Structural Changes in β 2 -Glycoprotein I and the Antiphospholipid Syndrome

This effect of anti– (cid:451) 2 GPI antibodies is known as lupus anticoagulant (LA) activity. A LA could also be seen with open (cid:451) 2 GPI (Agar et al., 2010). When closed native (cid:451) 2 GPI was added to normal plasma or (cid:451) 2 GPI-depleted plasma, no effect on an activated partial thromboplastin time (aPTT)-based clotting assay was observed. The antiphospholipid syndrome has been described for the first time by Graham Hughes in 1983 as a condition connected with thromboses or foetal losses and antiphospholipid antibodies presence. Form that time there has been a great progress in knowledge, including antiphospholipid antibodies characterisation, their probable and also possible action, clinical manifestations, laboratory detection and treatment possibilities . This book provides a wide spectrum of clinical manifestations through Chapters written by well known researchers and clinicians with a great practical experience in management of diagnostics or treatment of antiphospholipid antibodies' presence. 978-953-51-0526-8,

2 GPI was identified as the most important antigen in the antiphospholipid syndrome, which is amongst others characterized by the presence of antibodies directed to 2 GPI (McNeil et al., 1990;Galli et al., 1990). In 2010, a second three-dimensional conformation of 2 GPI was identified (Ağar et al., 2010) besides the known fishhook-like conformation that was suggested by the crystal structure of the protein (Bouma et al., 1999;Schwarzenbacher et al., 1999). In this chapter we will focus on this novel conformation of 2 GPI and discuss the consequences of the transition between the two conformations for 2 GPI on past but also on present findings.

Biochemistry of β 2 -glycoprotein I
2 GPI is a 43 kDa protein and consists of 326 amino acid residues (Lozier et al., 1984). 2 GPI is synthesized in the liver and it circulates in blood at variable levels (Rioche et al., 1974). 2 GPI is an anionic phospholipid binding glycoprotein composed of five homologous complement control protein repeats, CCP-I to CCP-V (Bouma et al., 1990;Schwarzenbacher et al., 1990). These CCPs are generally found in proteins from the complement system and mediate binding of complement factors to viruses and bacteria (Breier et al., 1970;Pangburn & Rawal, 2002). The first four domains contain approx. 60 amino acids each, whereas the fifth domain has a 6 residue insertion and an additional 19 amino acid C-terminal extension. The extra amino acids are responsible for the formation of a large positive charged patch within the fifth domain of 2 GPI (Hunt et al., 1993) that forms the binding site for anionic www.intechopen.com Antiphospholipid Syndrome 24 phospholipids ( Figure 1).The crystal structure of 2 GPI has been solved in 1999 by two groups (Bouma et al., 1990;Schwarzenbacher et al., 1990), and revealed a structure that resembles a J-shaped fishhook. The phospholipid binding site is located at the bottom side of CCP-V and consists of two major parts, a large positive patch of 14 charged amino acid residues and a flexible hydrophobic loop. This flexible loop has the potential to insert into membranes (Planque et al., 1999).

β 2 -Glycoprotein I and the antiphospholipid syndrome
The antiphospholipid syndrome (APS) is an auto-immune disease defined by the presence of antiphospholipid antibodies in blood of patients in combination with thrombotic complications in arteries or veins as well as pregnancy-related complications (Miyakis et al., 2006). In APS patients, the most common venous event is deep vein thrombosis and the most common arterial event is stroke. In pregnant women with APS early and late miscarriages can occur (Eswaran & Rosen, 1985). Next to miscarriages also placental infarctions, early deliveries and stillbirth have been reported (Lockshin et al., 1985;Branch et al., 1989;Birdsall et al., 1992). Antiphospholipid antibodies often occur transiently after infectious diseases, but there is a general consensus that these transient auto-antibodies are not related to an increased thrombotic risk (Miyakis et al., 2006). The syndrome occurs more in women than in men, and is most common in young to middle-aged adults but can also occur in children and the elderly. Among patients with systemic lupus erythematodes or lupus, the prevalence of antiphospholipid antibodies ranges from 12 to 30% for anticardiolipin antibodies, and 20 to 35% for lupus anticoagulant antibodies (Gezer et al., 2003). It is now generally accepted that the relevant auto-antibodies are not directed against phospholipids, but towards proteins bound to these phospholipids McNeil et al., 1990;. 2 GPI has a relative low affinity towards these negatively charged phospholipids but its affinity increased more than 100-fold in the presence of autoantibodies. Anti-2 GPI antibodies were found to be the most prominent auto-antibodies in APS (de Groot & Meijers, 2011). Recently, three independent groups have shown the importance of antibodies against 2 GPI (Pierangelli et al., 1999;Jankowski et al., 2003;Arad et al., 2011) Mice that were challenged by injection of antiphospholipid antibodies had increased thrombus formation and foetal resorption (García et al., 1997;Ikematsu et al., 1998). Despite the wealth of data on the role of 2 GPI in the pathophysiology of APS, there were no convincing indications that help our understanding of the function of 2 GPI in normal physiology.

