31 Antiphospholipid Antibody–Induced Pregnancy Loss and Thrombosis

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 anti- body, 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

-glycoprotein I bound to resting endothelial cells, although the basis for the inter- action of β

-glycoprotein I with viable endothelial cells remains unclear [12, 13]. As β

-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

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

+C3 C3a

C3b

+C3

C3bBbP

(C5 convertase) C4b2a3b C3bBb3bP

C5

C5b

C5b-8 C5a

+C6,C7,C8

+(C9)n

MAC C5b-9

Cell Iysis endothelial activation

Recruitment and activation of inflammatory

cells

Figure 31.1. Proposed mechanism for the pathogenic effects of aPL antibodies on tissue injury. First, aPL anti- bodies 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.

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 pro- teases, 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, are ligands for complement receptors 1 and 2 (CR1 and CR2) and the β

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 comple- ment 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 gen-

eration 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-

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 clusterin. There are also soluble complement inhibitors, including C1 inhibitor (inhibits C1r and C1s) and factor H and C4 binding protein (inhibitors of C3 and C4, respectively) [29].

Complement and Pregnancy Loss

Recent murine studies underscore the importance of complement regulation in fetal control of maternal processes that mediate tissue damage. In mice, Crry is a mem- brane bound intrinsic complement regulatory protein with function similar to MCP and DAF. Crry blocks C3 and C4 activation on self membranes, inhibiting the classi- cal and alternative pathway C3 convertases [30]. The absolute necessity for appro- priate complement inhibition in a normal pregnancy has been demonstrated by the finding that Crry deficiency in utero leads to progressive embryonic lethality [31].

Importantly, Crry

embryos are completely rescued from this 100% lethality and live pups are born at a normal Mendelian frequency when the Crry

parents are inter-crossed with C3

mice to generate C3

, Crry

embryos, suggesting that the Crry

embryos die in utero due to their inability to suppress complement activa- tion and tissue damage mediated by C3. Based on these findings, we proposed that aPL antibodies activate complement within decidual tissue, overwhelm the nor- mally adequate inhibitory mechanisms described above, and induce inflammation and fetal damage.

To examine the role of complement in aPL antibody–induced pregnancy loss, we used a murine model of antiphospholipid syndrome (APS) induced by passive transfer of human aPL antibodies (aPL–IgG). Using this model, we observed that aPL–IgG induces complement activation and that by blocking this activation we can prevent fetal loss and growth restriction [32, 33]. In our studies, we found that F(ab´)2 fragments of aPL–IgG do not mediate fetal injury, indicating that the Fc portion of IgG is necessary for aPL antibody–mediated injury [32]. Do aPL antibod- ies initiate inflammation and fetal demise by cross-linking stimulatory Fcg recep- tors (Fc γRs) expressed on monocytes, neutrophils, platelets, or mast cells, or by activating the classical pathway of complement? When we examined the effects of aPL–IgG in mice with a targeted deletion of the common γ subunit that lack stimu- latory Fc receptors (Fcγ

), we found that they were not protected from poor preg- nancy outcomes after passive transfer of aPL–IgG. Thus, aPL–IgG initiates fetal damage in the absence of activating FcγRs. Yet, the Fc portion of aPL–IgG is required, supporting a role for activation of the classical pathway in fetal injury.

Indeed, mice lacking C4 were protected.

To further study the role of complement in aPL antibody–induced pregnancy

loss, we inhibited C3 activation with Crry–Ig, an exogenously administered

inhibitor of C3 activation. Crry–IgG prevented aPL–IgG induced complement depo-

sition within the deciduas and protected mice from pregnancy complications. We observed similar results when C3-deficient mice were treated with aPL–IgG; there was no pregnancy loss or growth restriction [33]. While the nature of the antigens recognized by aPL antibodies is clearly important, complement activation seems to be the major effector mechanism by which these antibodies mediate tissue injury.

