cause conformational changes involving disulfide bond rearrangements that lead to integrin activation. Another disulfide bond in bTD that induces aIIbb3 activation when disrupted is the Cys663–Cys687 bond. Mutating cysteine 687 or both cysteine 663 and 687 to alanine yields constitutively active aIIbb3, suggesting that this bond has a role in stabilizing the inactive aIIbb3 conformer (12). This bond has an - RHStaple allosteric configuration (Table 1) and Cys687 is very exposed to solvent (Fig. 7), which suggests that it has a functional role. Cysteine substitutions in other b3 domains. Other cysteine substitutions located outside the EGF domains and bTD, Cys5Ala and Cys435Ala, induce aIIbb3 activation (119). These cysteines were previously thought to form a long range disulfide bond connecting the PSI domain to the first EGF domain. However, when the crystal structure of avb3 was published, it was realized that these cysteines do not form a disulfide bond with each other but rather form Cys5–Cys23 and Cys13–Cys435 disulfide bonds. Interestingly, substitution of cysteine 13 to glycine abrogates aIIbb3 formation, suggesting a structural role for this bond (99). However, mutation in its partner cysteine, Cys435Ala, did not impair integrin expression but rather activated it. Thus, the profound effect of the Cys13Gly mutation may be related to an effect on protein structure that is separate from its effect on the disulfide bond. Further investigation is needed to elucidate the role of the Cys5–Cys23 and Cys13–Cys435 disulfide bonds in aIIbb3 structure and function. Taken together, the evidence provided so far from mutational studies in aIIbb3 and avb3 suggests that disulfide bonds in the b3 subunit have various roles in integrin structure or function. They may stabilize the structure of the integrins, stabilize the inactive conformer of the integrins, or regulate their function. Cysteine substitutions in other integrins. Importantly, not only aIIbb3 and avb3 integrins contain functional disulfide bonds. In a set of cysteine-to-alanine substitutions in the b2 subunit and expression of the mutated aLb2 (LFA-1), aMb2 (Mac-1), and aXb2 (p150,95) integrins in COS-7 cells, it was shown that the integrins display enhanced adhesion to their ligands when the Cys514–Cys537 disulfide bond was disrupted (96). This bond is the 1–5 disulfide bond in the b2 EGF-3 domain and is homologous to the Cys523–Cys544 bond in b3 EGF-3, suggesting that this integrin unique bond is functional in both b3 and b2 integrins. A recent study showed that disruption of the b7 Cys494–Cys526 disulfide bond as well as the a4 Cys589–Cys594 disulfide bond resulted in increased ligand-binding affinity of the a4b7 integrin and outside-in signaling of the integrin independent of ligand binding (150). These bonds were also shown to be specifically reduced by low concentrations of DTT, giving rise to a unique non-extended active conformation of the


integrin that is different from the extended conformation of the integrin activated by inside-out signaling or Mn2 + . Cys494–Cys526 is the 1–5 integrin unique bond of b7 EGF2. It is homologous to the Cys473–Cys503 integrin unique 1–5 disulfide bond in b3 EGF-2 that was also shown to activate aIIbb3 on its disruption. A model structure of the a4b7 integrin showed that similar to the homologous cysteines in aIIbb3 and avb3, the b7 Cys494–Cys526 and the a4 Cys589– Cys594 disulfide bonds are located in the integrin knee, implying that the knee region and disulfide bonds in it are important for the regulation of integrin activation. The authors suggested a novel mechanism for integrin affinity and signaling regulation through reduction of the two disulfide bonds at the knees of integrin (150). Cysteine substitutions that elevate ligand binding affinity were also identified in the homologous b subunit from Drosophila (bPS), and were located in the EGF-1, EGF-3, PSI, and hybrid domain of bPS subunit (66). Thus, functional disulfide bonds in integrins are probably general regulators of integrin function. Redox Regulation of Integrin Function During Thrombus Formation

Various proteins are regulated by cleavage/formation of specific disulfide bonds, including blood proteins such as angiotensinogen, b2-glycoprotein I, interleukin receptor subunit c, plasminogen, vWF, tissue factor, and Factor XI (10). As mentioned earlier, functional disulfide bonds were shown to be involved in the regulation of integrin function and particularly in integrins involved in thrombus formation such as aIIbb3, avb3, and a2b1. It remains to be established whether and how these integrins are regulated by redox modulation during thrombus formation. Several studies suggested that physiological redox modulators act directly on integrins in platelets, thereby regulating platelet activation and thrombus formation. Redox regulation of aIIbb3 by LMW thiols

LMW thiols in plasma include glutathione, cysteine, cysteine–glycine, and homocysteine. Glutathione appears in either a reduced form (GSH) or an oxidized form (GSSG). Intracellular glutathione was shown to exhibit an important role in regulating cell function, including platelet activation. Platelets contain millimolar concentrations of glutathione, mostly ( > 90%) in the reduced form (8, 43, 82). Glutathione is also present in micromolar concentrations in plasma, also largely in the reduced form, with GSH/GSSG ratio of 4:1 to 13:1 (2, 73, 74, 80). Interestingly, physiological concentrations of GSH (10 lM) potentiate platelet aggregation induced by subthreshold concentrations of agonists and increased labeling of thiols in the b3 subunit of aIIbb3. GSH also potentiates aggregation by low concentrations of the direct aIIbb3-activating peptide LSARLAF, which implies a direct effect of GSH on aIIbb3 activation. Moreover, addition of a small amount of GSSG in a ratio of 5:1 GSH/GSSG enhances platelet aggregation relative to GSH alone (37). These results suggest that the stimulatory effect of glutathione on aggregation is not simply by reducing disulfide bonds by GSH. There is rather a requirement for a certain redox potential for the stimulatory effect enabling disulfide cleavage in integrin aIIbb3. Apart from glutathione, other LMW thiols such as cysteine, cysteinylglycine, and homocysteine also potentiate platelet aggregation but in concentrations that are higher than