β 2 GPI exists in two conformations
There is overwhelming evidence that antibodies against 2 GPI can induce thrombosis in animal models (Blank et al., 1991;Pierangelli et al., 1999;Jankowski et al., 2003, Arad et al. 2011), but it was unclear which metabolic pathway was disturbed by the auto-antibodies. Nevertheless, the 2 GPI protein itself must hold an important functional clue that could lead us to both its function and its role in the antiphospholipid syndrome. Since patients with antiphospholipid antibodies do not have circulating antibody-antigen complexes despite the presence of large amounts of 2 GPI and antibodies in the circulation, the epitope www.intechopen.com Structural Changes in β 2 -Glycoprotein I and the Antiphospholipid Syndrome 25 for the auto-antibodies on 2 GPI must be cryptic. As a consequence, the conformation of 2 GPI in plasma must be different from the one coated on an ELISA tray in tests for the detection of antibodies. The crystal structure of 2 GPI revealed a fishhook-like shape of the molecule (Bouma et al., 1990;Schwarzenbacher et al., 1990) (Figure 1). Part of the epitope that is recognized by auto-antibodies is located in the first domain of 2 GPI (Iverson et al., 1998;de Laat et al., 2006). The crystal structure indicated that these amino acids are expressed on the surface of domain I of 2 GPI and should thus be accessible for autoantibodies. But the lack of binding of antibodies to 2 GPI in solution fits better with a circular structure of 2 GPI, a structure that was originally suggested by Koike et al. (1998). Electron microscopy (EM) studies showed that when antibodies were bound to 2 GPI, the protein indeed showed a fishhook-like shape, but native 2 GPI in the absence of antibodies showed a closed 'circular' shape ( Figure 2) in which domains I and V interact with each other (Agar et al., 2010).
In blue the negatively charged amino acids are depicted and in red the positively charged amino acids. In yellow the large positive charged patch is shown within the fifth domain of 2GP that forms the binding site for anionic phospholipids. Picture was made using Cn3D version 4.1, produced by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).  By changing pH and salt concentrations, it was possible to convert 2 GPI from the native closed conformation into the open conformation and back (Agar et al., 2010). Analysis of EM pictures showed that more than 99% of plasma 2 GPI was in a closed conformation. These observations suggested that plasma 2 GPI circulates in a circular (closed) conformation, whereas after interaction with antibodies 2 GPI undergoes a major conformational change into a fishhook-like (open) structure.

Effect of the conformation of β 2 GPI on coagulation
2 GPI is present in high concentrations in plasma, and depletion of 2 GPI from normal plasma does not influence the results of coagulation assays (Oosting et al., 1992;Willems et al., 1996). When antibodies toward 2 GPI were added to plasma, clotting times prolonged in a 2 GPI-dependent way. This effect of anti-2 GPI antibodies is known as lupus anticoagulant (LA) activity. A LA could also be seen with open 2 GPI (Agar et al., 2010). When closed native 2 GPI was added to normal plasma or 2 GPI-depleted plasma, no effect on an activated partial thromboplastin time (aPTT)-based clotting assay was observed.

