Overview

The AP is responsible for 80-95% of total complement activity. The AP is a complement activation pathway which is triggered by artificial surfaces such as LPS from Gram negative outer membranes and rabbit erythrocytes, zymosan from fungal and yeast cell walls, as well as from many pure polysaccharides, viruses, bacterial, animal tumor cells, parasites and damaged cells. The pathway is initiated mainly by cell surface constituents that are foreign to the host like gram negative and positive bacteria. The alternative pathway generates bound C5b, the same product that the classical pathway generates but it does so without the need for antigen-antibody complexes for initiation. Because no antibody is required, it is a component of the innate immune system. This pathway involves 4 serum proteins like factor B and D. The alternative pathway is also an ancient immune mechanism that is primarily activated onto the surface of pathogens by a process called “tickover”. This formation of C3(H2O) allows for the binding of plasma protein Factor B, which in turn allows Factor D to cleave Factor B into Ba and Bb.Tickover is facilitated by the presence of surfaces that lack complement regulatory proteins and which support the binding of activated C3. It occurs through the spontaneous cleavage of a thioester bond in C3 to form C3i or C3(H2O). Tickover is facilitated by the presence of surfaces that support the binding of activated C3 and/or have neutral or positive charge characteristics (e.g., bacterial cell surfaces). Potential therapeutic targets specific to the alternative pathway include factor B, factor D, and properdin.

C3b Binding to foreign surface: AP recognition occurs in the presence of C3b and an activating substance such as bacterial lipoprotein, surfaces of certain parasites, yeasts, viruses and other foreign body surfaces, such as biomaterials. C3b originates from classical pathway activation and/or from natural spontaneous hydrolysis of C3. The resulting C3b binds to the surface of the activating substance. C3b does not usually bind to mammalian cells because they contain high levels of naturally occurring n- and o-acyl derivative of the deoxy-amino sugar nueraminic (or sialic) acid on the cell membrane. Prokaryotes and parasites lack this cell surface constituent and are, therefore, highly susceptible to C3b binding. (Bansal, US 13/583879).

Binding of Factor B to C3b: Binding of factor B to immobilized C3b is dependent on Mg2+ (Hourcade, J. Biological Chemistry, 270(34), 19716-19722, 1995). Initial binding of B to C3b is believed to be effected through the cooperative binding of two low affinity sites; one of them is located on Ba and the other, which is Mg2+ dependent, on the Bb region of factor B. (Ueda, J. Immunology, 138(4), 1143-1149 1987). In the presence of magnesium, Factor B binds to the C3b which is bound to the activating surface. Once bound, the Factor B protein is susceptible to immediate hydrolysis by a sserine protease called Factor D. Factor D cleaves B, releasing the Ba fragment and forming C3bBb. Properdin stabilizes the C3bBb complex and protects it from decay. C3bBbP enzyme complex exhibits C3 convertase activity (in a manner similar to C4b2a in the classical pathway) and converts C3 to C3b, launching a positive feedback arrangement that significantly amplifies the genesis of C3b molecules. Deposition of these C3b molecules on the foreign cell surface enhances nuetrophil opsonizaiton of the cell. The released C3a analphylatoxin molecules mediate a localized, inflammatory, chemotactic response that recruits neutrophils to the area. The autocatalytic activity of C3bBb convertase results in the produciton of high levels of C3b that can attach to this convertase to produce a C3bB3b complex that demonstrates C5 convertase activity. Circulating C5 is bound by the C3b component and enzymatically hydrolyzed by the activated Bb serine protease into the products C5a and C5b. The the hydrolysis of C5 and the commencement of the formation of the membrane attack complex. (Pugsley, Cardiovascular Toxicology (2003), pp. 43-69). To say another way, cleavage of C3 results in the formation of C3bBb3b, the C5 convertase. This enzyme is also stabilized by P to form C3bBb3bP. C5 convertase can cleave many molecules of C5 into C5a and C5b. If the PC3bB complex can not form, then the MAC complex (C5b-9) will also not form (Bansal, US 13/583879).

To review, conversion of C3 to C3b produces a product that can combine with factor B, giving C3bB.  These complexes are acted upon by factor D to generate C3bBb, which is a C3 convertase capable of cleaving more C3 to C3b, leading to more C3bBb and even more C3 conversion. Under certain circumstances, the C3bBb complex is stabilized by association with the positive regulator properdin (P) by association of C3b and Bb. The C3 convertases can associate with an additional C3b subunit to form the C5 convertase, C3bBbC3b, which is active in the production of the C5-C9 MAC. C3a is an anaphylatoxin that attracts mast cells to the site of challenge, resulting in local release of histamine, vasodilation and other inflammatory effects. The nascent C3b has an ability to bind to surfaces around its site of generation and functions as a ligand for C3 receptors mediating, for example, phagocytosis. The rate limiting step of activation of the alternative pathway in humans is the enzymatic action of factor D on the cleavage of factor B to form the alternative pathway C3 convertase.