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their concentrations in the blood (37). The stimulatory effect of homocysteine is of special interest, as it is a known risk factor for vascular disease. Although the homocysteine concentrations found to potentiate platelet aggregation were higher than usually found in the blood, they might be found in hypercysteinemic conditions in end-stage renal disease with unusually high rates of cardiovascular morbidity and death (47). Since GSH and the other LMW thiols, by themselves, did not cause platelet aggregation, it appears that thiol generation does not activate aIIbb3 by itself, but rather primes the receptor for activation by a second step which possibly involves thiol/disulfide exchange. Regulatory role for NO in aIIbb3 function

NO generated and released from activated platelets and endothelial cells affords an inhibitory regulator of platelet recruitment to an injured vessel site. The major mechanism of NO inhibition is through activation of guanylate cyclase that increases platelet intracellular cyclic guanosine monophosphate concentrations which inhibit platelet activation (75). An additional mechanism by which NO can modulate platelet function is by reacting with thiols to form S-nitrothiols. S-nitrosylation of cysteine residues with diverse functional outcomes was shown for many proteins in a wide variety of cells and tissues, and is considered the prototypic redox-based signaling mechanism (46). Several reports suggest that S-nitrosylation of thiols in aIIbb3 also regulates its function. A combination of NO and GSH inactivated the active conformer of purified aIIbb3, which is associated with reducing the amount of thiols on the integrin from an average of 4.5 to only 0.5, suggesting a direct modification of cysteines in aIIbb3 (147). Another study showed that NO and GSH reverse aIIbb3 activation in thrombin activated platelets (130). The authors also used Raman spectroscopy to show Snitrosylation of purified aIIbb3. In the native resting integrin, most cysteines formed disulfide bonds and some of them were sterically strained. After activation of the integrin with Mn2 + , there was a reduction in the strained disulfides with generation of a few free thiols, suggesting a conversion of few disulfides to thiols, followed by shuffling of disulfide bonds. NO treatment converted aIIbb3 back to the resting conformation with partial recovery of the strained disulfide bonds and loss of some free thiols, suggesting a direct interaction of NO with cysteines in the integrin through Snitrosylation, which alters the integrin conformation back to its inactive state. Interestingly, there are several Snitrosylation motifs in the extracellular region of aIIbb3, at Cys38, Cys433, Cys523, and Cys581 (130). The three latter motifs are grouped closely together in the cysteine-rich EGF region, the same region for which most of the evidence for redox regulation of disulfide bonds was obtained. Moreover, the Cys523 is engaged in the Cys523–Cys544 disulfide bond that was particularly suggested to be a regulatory bond, and Cys581 is engaged in the Cys567–Cys581 bond which was shown to be essential for aIIbb3 activation (88, 89). Redox regulation of integrin function by thiol isomerase activity

Enzymatically catalyzed disulfide exchanges are involved in the regulation of integrin function. One of the main thiol isomerases suggested to catalyze the disulfide bond exchange

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reaction is PDI. PDI is the prototypic thiol isomerase that includes at least 21 family members in mammals and five in yeast (7). The active sites of these enzymes contain CXXC motifs that can mediate oxidation, reduction or thiol/disulfide exchanges of substrate disulfide bonds. PDI includes two atype thioredoxin domains, each containing a CXXC motif, and two b-type thioredoxin domains thought to contribute to its chaperone function (33, 132). PDI is ubiquitously expressed and is highly concentrated in the oxidizing environment of the ER, where it ensures proper disulfide formation of newly formed proteins (7, 135). However, PDI is also found on the surface of various cells. Within the vasculature, extracellular PDI is found on the surface of platelets, endothelial cells, and leukocytes (34, 54, 72, 110). During the last two decades, a lot of information has accumulated regarding the important role of surface PDI in thrombus formation. Platelets and endothelial cells were shown to release PDI on activation (16, 54), and inhibition of PDI blocked agonist-induced platelet aggregation (35, 36). Anti-PDI antibodies were shown to inhibit thrombus formation in a model of carotid artery ligation and wire-induced endothelial disruption (106). Intravital microscopy using anti-PDI antibodies demonstrated that PDI is necessary for both platelet accumulation and fibrin generation in laser and FeCl3-induced injuries (22, 59). Efforts were made to identify the cell surface proteins that are substrates for PDI during thrombus formation. PDI was suggested to participate in activation (decryption) of tissue factor by forming an allosteric disulfide bond between Cys186 and Cys209 (18). PDI was also shown to be involved

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in GPIb regulation (9) and a2b1 adhesion to collagen (71). Most observations linked PDI to aIIbb3 function; PDI inhibitors inhibit platelet aggregation by various agonists and by calcium ionophore. Anti-PDI inhibitors act only on the second irreversible phase of aggregation induced by weak agonists and do not act on the reversible primary aggregation step (35, 39, 70). These results suggest that PDI mediates persistent fibrinogen binding to aIIbb3 and not the initial ligand/receptor interaction. Flow cytometry using fibrinogen or PAC-1, an antibody that recognizes the activated conformation of aIIbb3, showed that anti-PDI antibodies inhibit aIIbb3 activation by either agonist stimulation or the signaling independent activator Mn2 + (70, 79). Inhibition of PDI was also shown to inhibit platelet b3 and b1 integrinmediated adhesion (70, 71). Cho et al. (23) showed that Chinese Hamster Ovary cells that express aIIbb3 or avb3, as well as purified aIIbb3 or b3, bound recombinant PDI after Mn2 + stimulation, demonstrating a direct interaction between PDI and aIIbb3 through its b3 subunit. Furthermore, in contrast to WT mice, b3 - / - mice failed to accumulate PDI and generate fibrin after laser injury. Reciprocal bone marrow transplantation studies showed that b3 from both aIIbb3 on platelets and avb3 on endothelial cells are required for PDI accumulation after vascular injury (58). Using megakaryocyte- and platelet-specific PDI-deficient mice, it was shown that platelet PDI regulate aIIbb3 activation without affecting platelet activation and inside-out integrin signaling. PDI binding to mouse platelets in response to thrombin was significantly reduced in b3 - / - platelets relative to WT platelets

FIG. 8. A model for redox regulation of platelet integrin aIIbb3. Integrin aIIbb3 on platelets surface is shown in three different conformations. Representative disulfide bonds and thiols in the four EGF domains of b3 (colored in dark gray) are shown. The non-activated conformation in the left side does not bind fibrinogen, and most cysteines in the EGF domains are disulfide bonded. Agonist induced inside-out signaling alters integrin conformation to an intermediate active state, resulting in an initial binding of fibrinogen and primary platelet aggregation. This step involves reduction of disulfide bonds that can be stimulated by low-molecular-weight thiols in the plasma such as GSH and can be inhibited by NO. A further change in integrin conformation to a full active state results in firm fibrinogen binding and induces outside-in signaling, leading to secondary irreversible platelet aggregation. This step involves thiol/disulfide bond exchange catalyzed by thiol isomerases such as PDI, Erp57, and Erp5. GSH, reduced glutathione; NO, nitric oxide; PDI, protein disulfide isomerase.