A B
www.intechopen.com When open 2 GPI was added to plasma or 2 GPI-depleted plasma, the aPTT prolonged. Addition of antibody and open 2 GPI together to normal plasma gave an additional anticoagulant effect on top of the effect of open 2 GPI alone (Agar et al., 2010). The presence of 2 GPI in a certain conformation is dependent on the presence of anionic surfaces but also on the method of purification of 2 GPI. As was described by Agar et al (2010), the conformation of 2 GPI is dependent on the pH and salt concentration used during the purification of native 2 GPI. The structure of 2 GPI needs confirmation before doing experiments with purified 2 GPI. Schousboe (1985) was the first who described 2 GPI as a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. With the current knowledge of the two conformations (Agar et al., 2010), these studies were most likely performed with the open conformation of 2 GPI, the only conformation that gives a prolongation in an aPTT. Furthermore, it was suggested that 2 GPI inhibited coagulation by inhibition of activation of coagulation factor XII (Henry et al., 1988) and coagulation factor XI (Shi et al., 2004). It is likely, that also these observations were obtained with the open 'activated' conformation of 2 GPI. The purification method that all these groups used included the use of perchloric acid, and these harsh conditions may have induced a conformational change of closed native 2 GPI into the open activated conformation of 2 GPI. It is highly recommended that the conformation of 2 GPI is confirmed in coagulation tests or by electron microscopy. It is expected that in the near future specific immunological assays will become available to determine the specific conformational state of 2 GPI.

Functional consequence of the conformations of β 2 GPI
Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, plays a role in activating the hosts' immune response by binding to white blood cells (van der Poll & Opal, 2008). Analysis of electron microscopy pictures of 2 GPI incubated with LPS, showed that LPS was bound to domain V of 2 GPI and thereby induced a conformational change to the open conformation of 2 GPI ( Figure 3) and showed the same fishhook-like shape when 2 GPI was bound to anionic surfaces or antibodies (Agar et al., 2011a).
A functional consequence of 2 GPI binding to LPS was investigated in an in vitro cellular model of LPS-induced tissue factor (TF) expression. Native plasma-purified 2 GPI dosedependently inhibited LPS induced TF expression both in monocytes and endothelial cells (Agar et al., 2011a). Furthermore, in an ex vivo whole blood assay 2 GPI inhibited LPS induced interleukin-6 expression, an inflammatory marker in innate immunity. Furthermore, an in vivo relevance was found of the interaction between 2 GPI and LPS in plasma samples of 23 healthy volunteers intravenously challenged with LPS (de Kruif et al., 2007). A reduction of 25% of baseline values was observed of 2 GPI immediately after LPS injection, suggesting an in vivo interaction between 2 GPI and LPS. Also, a highly significant, negative association was found between plasma levels of 2 GPI and plasma levels of inflammatory markers TNF , IL-6 and-IL-8 after the LPS challenge. In agreement to this, there was a highly significant inverse relation between the baseline 2 GPI levels and the observed temperature rise upon LPS challenge (Agar et al., 2011a). Subsequently, a significant difference in 2 GPI levels was observed between non-sepsis www.intechopen.com and sepsis patients in the intensive care unit. 2 GPI levels returned to normal after recovery, again suggesting an in vivo interaction between 2 GPI and LPS. The reduction in 2 GPI levels after LPS challenge coincided with an uptake of 2 GPI by monocytes. When 2 GPI binds to LPS, it changes conformation after which the LPS-2 GPI complex is taken up by monocytes. Interestingly, the binding of this complex could be dose-dependently inhibited by receptor associated protein, indicating that binding of 2 GPI is mediated via a receptor of the LRP-family (Lutters et al., 2003;Pennings et al., 2006;Urbanus et al., 2008).
The ability of native 2 GPI to inactivate LPS in vivo might offer opportunities to use 2 GPI for the treatment of sepsis. 2 GPI binds to LPS via domain V of 2 GPI. It seems logical to use domain V of 2 GPI, and not the whole molecule for sepsis treatment. The use of the whole protein could induce the formation of auto-antibodies against the cryptic epitope located in domain I, which could lead to the development of APS (McNeil et al., 1990). The use of only domain V could potentially avoid the development of the pathological auto-antibodies against 2 GPI.

Evolutionary conservation of the LPS binding site in β 2 GPI
A survey of the genome sequences of 40 vertebrates and of the fruit fly and roundworm, revealed a 14% amino acid homology with  2 GPI from the fruit fly Drosophila melanogaster and a 17% homology with the roundworm Caenorhabditis elegans, the most primitive organisms in which  2 GPI could be identified (Agar et al., 2011b). It was found that the majority of mammals showed 75% or higher homology for the complete human 2 GPI amino acid sequence. Remarkably, all mammals except the platypus, showed 100% homology for all 22 cysteine residues present in 2 GPI, which serve an important structural role in protein folding and stability.
Surface plasmon resonance experiments revealed that the peptide AFWKTDA comprising a hydrophobic loop within a large positively charged patch in CCP-V of 2 GPI, was able to compete for binding of 2 GPI to the LPS. This amino acid sequence within domain V was completely conserved in all mammals (Agar et al., 2011b). The same amino acid sequences also attenuated the inhibition by 2 GPI in a cellular model of LPS-induced tissue factor expression. This indicated that the AFWKTDA amino acid sequence found in the genome of all mammals is the LPS binding region within CCP-V of 2 GPI. From this it can be concluded that the LPS scavenging function is not only present in humans but evolutionary conserved throughout all mammals. This certainly emphasizes an important role for 2 GPI in biology, explains its high concentration in blood, its conformational change and suggests a general role in scavenging of unwanted toxic substances and cells.

