31 Antiphospholipid Antibody–Induced Pregnancy Loss and Thrombosis Guillermina Girardi and Jane E

31 Antiphospholipid Antibody–Induced Pregnancy Loss and Thrombosis Guillermina Girardi and Jane E. Salmon Antiphospholipid (aPL) antibodies are a family of autoantibodies that exhibit a broad range of target specificities and affinities, all recognizing various combina- tions of phospholipids, phospholipid binding proteins, or both. The first aPL antibody, a complement fixing antibody that reacted with extracts from bovine hearts, was detected in patients with syphilis in 1906 [1]. The relevant antigen was later identified as cardiolipin, a mitochondrial phospholipid [2]. The presence of aPL antibodies in serum has been associated with arterial and venous thrombosis and recurrent pregnancy loss [3–7], but the pathogenic mechanisms mediating these events are unknown. Several hypotheses have been proposed to explain the cellular and molecular mechanisms by which aPL antibodies induce thrombosis and fetal loss. There are reports that aPL antibodies activate endothelial cells, monocytes, and platelets [8–10]. In vivo and in vitro studies have shown that exposure to aPL antibodies induces activation of endothelial cells and a prothrombotic phenotype, as assessed by upregulation of the expression of adhesion molecules, secretion of cytokines, and the metabolism of prostacyclins [8, 10, 11]. aPL antibodies recognize β 2-glycoprotein I bound to resting endothelial cells, although the basis for the inter- β action of 2-glycoprotein I with viable endothelial cells remains unclear [12, 13]. As β 2-glycoprotein I is considered a natural anticoagulant [14], some authors propose that aPL antibodies interfere with or modulate the function of phospholipid binding proteins involved in the regulation of coagulation, activate platelets, or induce monocytes to express tissue factor [9]. That endothelial cell, monocyte, and platelet activation are associated with aPL antibodies and thrombophilia, and that these cell phenotypes may also occur as a consequence of complement activation products, suggested a role for complement activation in aPL antibody–induced tissue damage. The Complement System Complement is part of the innate immune system and provides one of the main effector arms of host defense. Complement was first identified as a heat labile prin- ciple in serum that “complemented” antibodies in the killing of bacteria. We now know that complement is a system of more than 30 proteins in plasma and on cell surfaces that act in concert to protect the host against invading organisms, initiates inflammation, and tissue injury [15]. 395 396 Hughes Syndrome There are 3 pathways of complement activation: the classical, mannose binding lectin, and alternative pathways (Fig. 31.1). These 3 initiation pathways converge at the point of cleavage of the third component of complement (C3) and the steps leading to the cleavage of C3 are amplifying cascades of enzymes, analogous to those in coagulation. The classical pathway is activated when natural or elicited antibodies bind to antigen and unleash potent effectors associated with humoral responses in immune mediated tissue damage. Activation of the classical pathway is initiated by the binding of the C1 complex to antibodies complexed to antigens on cell or bacterial surfaces. C1s first cleaves C4, which binds covalently to the cell (or bacterial) surface, and then cleaves C2, leading to the formation of a C4b2a enzyme complex, the C3 convertase of the classical pathway. Activation of the classical pathway by natural antibody plays a major role in the response to neoepitopes unmasked on ischemic endothelium, and thus may be involved in reperfusion injury [16]. In addition, the classical pathway is activated through the action of (C3 convertase) C4b2a Recruitment and activation C3a of inflammatory +C3 cells C5a (C5 convertase) C5 C3b C4b2a3b C3bBb3bP C5b +C3 +C6,C7,C8 C3bBbP C5b-8 +(C9)n Cell Iysis C5b-9 endothelial activation MAC Figure 31.1. Proposed mechanism for the pathogenic effects of aPL antibodies on tissue injury. First, aPL antibodies are preferentially targeted to the placenta where they activate complement via the classical pathway. The complement cascade is initiated; C3 and subsequently C5 are activated. C5a is generated and attracts and activates neutrophils, monocytes, and platelets, and stimulates the release of inflammatory mediators, including reactive oxidants, proteolytic enzymes, chemokines, cytokines, and complement factors. Complement activation is amplified by the alternative pathway. This results in further influx of inflammatory cells and ultimately fetal injury. Depending on the extent of Damage, either death in utero or fetal growth restriction ensues. PMN-neu- trophil; MF-monocyte–macrophage. (Adapted from