(67). PDI was also shown to directly bind to endothelial avb3, and this binding was accelerated by Mn2 + stimulation. In addition, PDI inhibitors reduced avb3 activation and ligand binding (120). Taken together, PDI directly binds and regulates aIIbb3 and avb3 functions, and this binding was also shown to be important for PDI accumulation during thrombus formation. The target disulfide bonds in these integrins for PDI activity have not yet been identified, but the demonstration that PDI interacts with the b3 subunit of the integrin and the suggested regulatory role for some bonds in the b3 EGF domains and bTD makes the disulfides in these regions possible targets for PDI. Notably, apart from aIIbb3, avb3, and a2b1, PDI was also shown to interact with aMb2 integrin on the surface of neutrophils and to regulate its adhesive function and neutrophil recruitment during venous inflammation (44), suggesting that integrins are general substrates for surface PDI on different cells. Apart from PDI, other members of the PDI family are also involved in b3 integrins function. Erp57 was shown to be secreted from activated platelets and to regulate platelet aggregation and aIIbb3 activation and signaling (51, 140). AntiErp57 antibodies were shown to inhibit thrombus formation in laser-induced and FeCl3 injury mouse models, showing a role for this thiol isomerase in thrombus formation in vivo (140). A recent study showed that platelet surface Erp57 directly interacts with aIIbb3, and this interaction is required for platelet incorporation into developing thrombi (133). Erp57 and PDI probably have distinct roles in aIIbb3 integrin function and platelet aggregation, as addition of function blocking antiErp57 antibodies to PDI-deficient platelets further decreased integrin activation and platelet aggregation (67). Another PDI family member, Erp5, was also shown to be recruited to platelet surface after their activation and to regulate platelet aggregation, fibrinogen binding, and Pselectin exposure. Erp5 effect probably involves aIIbb3 regulation, as it was shown to associate with the integrin b3 subunit during platelet stimulation (61). Other members of the PDI family, Erp72, Erp44, and Erp29 were also shown to be released by activated platelets and relocate to their cell surface, but their role in platelet and integrin function has not yet been studied (52). Interestingly, purified aIIbb3 was shown to display an endogenous thiol isomerase activity, and this activity was induced in response to Mn2 + and accelerated by GSH and NO (79, 97, 107, 131). This activity was predicted from the presence of four highly conserved Cys–Gly–X–Cys (CGXC) motifs in each of the b3 EGF domains (97). The first cysteine in this motif is the fifth cysteine of the EGF forming the unique 1–5 disulfide bonds (Cys437–Cys457 in EGF-1, Cys473–Cys503 in EGF-2, Cys523–Cys544 in EGF-3, and Cys560–Cys583 in EGF-4) that were suggested to have a regulatory role in integrin function. Thus, these unique bonds may regulate the thiol isomerase activity of the integrin. The relevance of this endogenous thiol isomerase activity to thrombus formation and the potential targets for this activity remain to be elucidated. Summary and a Model for Redox Regulation of Integrin Function

Over the last three decades, there has been an increasing interest in assessing the importance of functional disulfide

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bonds as redox regulators of protein function. Integrins, and in particular those involved in thrombosis and hemostasis, were suggested to be regulated by redox modulation of functional disulfides in their extracellular portion in addition to the known regulation by intracellular factors. Integrin aIIbb3 plays a major role in thrombus formation and was, therefore, extensively studied, providing broad data about the involvement of functional disulfide bonds in redox regulation of its function. A model for redox regulation of this integrin is shown in Figure 8. In summary, aIIbb3 contains many cysteines that are located mainly in the EGF domains of the b3 subunit. In its resting state, most of these cysteines are engaged in disulfide bonds, and a few are possibly present as free thiols. aIIbb3 activation by agonist-induced inside-out signaling induces a conformational change in the receptor and initial binding of fibrinogen that leads to primary aggregation. This step probably involves disulfide bond reduction that can be mediated by small-molecule and protein reductants and influenced by reduced glutathione and NO, the latter reversing integrin activation. Ligand binding further changes the conformation of the integrin, leading to firm ligand binding, and induces outside-in signaling, leading to secondary irreversible aggregation. This step involves thiol/ disulfide bond exchange and is probably catalyzed by PDI. Other thiol isomerases, such as Erp57 and Erp5, are possibly also involved in catalyzing disulfide bond reduction and/or thiol/disulfide bond exchanges. Understanding the redox regulation of aIIbb3 and other integrins involved in thrombus formation, such as avb3 and a2b1, can provide a useful tool for developing new antithrombotic drugs that target redox sites in the integrins or in the redox agents and enzymes which regulate those sites. Indeed, PDI inhibitors are currently studied for potential antithrombotic therapy (40). Acknowledgments

The author is indebted to Adva Yeheskel from the Bioinformatics Unit, G.S.W. Faculty of Life Sciences, Tel Aviv University, who assisted in designing and preparing the figures and to Prof. Uri Seligsohn and Dr. Rima Dardik from the Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer who critically read the review. References

1. Ambo H, Kamata T, Handa M, Taki M, Kuwajima M, Kawai Y, Oda A, Murata M, Takada Y, Watanabe K, and Ikeda Y. Three novel integrin beta3 subunit missense mutations (H280P, C560F, and G579S) in thrombasthenia, including one (H280P) prevalent in Japanese patients. Biochem Biophys Res Commun 251: 763–768, 1998. 2. Anderson ME and Meister A. Dynamic state of glutathione in blood plasma. J Biol Chem 255: 9530–9533, 1980. 3. Arnaout MA. Integrin structure: new twists and turns in dynamic cell adhesion. Immunol Rev 186: 125–140, 2002. 4. Bassler N, Loeffler C, Mangin P, Yuan Y, Schwarz M, Hagemeyer CE, Eisenhardt SU, Ahrens I, Bode C, Jackson SP, and Peter K. A mechanistic model for paradoxical platelet activation by ligand-mimetic alphaIIb beta3 (GPIIb/IIIa) antagonists. Arterioscler Thromb Vasc Biol 27: e9–e15, 2007.