Components of the AP

Factor B: 

Human factor B is required for the initiation and propagation of the complement alternative pathway (AP). It also participates in the amplificaiton of the complementCP. Alone, factor B is a zymogen with little known biochemical activity, but in the context of the AP covertases, the factor B serine protease is activated in a process that first involves the association with C3b and subsequently the cleavage of factor B into two fragments, Ba and Bb. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995). 

Complement Protein B is a single polypeptide chain serum glycoprotein with about a 90k MS. It carries the catalytic center of the AP C3 convertase and represents a novel type of serine protease characterized by an unusual structure in the NH2 terminal region of its catalytic fragment Bb, when compared to otehr serine proteases. Assembly of the bimolecular, C3bBb, C3 convertase proceeds in two well defined steps. First, B binds stoichiometrically to C3b in a reaction requiring Mg2+ or Ni2+ ions. Second, complement protein D catalyzes the cleavage of a single arginyllysyl peptide bond of B, resulting in the relase of fragment Ba and the formation of the C3bBb protease. Ueda (J. Immunology) 138(4), 1143-1149, 1987)

Factor B is a tightly regulated, highly specific serine protease. It is the zymogen of the alternative pathway C3/C5 convertase, and is activated when it is split by factor D into two fragments, Ba and Bb, after it has formed a complex with C3b.The interaction between factor B and surface-bound C3b triggers a conformational change in factor B that ultimately creates the C3 convertase (PC3bBb) of the alternative complement pathway. The activation of the AP hinges on a Mg ion-enhanced interaction between factor B and C3b. Upon binding, factor B is rendered susceptible to proteolytic cleavage by factor D, forming fragments Ba and Bb. Bb, in association with C3b, comprises the AP C3 convertase. This complex has serine protease activity and functions to cleave native C3 in C3a and C3b.tic cleavage by factor D, forming Ba and Bb. Bb, in association with C3b comprises the APC3 convertase. This complex has serine protease activity and functions to cleave native C3 into C3a and C3b. 

Binding of factor B to immobilized C3b is depndent on Mg2+. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995).

More specifically, Facto B is a 90 kDa protein consisting of threee domains: a 3 module complement control protein (CCP1, CCP2 and CCP3), a von Willebrand factor A domain, and a C terminal serine protease domain that adopts a default inactive  (zymogen) conformation. The interaction between factor B and surface bound C3b triggers a conformational change in factor B that ultimately creates the C3 convertase (PC3bBb)of the AP. The activation of the AP hinges on a Magensium ion enchanced interaction between factor B and C3b. Upon binding, facotr B is rendered susceptible to proteolytic cleavage by factor D, forming fragments Ba and Bb. Bb, in association with C3b, comprises the AP C3 convertase which has serine protease activity and functions to cleave native C3 into C3a and C3b (Bansal, US 12/675, 220). 

To state yet another way, the first step in the assembly of the AP C3 convertase is the association of factor B with C3b. In this context, factor B can be cleaved by factor D, resulting in Ba and Bb, a process that requires a divalent cation. Ba then dissociates from the complex while Bb remains bound to C3b. C3bBb can be partially stabilized by assocaition with properdin. C3bBb and C3bBbP are active enzymes that cleave C3 at a single point, generating more C3b and ultimately more convertases. AP C5 convertase activity occurs thorugh the association of C3 convertase and additional C3b. In all cases the dissociation of Bb from the convertases is inevitable, irreversible, and followed by inactivation of proteolytic funciton. Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722. 

–Ba fragment: Ba, the NH2 terminal fragment of Factor B, is composed mainly of three tandem short consensus repeats, globular domains found in other complement proteins. It dissociates from the convertase during assembly, leaving the active C3 convertase, C3bBb. The Ba fragment has affinity for C3b and some monoclonal antibodies directed against Ba block factor B-C3b interations. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995).

Factor D: is a highly specific serine protease essential for activation of the AP. (Fung US2002/0081293). Factor D’s only known natural substrate is factor B bound to C3b. It is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor activation by a serpin for its control. It is a highly specific serine protease and cleaves factor B bound to C3b, generating the C3bBb enzyme which is the alternative pathway C3 convertase. 