Complement components downstream from C3 are involved in aPL antibody- induced tissue injury [32]. Blockade of C5 activation with anti-C5 monoclonal anti- bodies, as well as experiments in C5-deficient mice, indicate that C5 split products are required for pregnancy complications in APS. In fact, the pro-inflammatory sequelae of C5a–C5aR interactions and the recruitment of neutrophils are critical intermediates linking pathogenic aPL antibodies to fetal damage. After aPL–IgG treatment, embryos die surrounded by massive leukocyte infiltration. C5a, a potent anaphylatoxin that recruits and activates neutrophils by interacting with C5aR, plays an important role in aPL–IgG induced tissue injury. Absence of C5aR or blockade of C5aR with specific antagonist peptides protects mice from pregnancy complications of aPL–IgG treatment [32]. Activation of complement is amplified by the alternative pathway, evidenced by the protection afforded by factor B deficiency or treatment with anti-factor B monoclonal antibodies [32, 34]. Neutrophil deple- tion with monoclonal antibodies also prevented aPL–IgG induced embryo injury, confirming the importance of inflammation in this model [32].

We propose the following mechanism for the pathogenic effects of aPL antibodies on tissue injury (Fig. 31.2). First, aPL are preferentially targeted to the placenta where they activate complement via the classical pathway. C3 and subsequently C5 are activated. C5a is generated and attracts and activates neutrophils, monocytes, and mast cells, and stimulates the release of inflammatory mediators, including reactive oxidants, proteolytic enzymes, chemokines, and cytokines. Proteases secreted by inflammatory cells, particularly neutrophils, can also increase C5a gen- eration by directly cleaving C5 [35], leading to autocrine and paracrine stimulation and further recruitment of leukocytes.

Complement and Thrombosis

Complement activation and thrombophilia are linked in inflammatory diseases.

Using an in vivo microcirculation model, we showed that aPL–IgG antibodies induce endothelial cell activation, and enhanced and accelerated thrombus forma- tion in the presence of a vascular injury [11, 36, 37]. The average size of injury- induced thrombi in mice treated with aPL–IgG was 5 times greater than that of mice treated with IgG from healthy individuals. Administration of Crry–Ig significantly decreased aPL–IgG induced enhancement of thrombosis, resulting in values near those of the controls [32]. Thus, complement activation also appears to play a central role in aPL antibody–induced thrombophilia.

But how does complement activation cause a prothrombotic phenotype?

Complement fragments (such as C3a or C5a) can directly activate endothelial cells

by binding to cell surface receptors or indirectly activate endothelial cells by

binding to receptors on neighboring phagocytes or platelets; complement split

products may thereby induce a prothrombotic phenotype [38, 39]. C5a–C5aR inter-

actions can trigger thrombosis. In a rat model of antibody mediated thrombotic

glomerulonephritis, C5aR blockade prevents thrombus formation and leukocyte

accumulation and, similar to our findings, depletion of neutrophils prevents glomerular thrombosis, despite the presence of C3 and MAC [40]. C5a also recruits and stimulates other inflammatory cells, such as mast cells, monocytes, macrophages, and eosinophils. Enzymes in eosinophil granules function as power- ful procoagulants [41]. C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils which may be triggers of thrombosis [42]. Mast cells also release tumor necrosis factor α (TNF-α) stored in granules upon stimulation by C5aR, and TNF-α induces tissue factor expression on endothe- lial cells and monocytes [43]. Finally, C5a can directly induce release of tissue factor on endothelial cells, demonstrating an important aspect of interrelationship between the inflammatory and coagulation cascades [44]. It is therefore not sur- prising that in C5 deficient mice, aPL–IgG did not cause thrombophilia [45].

Formation of C5b-9 MAC involves the sequential assembly of the 5 terminal com- plement proteins into a heteropolymeric complex. MAC can activate pro-inflam- matory and prothrombotic signaling pathways through the interaction of membrane associated MAC proteins with heterotrimeric G proteins [24, 46]. The insertion of MAC into the membrane causes considerable perturbation of the lipid bilayer. MAC may cause tissue necrosis by lysing cells. However, most nucleated cells are resistant to lysis and non-lethal effects of the MAC which trigger cell activa- tion are likely to be more important to pathology [44, 47]. MAC activation is regu- lated by CD59, which interferes with its assembly and generation of the MAC pore.