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5. Beer J and Coller BS. Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage. J Biol Chem 264: 17564–17573, 1989. 6. Beglova N, Blacklow SC, Takagi J, and Springer TA. Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation. Nat Struct Biol 9: 282–287, 2002. 7. Benham AM. The protein disulfide isomerase family: key players in health and disease. Antioxid Redox Signal 16: 781–789, 2012. 8. Bosia A, Spangenberg P, Losche W, Arese P, and Till U. The role of the GSH-disulfide status in the reversible and irreversible aggregation of human platelets. Thromb Res 30: 137–142, 1983. 9. Burgess JK, Hotchkiss KA, Suter C, Dudman NP, Szollosi J, Chesterman CN, Chong BH, and Hogg PJ. Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane. J Biol Chem 275: 9758–9766, 2000. 10. Butera D, Cook KM, Chiu J, Wong JW, and Hogg PJ. Control of blood proteins by functional disulfide bonds. Blood 123: 2000–2007, 2014. 11. Butera D, Wind T, Lay AJ, Beck J, Castellino FJ, and Hogg PJ. Characterization of a reduced form of plasma plasminogen as the precursor for angiostatin formation. J Biol Chem 289: 2992–3000, 2014. 12. Butta N, Arias-Salgado EG, Gonzalez-Manchon C, Ferrer M, Larrucea S, Ayuso MS, and Parrilla R. Disruption of the beta3 663–687 disulfide bridge confers constitutive activity to beta3 integrins. Blood 102: 2491–2497, 2003. 13. Calvete JJ, Henschen A, and Gonzalez-Rodriguez J. Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the beta-subunits of the integrin family. Biochem J 274 (Pt 1): 63–71, 1991. 14. Calzada MJ, Alvarez MV, and Gonzalez-Rodriguez J. Agonist-specific structural rearrangements of integrin alpha IIbbeta 3. Confirmation of the bent conformation in platelets at rest and after activation. J Biol Chem 277: 39899–39908, 2002. 15. Chen J, Diacovo TG, Grenache DG, Santoro SA, and Zutter MM. The alpha(2) integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol 161: 337–344, 2002. 16. Chen K, Lin Y, and Detwiler TC. Protein disulfide isomerase activity is released by activated platelets. Blood 79: 2226–2228, 1992. 17. Chen P, Melchior C, Brons NH, Schlegel N, Caen J, and Kieffer N. Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation. J Biol Chem 276: 38628–38635, 2001. 18. Chen VM, Ahamed J, Versteeg HH, Berndt MC, Ruf W, and Hogg PJ. Evidence for activation of tissue factor by an allosteric disulfide bond. Biochemistry 45: 12020–12028, 2006. 19. Chen VM and Hogg PJ. Allosteric disulfide bonds in thrombosis and thrombolysis. J Thromb Haemost 4: 2533– 2541, 2006. 20. Chen YP, O’Toole TE, Leong L, Liu BQ, Diaz-Gonzalez F, and Ginsberg MH. Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3. Blood 86: 2606–2615, 1995.

MOR-COHEN

21. Chigaev A, Zwartz GJ, Buranda T, Edwards BS, Prossnitz ER, and Sklar LA. Conformational regulation of alpha 4 beta 1-integrin affinity by reducing agents. ‘‘Inside-out’’ signaling is independent of and additive to reductionregulated integrin activation. J Biol Chem 279: 32435– 32443, 2004. 22. Cho J, Furie BC, Coughlin SR, and Furie B. A critical role for extracellular protein disulfide isomerase during thrombus formation in mice. J Clin Invest 118: 1123– 1131, 2008. 23. Cho J, Kennedy DR, Lin L, Huang M, Merrill-Skoloff G, Furie BC, and Furie B. Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins. Blood 120: 647–655, 2012. 24. Cho MJ, Liu J, Pestina TI, Steward SA, Jackson CW, and Gartner TK. AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets. J Thromb Haemost 1: 363–373, 2003. 25. Coller BS, Cheresh DA, Asch E, and Seligsohn U. Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel. Blood 77: 75–83, 1991. 26. Cook KM and Hogg PJ. Post-translational control of protein function by disulfide bond cleavage. Antioxid Redox Signal 18: 1987–2015, 2013. 27. Cook KM, McNeil HP, and Hogg PJ. Allosteric control of betaII-tryptase by a redox active disulfide bond. J Biol Chem 288: 34920–34929, 2013. 28. Davis GE and Camarillo CW. Regulation of integrinmediated myeloid cell adhesion to fibronectin: influence of disulfide reducing agents, divalent cations and phorbol ester. J Immunol 151: 7138–7150, 1993. 29. Depuydt M, Messens J, and Collet JF. How proteins form disulfide bonds. Antioxid Redox Signal 15: 49–66, 2011. 30. Derrick JM, Shattil SJ, Poncz M, Gruppo RA, and Gartner TK. Distinct domains of alphaIIbbeta3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an alphaIIbbeta3 interacting peptide. Thromb Haemost 86: 894–901, 2001. 31. Du X, Gu M, Weisel JW, Nagaswami C, Bennett JS, Bowditch R, and Ginsberg MH. Long range propagation of conformational changes in integrin alpha IIb beta 3. J Biol Chem 268: 23087–23092, 1993. 32. Edwards BS, Curry MS, Southon EA, Chong AS, and Graf LH, Jr. Evidence for a dithiol-activated signaling pathway in natural killer cell avidity regulation of leukocyte function antigen-1: structural requirements and relationship to phorbol ester- and CD16-triggered pathways. Blood 86: 2288–2301, 1995. 33. Ellgaard L and Ruddock LW. The human protein disulphide isomerase family: substrate interactions and functional properties. EMBO Rep 6: 28–32, 2005. 34. Essex DW, Chen K, and Swiatkowska M. Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane. Blood 86: 2168–2173, 1995. 35. Essex DW and Li M. Protein disulphide isomerase mediates platelet aggregation and secretion. Br J Haematol 104: 448–454, 1999. 36. Essex DW and Li M. A polyclonal antibody to protein disulfide isomerase induces platelet aggregation and secretion. Thromb Res 96: 445–450, 1999. 37. Essex DW and Li M. Redox control of platelet aggregation. Biochemistry 42: 129–136, 2003.