β 2 GPI as an overall scavenger
Analysis of the structure and function of 2 -GPI has induced a turn into our understanding of the antiphospholipid syndrome. For the last two decades the protein During the last years, more and more evidence has become available that 2 GPI is a more general scavenger in our circulation. Maiti et al (2008) showed a 2 GPI-dependent phosphatidylserine (PS) expressing apoptotic cell uptake by macrophages. Binding of 2 GPI to these cells caused recognition and uptake of the 2 GPI-apoptotic cell complex by the LRP receptor on macrophages. The receptor for binding to apoptotic cells was determined to be the Ro 60 receptor (Reed et al., 2008(Reed et al., , 2009). Furthermore, another study suggested that the binding of 2 GPI to PS-expressing procoagulant platelet microparticles might promote their clearance by phagocytosis (Abdel-Monem et al., 2010).
Blood contains microparticles (MPs) derived from a variety of cell types, including platelets, monocytes, and endothelial cells. MPs are formed from membrane blebs that are released from the cell surface by proteolytic cleavage of the cytoskeleton (Owens & Mackman, 2011). MPs may be procoagulant because they provide a membrane surface for the assembly of components of the coagulation protease cascade. Importantly, procoagulant activity is increased by the presence of anionic phospholipids, particularly phosphatidylserine (PS), and the procoagulant protein tissue factor (TF), which is the major cellular activator of the clotting cascade (Owens & Mackman, 2011). Since microparticles are considered to be important in coagulation, the efficient recognition and removal of these particles is critical for the maintenance of homeostasis and resolution of inflammation.
It has been shown that autoantibodies to 2 GPI inhibit this uptake of microparticles and bound TF to induce a procoagulant state (Abdel-Monem et al., 2010). The role of TF was studied in a renal injury mouse model that shared many features with thrombotic microvascular disease (Seshan et al,. 2009). Both complement-dependent and complementindependent mechanisms were found to be responsible for endothelial activation and microvascular disease induced by antiphospholipid antibodies obtained from APS patients. The presence of antibodies against 2 GPI showed a disturbed uptake of microparticles leading to increased TF in the circulation, which on its turn caused renal injury. It was also shown that mice expressing low levels of TF were protected against this injury induced by the presence of the antiphospholipid antibodies (Seshan et al., 2009).

Conclusions and future perspective
In conclusion, over the last few years a wealth of interesting data has become available that increased our knowledge on 2 GPI. The discovery of the native state of 2 GPI as a circular protein explained the important paradox that antibodies and protein could circulate separately in blood. Also, other previously ascribed functions were probably functions of the protein induced by the harsh method of purification after which only the open, activated, conformation of the protein was obtained. Furthermore, the potential of antibodies, anionic phospholipids, LPS and probably even more agents to switch the conformation of 2 GPI from a circular to an open activated conformation has given a clue to its role as scavenger, since the open conformation can bind to and subsequently be taken up by cells such as monocytes and macrophages. A role of 2 GPI as overall scavenger in coagulation and innate immunity, two complex processes that are highly intertwined, may provide clues to one of the remaining questions in APS research, namely how binding of antiphospholipid antibodies to 2 GPI result in thrombosis. An interesting option is that By identifying agents that can switch the conformation of 2 GPI, other areas of biology can be identified where 2 GPI might play a role. With the evolutionary conservation that was observed for the protein, its role in biology must be significant. It goes beyond saying that the appreciation of the different conformations and the ability to switch conformation has provided an important basis for research in this area.

Acknowledgments
This research was supported in part by a grant from the Netherlands Organization for Scientific Research (ZonMW 91207002; JCM Meijers and PG de Groot) and an Academic Medical Center stimulation grant (JCM Meijers).