Properdin:  is a serum glycoprotein which stabilizes the labile C3 convertase (C3bBb) of the AP of the complement system. Properdin binding to the complex C3bB promotes factor D induced cleavage of factor B. Properdin is central to deposition of the activated complement fragment C3b on the surfaces of pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. Individuals with properdin frequently develop meningococcal disease. In the absence of properdin, the C3bB complex cannot be formed and cleaved with factor D. Only oligomeric forms of properdin are considered active. Oligomeric properdin binds C3b and initiates the AP activation. Human properdin is a 469 amino acid protein that includes a signal peptide (amiono  acids 1-28), and six, non-identical thrombospondin type 1 repeates (TSR) of about 60 amiono acids each, as follows: amino acids 80-134 (TSR), amino acids 139-191 (TSR2), amino acids 196-255 (TSR3), amino acids 260-313 (TSR4), amino acids 318-377 (TSR5), and amino acids 382-462 (TSR6). Properdin is formed by oligomerization of a rod-like monomer into cyclic dimers, trimers, and tegramers.

The amino acid sequences of mammalian properdin as well as human properdin are known. Properdin has an unusual structure formed by oligomerisation of a rod like monomer into cyclic dimers, trimers and teramers.  The monomer contains a N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids called “thrombospondin type 1 repeats” or TSR modules. Polyclonal antibodies raised against each TSR have been found to be module specific. Perdikoulis, Biochimica et Biophysica Acta 1548 2001) pp. 265-277. Human properdin is a 469 amino acid protein that includes a signal peptide (amino acids 1-28), 6 non-identical thrombospondin type 1 repeats (TSR) of about 60 amino acids each as follows: amino acics 80-134 (TSR1), amino acids 139-191 (TSR2), amino acids 196-255 (TSR3), amino acids 260-313 (TSR4), amino acids 318-377 (TSR5) and amino acids 3829-462 (TSR6). Bansal US13/849092) discloses that monoclona antibodies that specifically bind to an epitope of the N terminal region consisting of amino acids 71-110 of human properdin which can inhibit properdin function. 

Properdin is composed of multiple identical protein subunits, with each subunit carrying a separate ligand-binding site. Previous reprots suggest that properdin function depends on multiple interactions between its subunits with its ligands. Hourcade (J Biological Chemistry, 281(4), pp. 2128-2132). The amino acid sequences of mammalina properdin are known (GenBank database under Accession No. AAA36489). Human properdin is a 469 amino acid protein that includes a signal peptide (amino acids 1-28), and six-non-identical thrombospondin type 1 repeats (TSR) of about 60 amino acids each. All six TSRs of properdin have different function with TSR5 being involved in properdin function. The N-terminal region of properdin including the first half of the TSR1 are important for properdin functions and monoclonal antibodies which specifcally bind to an peitope of the N-terminal region can inhibit properdin function (Bansal (12/920,997, now US 8,435,512; see also US 13/849092). 

Amplification Loops: 

The AP has a critical role in amplifying the complement response independent of the initiating pathway and in exacerbating inflammatory pathologies. When a C3b molecule is deposited on a activating surface, either by the CP and LP C3 covertases or as a result of bystander effects or tick over, the subsequent binding of the serine proteases factor B (FB) and factor D (FD) leads to the assembly of the AP C3 convertase (C3bBb complex) that cleaves C3 into C3a and additional C3b, which can participiate in the formation of new convertases. In many settings, this amplificaiton loop is the major source of opsonization and feeds all effect arms of the complement system, including inflammatory and adaptive signalling, phagocytosis and the formation of C3 covertases with subsequent MAC assembly. (Lambris, “The renaissance of complement therapeutics” (2018))

The AP involves amplification loops utilizing C3b produced by the CP and LP. Some molecules of C3b generated by the CP C3 convertase are funneled into the AP. Surface bound C3b binds Factor B to yield C3bB, which becomes a substrate for Factor D. Factor D is a serine esterase that cleaves the Ba fragment, leaving C3bBb bound to the surface of the target cell. C3bBb is stabilized by properdin (P), forming the complex C3bBbP, which acts as the AP C3 convertase. This C3 convertase participates in an amplification loop (the AP amplification loop”) to cleave many C3 molecules, resulting in the deposition of C3b molecules on the target cell. In other words, the C3b formed by the AP pathway makes AP C3 convertase which in turn cleaves C3 and generates even more C3b, which feeds back into the loop. This self-perpetuating cycle of reactions generates large amounts of C3b. Some of these C3b molecules bind back to C3bBb to form C3bBbC3b, the AP C5 convertase. C5 convertase cleaves C5 into C5a and C5b. C5b binds to the surface of the cell to initiate the formation of the membrane attack complex.