38. Essex DW and Li M. Redox modification of platelet glycoproteins. Curr Drug Targets 7: 1233–1241, 2006. 39. Essex DW, Li M, Miller A, and Feinman RD. Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation. Biochemistry 40: 6070–6075, 2001. 40. Flaumenhaft R. Protein disulfide isomerase as an antithrombotic target. Trends Cardiovasc Med 23: 264–268, 2013. 41. Frelinger AL, 3rd, Du XP, Plow EF, and Ginsberg MH. Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function. J Biol Chem 266: 17106–17111, 1991. 42. Ganderton T, Wong JW, Schroeder C, and Hogg PJ. Lateral self-association of VWF involves the Cys2431Cys2453 disulfide/dithiol in the C2 domain. Blood 118: 5312–5318, 2011. 43. Giustarini D, Campoccia G, Fanetti G, Rossi R, Giannerini F, Lusini L, and Di Simplicio P. Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress. Arch Biochem Biophys 380: 1–10, 2000. 44. Hahm E, Li J, Kim K, Huh S, Rogelj S, and Cho J. Extracellular protein disulfide isomerase regulates ligandbinding activity of alphaMbeta2 integrin and neutrophil recruitment during vascular inflammation. Blood 121: 3789–3800, S3781–S3715, 2013. 45. Handa M, Watanabe K, Kawai Y, Kamata T, Koyama T, Nagai H, and Ikeda Y. Platelet unresponsiveness to collagen: involvement of glycoprotein Ia-IIa (alpha 2 beta 1 integrin) deficiency associated with a myeloproliferative disorder. Thromb Haemost 73: 521–528, 1995. 46. Hess DT, Matsumoto A, Nudelman R, and Stamler JS. Snitrosylation: spectrum and specificity. Nat Cell Biol 3: E46–E49, 2001. 47. Hoffer LJ, Robitaille L, Elian KM, Bank I, Hongsprabhas P, and Mamer OA. Plasma reduced homocysteine concentrations are increased in end-stage renal disease. Kidney Int 59: 372–377, 2001. 48. Hogg PJ. Disulfide bonds as switches for protein function. Trends Biochem Sci 28: 210–214, 2003. 49. Hogg PJ. Contribution of allosteric disulfide bonds to regulation of hemostasis. J Thromb Haemost 7 Suppl 1: 13–16, 2009. 50. Hogg PJ. Targeting allosteric disulphide bonds in cancer. Nat Rev Cancer 13: 425–431, 2013. 51. Holbrook LM, Sasikumar P, Stanley RG, Simmonds AD, Bicknell AB, and Gibbins JM. The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function. J Thromb Haemost 10: 278–288. 52. Holbrook LM, Watkins NA, Simmonds AD, Jones CI, Ouwehand WH, and Gibbins JM. Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation. Br J Haematol 148: 627–637, 2010. 53. Hotchkiss KA, Chesterman CN, and Hogg PJ. Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase. Biochemistry 35: 9761–9767, 1996. 54. Hotchkiss KA, Matthias LJ, and Hogg PJ. Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase. Biochim Biophys Acta 1388: 478–488, 1998. 55. Hynes RO. Integrins: a family of cell surface receptors. Cell 48: 549–554, 1987.

13

56. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687, 2002. 57. Jackson SP, Nesbitt WS, and Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 1: 1602–1612, 2003. 58. Jasuja R, Furie B, and Furie BC. Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo. Blood 116: 4665–4674, 2010. 59. Jasuja R, Passam FH, Kennedy DR, Kim SH, van Hessem L, Lin L, Bowley SR, Joshi SS, Dilks JR, Furie B, Furie BC, and Flaumenhaft R. Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents. J Clin Invest 122: 2104–2113, 2012. 60. Jordan PA and Gibbins JM. Extracellular disulfide exchange and the regulation of cellular function. Antioxid Redox Signal 8: 312–324, 2006. 61. Jordan PA, Stevens JM, Hubbard GP, Barrett NE, Sage T, Authi KS, and Gibbins JM. A role for the thiol isomerase protein ERP5 in platelet function. Blood 105: 1500–1507, 2005. 62. Jung SM and Moroi M. Platelets interact with soluble and insoluble collagens through characteristically different reactions. J Biol Chem 273: 14827–14837, 1998. 63. Jung SM and Moroi M. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1). J Biol Chem 275: 8016–8026, 2000. 64. Jung SM and Moroi M. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets. Trends Cardiovasc Med 10: 285–292, 2000. 65. Kamata T, Ambo H, Puzon-McLaughlin W, Tieu KK, Handa M, Ikeda Y, and Takada Y. Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit. Biochem J 378: 1079–1082, 2004. 66. Kendall T, Mukai L, Jannuzi AL, and Bunch TA. Identification of integrin beta subunit mutations that alter affinity for extracellular matrix ligand. J Biol Chem 286: 30981–30993, 2011. 67. Kim K, Hahm E, Li J, Holbrook LM, Sasikumar P, Stanley RG, Ushio-Fukai M, Gibbins JM, and Cho J. Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice. Blood 122: 1052–1061, 2013. 68. Kuijpers MJ, Schulte V, Bergmeier W, Lindhout T, Brakebusch C, Offermanns S, Fassler R, Heemskerk JW, and Nieswandt B. Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo. FASEB J 17: 685–687, 2003. 69. Lahav J, Gofer-Dadosh N, Luboshitz J, Hess O, and Shaklai M. Protein disulfide isomerase mediates integrindependent adhesion. FEBS Lett 475: 89–92, 2000. 70. Lahav J, Jurk K, Hess O, Barnes MJ, Farndale RW, Luboshitz J, and Kehrel BE. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange. Blood 100: 2472–2478, 2002. 71. Lahav J, Wijnen EM, Hess O, Hamaia SW, Griffiths D, Makris M, Knight CG, Essex DW, and Farndale RW. Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin alpha2beta1. Blood 102: 2085–2092, 2003.

