Complement-Mediated Coagulation Disorders: Paroxysmal Nocturnal Hemoglobinuria and Atypical Hemolytic Uremic Syndrome

Introduction

Complement is an elaborate system that participates in the innate immune response. Mutations and autoantibodies leading to unregulated complement activation are implicated in the pathogenesis of a variety of human diseases. Among them, paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) are well-established disorders of complement dysregulation. Diseases associated with complement dysregulation are often associated with thrombosis, highlighting the close interaction between the complement and the coagulations cascades. In light of recent advances in complement diagnostics and therapeutics, complement dysregulation emerges as an important driver of human disease that may be effectively treated by complement inhibition.

Brief Historic Perspective

The complement system was first described in the late 19th century. In 1891, Buchner and colleagues discovered a heat labile factor capable of killing bacteria. This factor was named “alexin” after the Greek word for “protection.” A few years later, Paul Ehrlich introduced the term “complement” to describe a heat labile antimicrobial factor that is capable of attacking and killing bacteria. Complement was further understood not just as single factor but as two fractions, midpiece and endpiece (now renamed as C1 and C2, respectively) by Ferrata and Brand. Between 1914 and 1921, complement components C1, C3, and C4 were characterized.

By the early 1930s, complement had emerged as a complex system comprising four factors which act sequentially, C1, C4, C2, and C3. Later, Mayer and colleagues from the Johns Hopkins University proposed the “one-hit” theory, suggesting that a single complement reaction could lead to red cell lysis. The same group also described the classical pathway cascade. It wasn't until 1955 that Pillemer and colleagues introduced the highly controversial properdin system, now known as the alternative pathway of complement (APC). More than 30 years later, another pathway of complement activation—the lectin pathway—was described.

The ongoing research for more than a century resulted in the discovery of the first complement inhibitor, eculizumab. The introduction of eculizumab, a monoclonal that blocks terminal complement by binding to C5, into clinical practice, renewed interest in complement therapeutics and paved the way for future research into precision medicine pertaining to complement inhibition.

Complement for the Clinician

The complement system comprises over 30 soluble and membrane-bound proteins and provides an important line of defense against bacteria, fungi, and viruses. Activation of the complement cascade may be initiated by the classical, the alternative, and the lectin pathway, which will be described in detail. Other ways of complement activation have been postulated, mainly involving the crosstalk between complement and thrombosis, and these will be described separately. However, it is important to bear in mind that the alternative pathway serves as an amplification loop for the lectin and classical pathway. Accordingly, it has it has been estimated that the APC accounts for 80% of complement activation products even when the complement cascade is initiated by the classical and lectin pathways. In order to prevent unwanted complement activation on host cells, the complement system includes numerous regulatory proteins. Complement regulatory proteins are either membrane bound or soluble. Among multiple complement regulators, the ones most relevant with complement-mediated coagulation disorders will be highlighted below.

Membrane-Bound Complement Regulators

The following complement regulators on the cell membrane have been linked to complement dysregulation in human diseases:

1.
CD55 or decay accelerating factor (DAF) is a widely expressed glycosylphosphatidylinositol (GPI) anchor membrane protein that accelerates the decay of cell-surface-bound C3 convertases, thus limiting the formation of the C5 convertase and ultimately the formation of the membrane attack complex (MAC). Because CD55 is a membrane protein, it only inhibits complement activation on cells that express it.
2.
CD59 is another widely expressed GPI-anchored membrane protein and is the major inhibitor of the terminal complement pathway. It functions by binding to C8 and C9 in the assembling MAC, thereby inhibiting pore formation of the MAC.
3.
CD46 (membrane cofactor protein or MCP) is a transmembrane cell surface protein that also accelerates the decay of C3 convertases. CD46 is expressed on most cell types but not on erythrocytes. In conjunction with soluble factor I, CD46 also inactivates C3b to iC3b, thereby preventing reformation of the C3 convertase.
4.
Thrombomodulin is an endothelial cell receptor that modulates the generation of thrombin via its cofactor role in the activation of protein C; however, it also regulates factor I-mediated C3b inactivation.

Soluble Complement Regulators

1.
Factor H regulates the alternative pathway in the fluid phase and on cell surfaces. It can bind directly to C3b and disrupt the C3 convertase of the alternative pathway. It also serves as an important cofactor for factor I to cleave and inactivate C3b.
2.
Factor I is synthesized in the liver and regulates the classical, alternative, and lectin pathways. In the alternative pathway, it cleaves and inactivates C3b.
3.
Factor B is cleaved by factor D to generate the C3 convertase of the alternative pathway C3(H20)Bb.