C3a

C1,C4,C2 aPL

C3 Factor B

ALTERNATIVE PATHWAY AMPLIFICATION

PMN MØ

C5aR

C5aR platelets

INFLAMMATION THROMBOSIS TISSUE DAMAGE

FETAL DEATH

MAC C5aR c3b

C5aR

Figure 31.2. Schematic diagram of the 3 complement activation pathways.

CD59 has been shown to protect glomerular endothelium from thrombosis, whereas blockade of CD59 with a monoclonal antibody was associated with C5b-9 formation in glomeruli and increased platelet and fibrin deposition [48]. Mice express 2 CD59 genes (mCd59a and mCd59b); mCd59b knockout mice present a strong phenotype characterized by hemolytic anemia, platelet activation, and thrombophilia [49].

Although the cause of tissue injury in APS is likely multifactoral, complement activation is an absolute requirement for 2 of the most deleterious phenotypic out- comes in this condition. Blockade of complement is effective in preventing fetal injury and thrombosis in experimental models of APS and may have therapeutic implications in patients. Complement inhibitors are now being tested in patients with inflammatory, ischemic, and autoimmune diseases. Identifying the comple- ment components involved in aPL antibody–induced pregnancy complications in patients may define targets for interventions that prevent, arrest, or modify APS.

References

1. Wassermann A, Neisser A, Bruck C. Eine serodiagnostiche Reaktion bei Syphilis. Deutsche Med Wochenschr 1906;32:745–746.

2. Pangborn MC. A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med 1941;48:484–486.

3. Asherson RA, et al. The “primary” antiphospholipid syndrome: major clinical and serological fea- tures. Medicine (Baltimore) 1989;68:366–374.

4. Lockshin MD. Antiphospholipid antibody and antiphospholipid antibody syndrome. Curr Opin Rheumatol 1991;3:797–802.

5. Lockwood CJ, Rand JH. The immunobiology and obstetrical consequences of antiphospholipid anti- bodies. Obstet Gynecol Surv 1994;49:432–441.

6. Sammaritano LR, Gharavi AE, Lockshin MD. Antiphospholipid antibody syndrome: immunologic and clinical aspects. Semin Arthritis Rheum 1990;20:81–96.

7. Shapiro SS. The lupus anticoagulant/antiphospholipid syndrome. Annu Rev Med 1996;47:533–553.

8. Del Papa N, et al. Endothelial cells as target for antiphospholipid antibodies. Human polyclonal and monoclonal anti-beta 2-glycoprotein I antibodies react in vitro with endothelial cells through adher- ent beta 2-glycoprotein I and induce endothelial activation. Arthritis Rheum 1997;40:551–561.

9. Roubey RA. Tissue factor pathway and the antiphospholipid syndrome. J Autoimmun 2000;15:217–220.

10. Simantov R, et al. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995;96:2211–2219.

11. Pierangeli SS, et al. Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo. Circulation 1999;99:1997–2002.

12. Ma K, et al. High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II. J Biol Chem 2000;275:15541–15548.

13. Meroni PL, et al. Beta2-glycoprotein I as a “cofactor” for anti-phospholipid reactivity with endothe- lial cells. Lupus 1998;7(suppl 2):S44–S47.

14. Kandiah DA, Krilis SA. Beta 2-glycoprotein I. Lupus 1994;3:207–212.

15. Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:1058–1066.

16. Weiser MR, et al. Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement. J Exp Med 1996;183:2343–2348.

17. Korb LC, Ahearn JM. C1q binds directly and specifically to surface blebs of apoptotic human ker- atinocytes: complement deficiency and systemic lupus erythematosus revisited. J Immunol 1997;158:4525–4528.

18. Mevorach D, et al. Complement-dependent clearance of apoptotic cells by human macrophages. J Exp Med 1998;188:2313–2320.

19. Hugli TE. Structure and function of C3a anaphylatoxin. Curr Top Microbiol Immunol 1990;153:181–208.

20. Brown EJ. Complement receptors and phagocytosis. Curr Opin Immunol 1991;3:76–82.

21. Holers VM. In: Rich R, ed. Complement, principles and practices of clinical immunology. St. Louis, MO: Mosby, 1995:363.

22. Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin. Nature 1991;349:614–617.

23. Wetsel RA. Structure, function and cellular expression of complement anaphylatoxin receptors. Curr Opin Immunol 1995;7:48–53.

24. Morgan BP, Meri S. Membrane proteins that protect against complement lysis. Semin Immunopathol 1994;15:369–396.

25. Shin ML, Rus HG, Nicolescu FI. Membrane attack by complement: assembly and biology of terminal complement complexes. Biomembranes 1996;4:123–149.

26. Hourcade D, Holers VM, Atkinson JP. The regulators of complement activation (RCA) gene cluster.

Adv Immunol 1989;45:381–416.

27. Lublin DM, Atkinson JP. Decay-accelerating factor and membrane cofactor protein. Curr Top Microbiol Immunol 1989;153:123–145.

28. Oglesby TJ, et al. Membrane cofactor protein (CD46) protects cells from complement-mediated attack by an intrinsic mechanism. J Exp Med 1992;175:1547–1551.

29. Holers VM. Complement as a regulatory and effector pathway in human diseases. In: Lambris JD, Holers VM, eds. Therapeutic interventions in the complement system. Totowa, NJ: Humana Press:

2000;1–32.

30. Kim YU, et al. Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein. J Exp Med 1995;181:151–159.

31. Xu C, et al. A critical role for murine complement regulator crry in fetomaternal tolerance. Science 2000;287:498–501.

32. Girardi G, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospho- lipid syndrome. J Clin Invest 2003;112:1644–1654.

33. Holers VM et al. Complement C3 activation is required for antiphospholipid antibody-induced fetal loss. J Exp Med 2002;195:211–220.

34. Thurman T, et al. A novel inhibitor of the alternative pathway of complement protects mice from fetal injury in the antiphopsholipid syndrome. Mol Immunol 2004;41:318.

35 Huber-Lang M, et al. Generation of C5a by phagocytic cells. Am J Pathol 2002;161:1849–1859.

36. Pierangeli SS, et al. Effect of human IgG antiphospholipid antibodies on an in vivo thrombosis model in mice. Thromb Haemost 1994;71:670–674.

37. Pierangeli SS, et al. Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobu- lins from patients with the antiphospholipid syndrome. Thromb Haemost 1995;74:1361–1367.

38. Benzaquen LR, Nicholson-Weller A, Halperin JA. Terminal complement proteins C5b–9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells. J Exp Med 1994;179:985–992.

39. Hattori R, et al. Complement proteins C5b–9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface. J Biol Chem 1989;264:9053–9060.

40. Kondo C, et al. The role of C5a in the development of thrombotic glomerulonephritis in rats. Clin Exp Immunol 2001;124:323–329.

41. Samoszuk M, Corwin M, Hazen SL. Effects of human mast cell tryptase and eosinophil granule pro- teins on the kinetics of blood clotting. Am J Hematol 2003;73:18–25.

42. Wojta J, et al. C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils. Blood 2002;100:517–523.

43. Shebuski RJ, Kilgore KS. Role of inflammatory mediators in thrombogenesis. J Pharmacol Exp Ther 2002;300:729–735.

44. Ikeda K, et al. C5a induces tissue factor activity on endothelial cells. Thromb Haemost 1997;77:394–398.

45. Pierangeli SS, et al. Complement C5 Activation is required for antiphospholipid antibody-induced thrombofilia. Arthritis Rheum 2003;48:S163.

46. Shin ML, et al. Membrane factors responsible for homologous species restriction of complement- mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9. J Immunol 1986;136:1777–1782.

47. Morgan BP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J 1989;264:1–14.

48. Nangaku M, et al. CD59 protects glomerular endothelial cells from immune-mediated thrombotic microangiopathy in rats. J Am Soc Nephrol 1998;9:590–597.

49. Qin X, et al. Deficiency of the mouse complement regulatory protein mCd59b results in spontaneous hemolytic anemia with platelet activation and progressive male infertility. Immunity 2003;18:217–227.