14

72. Lame MW, Jones AD, Wilson DW, and Segall HJ. Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells. Proteomics 5: 4398– 4413, 2005. 73. Lash LH and Jones DP. Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma. Arch Biochem Biophys 240: 583–592, 1985. 74. Lauterburg BH and Velez ME. Glutathione deficiency in alcoholics: risk factor for paracetamol hepatotoxicity. Gut 29: 1153–1157, 1988. 75. Loscalzo J. Nitric oxide insufficiency, platelet activation, and arterial thrombosis. Circ Res 88: 756–762, 2001. 76. Lu C, Ferzly M, Takagi J, and Springer TA. Epitope mapping of antibodies to the C-terminal region of the integrin beta 2 subunit reveals regions that become exposed upon receptor activation. J Immunol 166: 5629– 5637, 2001. 77. MacIntyre DE, Grainge CA, Drummond AH, and Gordon JL. Effect of thio reagents on platelet transport processes and responses to stimuli. Biochem Pharmacol 26: 319– 323, 1977. 78. Manickam N, Sun X, Hakala KW, Weintraub ST, and Essex DW. Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation. Br J Haematol 142: 457– 465, 2008. 79. Manickam N, Sun X, Li M, Gazitt Y, and Essex DW. Protein disulphide isomerase in platelet function. Br J Haematol 140: 223–229, 2008. 80. Mansoor MA, Svardal AM, and Ueland PM. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 200: 218–229, 1992. 81. Margaritis A, Priora R, Frosali S, Di Giuseppe D, Summa D, Coppo L, Di Stefano A, and Di Simplicio P. The role of protein sulfhydryl groups and protein disulfides of the platelet surface in aggregation processes involving thiol exchange reactions. Pharmacol Res 63: 77–84, 2011. 82. Matsuda S, Ikeda Y, Aoki M, Toyama K, Watanabe K, and Ando Y. Role of reduced glutathione on platelet functions. Thromb Haemost 42: 1324–1331, 1979. 83. Metcalf DG, Moore DT, Wu Y, Kielec JM, Molnar K, Valentine KG, Wand AJ, Bennett JS, and DeGrado WF. NMR analysis of the alphaIIb beta3 cytoplasmic interaction suggests a mechanism for integrin regulation. Proc Natl Acad Sci U S A 107: 22481–22486, 2010. 84. Metcalfe C, Cresswell P, Ciaccia L, Thomas B, and Barclay AN. Labile disulfide bonds are common at the leucocyte cell surface. Open Biol 1: 110010, 2011. 85. Mihel J, Sikic M, Tomic S, Jeren B, and Vlahovicek K. PSAIA - protein structure and interaction analyzer. BMC Struct Biol 8: 21, 2008. 86. Monod J, Wyman J, and Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 12: 88–118, 1965. 87. Mor-Cohen R, Rosenberg N, Averbukh Y, Seligsohn U, and Lahav J. Disulfide bond exchanges in integrins alphaIIbbeta3 and alphavbeta3 are required for activation and post-ligation signaling during clot retraction. Thromb Res 133: 826–836, 2014. 88. Mor-Cohen R, Rosenberg N, Einav Y, Zelzion E, Landau M, Mansour W, Averbukh Y, and Seligsohn U. Unique disulfide bonds in epidermal growth factor (EGF) domains of beta3 affect structure and function of alphaIIbbeta3 and

MOR-COHEN

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

alphavbeta3 integrins in different manner. J Biol Chem 287: 8879–8891, 2012. Mor-Cohen R, Rosenberg N, Landau M, Lahav J, and Seligsohn U. Specific cysteines in beta3 are involved in disulfide bond exchange-dependent and -independent activation of alphaIIbbeta3. J Biol Chem 283: 19235–19244, 2008. Mor-Cohen R, Rosenberg N, Peretz H, Landau M, Coller BS, Awidi A, and Seligsohn U. Disulfide bond disruption by a beta 3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active alpha IIb beta 3. Thromb Haemost 98: 1257–1265, 2007. Ni H, Li A, Simonsen N, and Wilkins JA. Integrin activation by dithiothreitol or Mn2 + induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain. J Biol Chem 273: 7981–7987, 1998. Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk JW, Zirngibl H, and Fassler R. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J 20: 2120–2130, 2001. Niewiarowski S, Norton KJ, Eckardt A, Lukasiewicz H, Holt JC, and Kornecki E. Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa. Biochim Biophys Acta 983: 91–99, 1989. Niiya K, Hodson E, Bader R, Byers-Ward V, Koziol JA, Plow EF, and Ruggeri ZM. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 70: 475–483, 1987. Niu H, Xu Z, Li D, Zhang L, Wang K, Taylor DB, Liu J, and Gartner TK. Peptide LSARLAF induces integrin beta3 dependent outside-in signaling in platelets. Thromb Res 130: 203–209, 2012. Nolan SM, Mathew EC, Scarth SL, Al-Shamkhani A, and Law SK. The effects of cysteine to alanine mutations of CD18 on the expression and adhesion of the CD11/CD18 integrins. FEBS Lett 486: 89–92, 2000. O’Neill S, Robinson A, Deering A, Ryan M, Fitzgerald DJ, and Moran N. The platelet integrin alpha IIbbeta 3 has an endogenous thiol isomerase activity. J Biol Chem 275: 36984–36990, 2000. Pearce AC, Wonerow P, Marshall SJ, Frampton J, Gartner TK, and Watson SP. The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa. Biochem J 378: 193–199, 2004. Peretz H, Rosenberg N, Landau M, Usher S, Nelson EJ, Mor-Cohen R, French DL, Mitchell BW, Nair SC, Chandy M, Coller BS, Srivastava A, and Seligsohn U. Molecular diversity of Glanzmann thrombasthenia in southern India: new insights into mRNA splicing and structure-function correlations of alphaIIbbeta3 integrin (ITGA2B, ITGB3). Hum Mutat 27: 359–369, 2006. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, and Ferrin TE. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612, 2004. Philippeaux MM, Vesin C, Tacchini-Cottier F, and Piguet PF. Activated human platelets express beta2 integrin. Eur J Haematol 56: 130–137, 1996.