Girardi G, et al Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003;112:1652, with copyright permission from the Journal of Clinical Investigation. Antiphospholipid Antibody–Induced Pregnancy Loss and Thrombosis 397 C-reactive protein (CRP) and serum amyloid P as they bind nuclear constituents released from necrotic or dying cells, or directly when apoptotic bodies derived from cells bind C1q [17, 18]. Activation of the mannose binding lectin pathway is triggered by binding of the complex of mannose binding lectin and the serine proteases, mannose binding lectin – associated proteases 1 and 2 (MASP1 and MASP2, respectively). MASP2 acts in a manner similar to C1s to lead to the formation of the C3 convertase enzyme. MASP1 may also be able to cleave C3 directly. Alternative pathway activation mechanisms differ in that they are initiated by the binding of spontaneously activated complement components to the surface of pathogens. Under normal physiologic conditions, C3 undergoes low-grade spontaneous hydrolysis. This pathway is antibody independent and is triggered by the activity of factor B, factor D, and properdin. Triggering of the alternative pathway is initiated by the covalent binding of a small amount of C3b to hydroxyl groups on cell surface carbohydrates and proteins and is activated by low-grade cleavage of C3 in plasma. This C3b binds factor B, a protein homologous to C2, to form a C3bB complex. Factor D cleaves factor B bound to C3b to form the alternative pathway C3 complex C3bBb. Properdin (P) binds to and stabilizes this enzyme complex. Convergence of the 3 complement activation pathways on the C3 protein results in a common pathway of effector functions. The initial step is generation of the fragments C3a and C3b. C3a, an anaphylatoxin that binds to receptors on leuko- cytes and other cells, causes activation and release of inflammatory mediators (reviewed in [19]). C3b and its further sequential cleavage fragments, iC3b and C3d, β are ligands for complement receptors 1 and 2 (CR1 and CR2) and the 2 integrins, CD11b/CD18 and CD11c/CD18, present on a variety of inflammatory and immune accessory cells (reviewed in [20, 21]). C3b is covalently bound to the site of complement activation and then binds to C4b or C3b in the convertase enzymes of the classical (C4b2a3b) and alternative (C3bBb3bP) pathways, respectively, forming C5 convertase enzymes. This C3b acts as an acceptor site for C5, which is cleaved by C5 convertase to C5b and anaphylatoxin C5a. C5a is a potent soluble inflammatory anaphylatoxic and chemotactic molecule that promotes recruitment and activation of neutrophils and monocytes and mediates endothelial cell activation through its receptor, C5a receptor (C5aR [CD88]), a member of the heptahelical 7 trans- membrane spanning protein family [22, 23]. Binding of C5b to the target initiates the non-enzymatic assembly of the C5b-9 membrane attack complex (MAC). MAC is a pore forming lipophilic complex that can destroy cells by permeabilization of the membranes and act as an ion channel that triggers cell activation. Insertion of C5b-9 MAC causes erythrocyte lysis through changes in intracellular osmolarity, while C5b-9 MAC damages nucleated cells primarily by activating specific signaling pathways through the interaction of the membrane associated MAC proteins with heterotrimeric G proteins [24, 25]. Because activated complement fragments have the capacity to bind and damage self tissues, it is imperative that autologous bystander cells be protected from the deleterious effects of complement. To avoid the potentially deleterious activities of complement acting on self tissues, the activation of the complement cascade is tightly controlled by membrane and soluble regulatory proteins. C3 is an impor- tant site of such complement regulation. Inhibition of C3 activation blocks the generation of most mediators of inflammation and tissue injury along the complement pathway. Two membrane bound proteins regulate the activation of C3 on the surface of host cells [26, 27]. Decay accelerating factor (DAF) and membrane co- 398 Hughes Syndrome factor protein (MCP) are expressed on most human cells and inactivate C3 conver- tases, thus limiting all 3 initiating pathways. DAF inhibits the assembly and acceler- ates the decay of the C3 convertase enzymes that activate C3 and amplify the classical and alternative complement pathways. MCP is a co-factor for factor I mediated degradation and inactivation of C3b and C4b [28]. A third protein, CD59, also membrane anchored, prevents assembly of C5b–9 MAC. CD59 inhibits both the insertion and polymerization of C9, blocking the MAC formation and thus pre- venting the terminal effector functions of complement [24]. The MAC is also inhib- ited by S protein and

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