half-life of the C4b • C2b complex is about 3 min at 37°C. Thus the enzyme spontaneously decays by dissociation of the C2b catalytic subunit. This rate of dissociation is increased by C4 binding protein (C4bp), which competes with C2 for a binding site on C4b. C4bp also acts as a cofactor for another control protein called factor I, which destroys the C4b by proteolytic degradation (Fig. 3).
The alternative pathway (AP) was originally discovered in the early 1950s by Pillemer et al. (1954). Pillemer's group studied the ability of a yeast cell wall preparation, called zymosan, to consume C3 without affecting the amount of CI, C2, or C4. A new protein, called properdin, was isolated and implicated in initiating C3 activation independent of the CP. This new scheme was called the properdin pathway. However, this work fell into disrepute when it was realized that plasma contains natural antibodies against zymosan, which implied CP involvement in Pillemer's experiments. The pathway was rediscovered in the late 1960s with the study of complement activation by bacterial lipopolysaccharide and with the discovery of a C4-deficient guinea pig. The 1970s witnessed the isolation and characterization of each of the proteins of this pathway until it was possible to completely reconstruct the entire AP by recombining each of the purified proteins (Schreiber et al., 1978). The AP is responsible for most of the complement activation produced by biomaterials, although the CP may also be involved to some degree.
The proteins of this pathway are described in Table 2. Their actions can be conceptually divided into three phases: initiation, amplification, and regulation (Fig. 4). Initiation is a spontaneous process that is responsible for the nonselective nature of complement. During this stage, a small portion of the C3 molecules in plasma (0.005%/min) undergo a conformational change that results in hydrolysis of the thioester group, producing a form of C3 called C3(H2G).
For a short period this C3(H20) looks like C3b and will bind to factor B in solution. This interaction requires Mg2+ and can be inhibited by E.DTA. The C3(HzO) -B complex is a substrate for factor D, which cleaves the B protein to form an alternative pathway C3 convertase: C3(H20) • Bb. When it is cleaved by D to form Bb, a cryptic serine protease site is exposed on the Bb protein. This C3(H20) - Bb enzyme then can act to cleave more C3 to form C3b. The C3b can then follow several paths. First, it may attach, through its thioester, to a surface (Figs. 3 and 4) and lead to more C3 convertase (amplification), or it may react with water and become metabolized. Since the half-life of the thioester bond is about 60 fx sec, most of the C3b produced is hydrolyzed and inactivated, a process that has been termed "C3 tickover."
The C3 tickover is a continuous and spontaneous process that ensures that whenever an activating surface presents itself, reactive C3b molecules will be available to mark the surface as foreign. Recognition of the C3b by factor B, cleavage by factor D, and generation of more C3 convertase leads to the amplification phase (Fig. 4). During this stage, many more C3b molecules are produced, bind to the surface, and in turn lead to additional C3b • Bb sites. Eventually, a C3b molecule attaches to one of the C3 convertase sites by direct attachment to the C3b protein component of the enzyme. This C3b—C3b • Bb complex is the alternative pathway C5 convertase and, in a manner reminiscent of the CP C5 convertase, converts C5 to C5b and C5a.
As with the classical pathway, there are a number of control mechanisms that operate to regulate this pathway. The intrinsic instability of the C3b thioester bond (half-life = 60 usee) ensures that most of the C3b (80—90%) is inactivated in the fluid phase. Once formed, the half-life of the C3 convertase (C3b • Bb complex) is only 1.5 to 4 min and this rate of dissociation is increased by factor H. After displacement from the C3b, Bb is relatively inactive. Aside from accelerating the decay of C3 convertase activity, factor H also promotes the proteolytic degradation of C3b by factor I (Figs. 3 and 4). Factors H and I also combine to limit the amount of active C3(H20) produced in the fluid phase.
In addition to factor H, there are several cell membrane-bound proteins that have similar activities and act to limit complement-mediated damage to autologous, bystander cells. Decay-accelerating factor, or DAF, displaces Bb from the C3 convertase and thus destroys the enzyme activity. DAF is found on all cells in the blood (bound to the plasma membrane through a unique lipid group) but is absent in a disease called proximal nocturnal hemoglobinuria (PNH), which manifests a high spontaneous rate of red blood cell lysis. In addition to DAF, there are two other cell-bound control proteins: membrane cofactor protein (MCP) and CR1 (complement receptor 1, see later discussion). MCP is found on all blood cells except erythrocytes, while CR1 is expressed on most blood cells. Both function like factor H to displace Bb from the C3 convertase and act as a cofactor for the factor I-mediated cleavage of C3b. A soluble recombinant form of CRT (sCRl) is now being evaluated for its efficacy in mitigating complement-mediated damage in several settings of acute inflammatory injury.
Properdin, the protein originally discovered by Pillemer, functions by binding to surface-bound C3b and stabilizing the C3 and C5 convertase enzymes. The normal half-life of the C3b ■ Bb complex is increased to 18-40 min. Although properdin is not necessary for activation of the alternative pathway,
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