102. Piguet PF, Vesin C, and Rochat A. Beta2 integrin modulates platelet caspase activation and life span in mice. Eur J Cell Biol 80: 171–177, 2001. 103. Piotrowicz RS, Orchekowski RP, Nugent DJ, Yamada KY, and Kunicki TJ. Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets. J Cell Biol 106: 1359–1364, 1988. 104. Plow EF and Byzova T. The biology of glycoprotein IIbIIIa. Coron Artery Dis 10: 547–551, 1999. 105. Plow EF, D’Souza SE, and Ginsberg MH. Ligand binding to GPIIb-IIIa: a status report. Semin Thromb Hemost 18: 324–332, 1992. 106. Reinhardt C, von Bruhl ML, Manukyan D, Grahl L, Lorenz M, Altmann B, Dlugai S, Hess S, Konrad I, Orschiedt L, Mackman N, Ruddock L, Massberg S, and Engelmann B. Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation. J Clin Invest 118: 1110–1122, 2008. 107. Robinson A, O’Neill S, Kiernan A, O’Donoghue N, and Moran N. Bacitracin reveals a role for multiple thiol isomerases in platelet function. Br J Haematol 132: 339– 348, 2006. 108. Ruiz C, Liu CY, Sun QH, Sigaud-Fiks M, Fressinaud E, Muller JY, Nurden P, Nurden AT, Newman PJ, and Valentin N. A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (alphaIIbbeta3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia-like phenotype. Blood 98: 2432–2441, 2001. 109. Sajid M and Stouffer GA. The role of alpha(v)beta3 integrins in vascular healing. Thromb Haemost 87: 187–193, 2002. 110. Santos CX, Stolf BS, Takemoto PV, Amanso AM, Lopes LR, Souza EB, Goto H, and Laurindo FR. Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages. J Leukoc Biol 86: 989–998, 2009. 111. Schaffner-Reckinger E, Brons NH, and Kieffer N. Evidence from site-directed mutagenesis that the cytoplasmic domain of the beta3 subunit influences the conformational state of the alphaVbeta3 integrin ectodomain. Thromb Haemost 85: 716–723, 2001. 112. Schmidt B, Ho L, and Hogg PJ. Allosteric disulfide bonds. Biochemistry 45: 7429–7433, 2006. 113. Schmidt B and Hogg PJ. Search for allosteric disulfide bonds in NMR structures. BMC Struct Biol 7: 49, 2007. 114. Shatsky M, Nussinov R, and Wolfson HJ. A method for simultaneous alignment of multiple protein structures. Proteins 56: 143–156, 2004. 115. Shattil SJ, Kashiwagi H, and Pampori N. Integrin signaling: the platelet paradigm. Blood 91: 2645–2657, 1998. 116. Sonnenberg A, Modderman PW, and Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature 336: 487–489, 1988. 117. Sosnoski DM, Emanuel BS, Hawkins AL, van Tuinen P, Ledbetter DH, Nussbaum RL, Kaos FT, Schwartz E, Phillips D, Bennett JS, et al. Chromosomal localization of the genes for the vitronectin and fibronectin receptors alpha subunits and for platelet glycoproteins IIb and IIIa. J Clin Invest 81: 1993–1998, 1988. 118. Stouffer GA and Smyth SS. Effects of thrombin on interactions between beta3-integrins and extracellular matrix in platelets and vascular cells. Arterioscler Thromb Vasc Biol 23: 1971–1978, 2003.

15

119. Sun QH, Liu CY, Wang R, Paddock C, and Newman PJ. Disruption of the long-range GPIIIa Cys(5)-Cys(435) disulfide bond results in the production of constitutively active GPIIb-IIIa (alpha(IIb)beta(3)) integrin complexes. Blood 100: 2094–2101, 2002. 120. Swiatkowska M, Szymanski J, Padula G, and Cierniewski CS. Interaction and functional association of protein disulfide isomerase with alphaVbeta3 integrin on endothelial cells. FEBS J 275: 1813–1823, 2008. 121. Takagi J, Beglova N, Yalamanchili P, Blacklow SC, and Springer TA. Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits. Proc Natl Acad Sci U S A 98: 11175–11180, 2001. 122. Takagi J, Petre BM, Walz T, and Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110: 599–611, 2002. 123. Thornton JM. Disulphide bridges in globular proteins. J Mol Biol 151: 261–287, 1981. 124. Ulmer TS, Yaspan B, Ginsberg MH, and Campbell ID. NMR analysis of structure and dynamics of the cytosolic tails of integrin alpha IIb beta 3 in aqueous solution. Biochemistry 40: 7498–7508, 2001. 125. Verhaar R, Dekkers DW, De Cuyper IM, Ginsberg MH, de Korte D, and Verhoeven AJ. UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3. Blood 112: 4935–4939, 2008. 126. Vinogradova O, Haas T, Plow EF, and Qin J. A structural basis for integrin activation by the cytoplasmic tail of the alpha IIb-subunit. Proc Natl Acad Sci U S A 97: 1450– 1455, 2000. 127. Vinogradova O, Vaynberg J, Kong X, Haas TA, Plow EF, and Qin J. Membrane-mediated structural transitions at the cytoplasmic face during integrin activation. Proc Natl Acad Sci U S A 101: 4094–4099, 2004. 128. Vinogradova O, Velyvis A, Velyviene A, Hu B, Haas T, Plow E, and Qin J. A structural mechanism of integrin alpha(IIb)beta(3) ‘‘inside-out’’ activation as regulated by its cytoplasmic face. Cell 110: 587–597, 2002. 129. Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, and Jordan RE. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood 88: 907–914, 1996. 130. Walsh GM, Leane D, Moran N, Keyes TE, Forster RJ, Kenny D, and O’Neill S. S-Nitrosylation of platelet alphaIIbbeta3 as revealed by Raman spectroscopy. Biochemistry 46: 6429–6436, 2007. 131. Walsh GM, Sheehan D, Kinsella A, Moran N, and O’Neill S. Redox modulation of integrin [correction of integin] alpha IIb beta 3 involves a novel allosteric regulation of its thiol isomerase activity. Biochemistry 43: 473–480, 2004. 132. Wang C, Yu J, Huo L, Wang L, Feng W, and Wang CC. Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a¢. J Biol Chem 287: 1139–1149, 2012. 133. Wang L, Wu Y, Zhou J, Ahmad SS, Mutus B, Garbi N, Hammerling G, Liu J, and Essex DW. Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the alphaIIbbeta3 integrin. Blood 122: 3642–3650. 134. Waterhouse AM, Procter JB, Martin DM, Clamp M, and Barton GJ. Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191, 2009.