Activation of the Classical Pathway

The classical pathway is mainly activated by antibody-antigen complexes recognized via C1q. Among antibody isotypes, immunoglobulin M (IgM) is the most effective in activating complement. Activation of complement with the four subclasses of IgGs varies as a function of steric hindrance by the Fab arms in the approach of C1q to the CH2 sites (IgG3>IgG1>IgG2>IgG4). Except for antibodies, C1q binds also directly to certain epitopes from microorganisms or apoptotic cells and to cell surface molecules, such as acute phase proteins that bind to pathogens or affected cells and activate complement. C1q subsequently cleaves C1r that activates C1s protease. Then, C1s cleaves C4 and C2, leading to the formation of classical pathway C3 convertase (C4bC2a). C3 cleavage by the C3 convertase generates the anaphylatoxin C3a and C5 convertase (C4bC2aC3b), which cleaves C5 into C5a and C5b that initiates the terminal pathway of complement. This process is shown in Fig. 25.1 .

In the terminal pathway of complement, C5b binds to C6 generating C5b-6, which, in turn, binds to C7 creating C5b-7, which is able to insert into lipid layers of the membrane. Then, C5b-7 binds C8 and C9, which unfolds in the membrane and binds several C9 molecules forming the MAC.

Activation of the Alternative Pathway of Complement

The alternative pathway is constitutively activated at low levels through the slow spontaneous hydrolysis of C3 that forms C3(H ₂ O). This process is called “tickover.” Therefore the APC is activated on any surface that has the ability to activate complement. The activated C3(H ₂ O) binds factor B, generating C3(H ₂ O)B. Factor B is subsequently cleaved by factor D generating the APC C3 convertase [C3(H ₂ O)Bb]. The C3 convertase then catalyzes the cleavage of additional C3 molecules to generate the anaphylatoxin C3a and C3b, which attach to cell surfaces. This initiates the amplification loop, where C3b pairs with factor B on cell surfaces, bound factor B is cleaved by factor D to generate a second form of the APC C3 convertase (C3bBb). Membrane-bound C3 convertase then cleaves additional C3 to generate more C3b deposits, establishing an amplification loop. The binding and cleavage of an additional C3 molecule to C3 convertase forms the APC C5 convertase (C3bBbC3b), that cleaves C5 to C5a and C5b. C5b initiates the terminal complement pathway that forms the MAC, as described above. The process from initial spontaneous C3 activation through amplification is depicted in Fig. 25.2 . Both C3 and C5 APC convertases are stabilized by properdin, which also serves as a selective pattern recognition molecule for de novo C3 APC convertase assembly.

Activation of the Lectin Pathway of Complement

Lectin pathway activation is initiated by mannose-binding lectins (MBLs) that recognize carbohydrate structures on microbial surfaces, such as viruses, protozoan parasites, fungi, and various bacteria. Other pattern recognition molecules involved in lectin pathway activation are ficolins and collectin 11. These molecules act through MBL-associated serine proteases (MASPs), which generate the C3 convertase (C4bC2a) in a process similar to that of the classical pathway.

Complement and Thrombosis

The complement and coagulation cascade have long been considered independent pathways. Over recent years, several studies have linked complement and thrombosis. Direct complement effects on the coagulation cascade are mediated by the anaphylotoxin C5a. C5a has been suggested to: increase inflammatory cytokines, downregulate ADAMTS-13, generate tissue factor and PAI1, decrease levels of protein S and increase protein C resistance because of increased factor VIII activity, and, most importantly, activate thrombin. On the other hand, coagulation factors are also able to activate the complement cascade. In particular, factor XII interacts with C1 of the classical pathway. Thrombin cleaves C3 and also generates C5a in the absence of C3. It is also well described that the fibrinolytic factors, plasmin and kallikrein, directly cleave C3.

Beyond direct effects, increased thrombotic tendency also has been observed as a result of intravascular hemolysis caused by complement dysregulation in complement-mediated anemias. Thus, free hemoglobin results in platelet activation, ADAMTS13 inhibition, release of endothelial and red cell procoagulant microparticles, increased levels of tissue factor and oxygen reactive species, and depletion of nitric oxide (NO). The deficiency of NO as a result of scavenging by free hemoglobin contributes to endothelial dysfunction and platelet activation causing vascular and prothrombotic abnormalities.

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