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135. Wilkinson B and Gilbert HF. Protein disulfide isomerase. Biochim Biophys Acta 1699: 35–44, 2004. 136. Wong JW and Hogg PJ. Analysis of disulfide bonds in protein structures. J Thromb Haemost 8: 2345, 2010. 137. Wouters MA, Fan SW, and Haworth NL. Disulfides as redox switches: from molecular mechanisms to functional significance. Antioxid Redox Signal 12: 53–91, 2010. 138. Wouters MA, George RA, and Haworth NL. ‘‘Forbidden’’ disulfides: their role as redox switches. Curr Protein Pept Sci 8: 484–495, 2007. 139. Wouters MA, Lau KK, and Hogg PJ. Cross-strand disulphides in cell entry proteins: poised to act. Bioessays 26: 73–79, 2004. 140. Wu Y, Ahmad SS, Zhou J, Wang L, Cully MP, and Essex DW. The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis. Blood 119: 1737– 1746, 2012. 141. Xiao T, Takagi J, Coller BS, Wang JH, and Springer TA. Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432: 59–67, 2004. 142. Xie C, Zhu J, Chen X, Mi L, Nishida N, and Springer TA. Structure of an integrin with an alphaI domain, complement receptor type 4. EMBO J 29: 666–679, 2010. 143. Xiong JP, Mahalingham B, Alonso JL, Borrelli LA, Rui X, Anand S, Hyman BT, Rysiok T, Muller-Pompalla D, Goodman SL, and Arnaout MA. Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/ beta transmembrane fragment. J Cell Biol 186: 589–600, 2009. 144. Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, and Arnaout MA. Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science 294: 339–345, 2001. 145. Xiong JP, Stehle T, Goodman SL, and Arnaout MA. New insights into the structural basis of integrin activation. Blood 102: 1155–1159, 2003. 146. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, and Arnaout MA. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science 296: 151–155, 2002. 147. Yan B and Smith JW. A redox site involved in integrin activation. J Biol Chem 275: 39964–39972, 2000. 148. Yan B and Smith JW. Mechanism of integrin activation by disulfide bond reduction. Biochemistry 40: 8861–8867, 2001. 149. Yang J, Ma YQ, Page RC, Misra S, Plow EF, and Qin J. Structure of an integrin alphaIIb beta3 transmembranecytoplasmic heterocomplex provides insight into integrin activation. Proc Natl Acad Sci U S A 106: 17729–17734, 2009. 150. Zhang K, Pan Y, Qi J, Yue J, Zhang M, Xu C, Li G, and Chen J. Disruption of disulfide restriction at integrin knees

MOR-COHEN

induces activation and ligand-independent signaling of alpha(4)beta(7). J Cell Sci 126: 5030–5041, 2013. 151. Zhu J, Boylan B, Luo BH, Newman PJ, and Springer TA. Tests of the extension and deadbolt models of integrin activation. J Biol Chem 282: 11914–11920, 2007. 152. Zhu J, Luo BH, Xiao T, Zhang C, Nishida N, and Springer TA. Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol Cell 32: 849–861, 2008. 153. Zucker MB and Masiello NC. Platelet aggregation caused by dithiothreitol. Thromb Haemost 51: 119–124, 1984.

Address correspondence to: Dr. Ronit Mor-Cohen The Amalia Biron Research Institute of Thrombosis and Hemostasis Chaim Sheba Medical Center Tel Hashomer 52621 Israel E-mail: [email protected] Date of first submission to ARS Central, October 1, 2014; date of acceptance, October 14, 2014 Abbreviations Used bPS ¼ b subunit from Drosophila bTD ¼ b-tail domain Biotin-BMCC ¼ 1-biotinamido-4-[4¢-(maleimidomethyl)cyclohexanecarboxamido]butane CT ¼ cytoplasmic tails DTT ¼ dithiothreitol EDTA ¼ ethylenediaminetetraaceticacid EGF ¼ epidermal growth factor ER ¼ endoplasmic reticulum GSH ¼ reduced glutathione GSSG ¼ oxidized glutathione GT ¼ Glanzmann thrombasthenia LH ¼ left handed LIBS ¼ ligand-induced binding site LMW ¼ low-molecular weight LPS ¼ lipopolysaccharide MPB ¼ 3-(N-maleimido-propionyl)biocytin NO ¼ nitric oxide pCMBS ¼ 4-(chloromercuri) benzenesulfonic acid PDI ¼ protein disulfide isomerase PSI ¼ plexins, semaphorins and integrins RH ¼ right handed TM ¼ transmembrane vWF ¼ von Willebrand factor WT ¼ wild-type

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