Pathogenesis of antiphospholipid syndrome

Introduction

The presence of antiphospholipid antibodies (aPL) is the defining feature of antiphospholipid syndrome (APS), a systemic disorder clinically characterized by widespread thrombosis and obstetric complications. Despite their name, the majority of aPL associated with APS are directed against phospholipid-binding plasma proteins, among them β2-glycoprotein I (β2GPI) and prothrombin are recognized as the most relevant antigenic targets.

In the laboratory, aPL can be broadly categorized into those antibodies that are detected using solid-phase assays such as anticardiolipin antibodies (aCL) or anti-β2GPI antibodies, and those antibodies detected by their ability to prolong phospholipid-dependent coagulation tests, known as lupus anticoagulant (LA).

The majority of aCL detected in patients with APS are directed against epitopes expressed on β2GPI, a plasma protein of 326 amino acids produced in the liver. β2GPI is composed of five domains; four are so called complement control protein modules (domain I to IV) followed by the phospholipid-binding domain (domain V). In the circulation, β2GPI exists in a circular form where domain I interacts with domain V. Upon binding of domain V to anionic phospholipids, β2GPI adopts a “hockey stick-like conformation” and antibody binding sites are exposed. Of special interest are antibodies targeting to domain I of β2GPI that showed significant correlations with thrombosis and pregnancy complications. When β2GPI interacts with anti-β2GPI antibody, β2GPI becomes dimerized and conformational changes are introduced in its structure enhancing its affinity for anionic phospholipids.

β2GPI binds to anionic phospholipids on the cell surfaces, and avidly to negatively charged phospholipids in the plasma such as circulating microparticles and oxidized low-density lipoproteins (oxLDL). Despite being a secreted protein circulating in plasma, β2GPI has also the ability to complex with human leukocyte antigen (HLA) class II molecules on cell surfaces.

Several other proteins including prothrombin, annexin V, protein S, protein C, and high-/low-molecular-weight kininogens, have been associated with APS. In particular, prothrombin is the “second” major antigenic target of autoimmune aPL. Two population of antiprothrombin antibodies have been identified, antibodies binding to prothrombin alone, aPT-A, and antibodies recognizing phosphatidylserine-prothrombin complexes known as phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT).

Prolongation of phospholipid-dependent coagulation tests (prothrombin time, activated partial thromboplastin time, kaolin clotting time, dilute Russell’s viper venom time) is caused by antibodies with different specificities against phospholipid-binding proteins, again, mainly β2GPI and prothrombin. In fact, some monoclonal anti-β2GPI and anti-prothrombin antibodies have LA activity in vitro .

Thrombotic complications in APS can occur in most vessels and numerous pathogenic mechanisms have been implicated ( Table 58.1 ). aPL promote thrombosis through inhibition of the natural anticoagulation pathways, impairment of the fibrinolytic system, activation of cells, including monocytes, endothelial cells, platelets, neutrophils and trophoblasts, and activation of the complement system. Pregnancy complications in APS are related to thrombotic or ischemic mechanisms, but also to inflammation, direct placental damage and perturbation of normal trophoblast functions. In this chapter, we detail the proposed pathogenic mechanisms of aPL-mediated induction of a hypercoagulable state in APS.

Table 58.1

Antiphospholipid antibodies (aPL)-mediated pathogenicity.

Effects of aPL on coagulation system

Protein C pathway

Contact activation pathway

β2-glycoprotein I/thrombin interaction

Decreased tissue factor pathway inhibitor activity

Effect of aPL on fibrinolysis

Up-regulation of plasminogen activator inhibitor -1

Interference with activated factor XII

Effects of lipoprotein(a)

Effect of aPL on cells

Induction of procoagulant activity (endothelial cells and monocytes)

Induction of proinflammatory activity ( endothelial cells and monocytes)

Stimulation of platelet activation and aggregation

Activation of neutrophils and NETs release

Disruption of the annexin A5 shield on vascular cells

T-cell interaction

Placental damage

Throphoblast perturbation

Endometrial angiogenesis

Release of extracellular vesicles by vascular cells ( endothelial cells and platelets )

Release of placenta-derived extracellular vesicles

aPL and atherothrombosis Increase uptake of oxLDL/β2GPI complexes by macrophages

Mitochondrial dysfunctions and oxidative stress
Complement activation

aPL , antiphospholipid antibodies; β2GPI , β2-glycoprotein I; oxLDL , oxidized-low density lipoprotein; NETs , neutrophil extracellular traps

Pathogenic mechanisms of aPL

Large in vitro and in vivo experimental evidence supports the pathogenic role of aPL in the clinical manifestations of APS. However, the pathophysiology of APS is only partially understood, and more than one mechanism might be involved. The effect of aPL on cells, via phospholipid-binding proteins, β2GPI and prothrombin, inducing inflammatory and procoagulant responses is considered to be more important pathogenic action than the modification of the function of the protein by aPL.

Despite of the persistently presence of aPL in APS, clinical manifestations occur only occasionally suggesting that aPL are necessary but no sufficient for the development of APS. A “second hit”, such as an inflammatory responses, is necessary to move the thrombotic/hemostatic balance in favor of thrombosis.

aPL and the coagulation system

The coagulation system is an amplification cascade of enzymatic reactions resulting in thrombin formation. Protein C is a major constituent of the anticoagulant system. Thrombin binds to thrombomodulin and activates protein C; activated protein C complexes with protein S on the surface of platelets or endothelial cells. These complexes function as an anticoagulant by proteolytically catalyzing the inactivation of activated factors V and VIII. Both protein C and protein S are phospholipid-binding plasma proteins.

Resistance, related to aPL, to activated protein C is one of the classical mechanisms responsible for thrombosis. The protein C system may be interfered by aPL in different ways. These antibodies inhibit both the activation of protein C by the thrombin/thrombomodulin complex and the inactivation of activated factor V by activated protein C. Rabbit polyclonal antibodies and a human monoclonal anti-β2GPI antibody inhibit activated protein C function. Most prothrombin–antiprothrombin antibodies immune complexes may predispose to thrombosis by interfering with the inactivation of activated factor V by activated protein C in the presence and absence of protein S. The effect of protein S in the protein C pathway may be modified by aPL. Decreased levels of protein S were found in plasma from APS patients, and some of the immunoglobulin (Ig) G that inhibit activated factor V degradation were directed not only to phospholipid-bound protein C but also to phospholipid-bound protein S.

The interference of aPL with the contact pathway of coagulation has been associated with thrombosis and pregnancy complications in APS. The contact activation pathway is initiated with activation of factor XII by negatively charged surfaces. Activated factor XII cleaves factor XI to activated factor XI in the presence of high-molecular-weight kininogen and prekalikrein. β2GPI inhibited the phospholipid-mediated autoactivation of factor XII and the contact activation pathway of coagulation. Moreover, β2GPI binds to factor XI and inhibits activation of factor XI by thrombin and activated factor XII; this inhibition attenuates thrombin generation. Monoclonal anti-β2GPI antibodies enhanced the inhibition of factor XI activation by β2GPI and thrombin complexes.

Thrombin is one of the most potent enzymes involved in the regulation of many biological functions in vivo including inflammation, angiogenesis, arteriosclerosis, neoplastic transformation, and tissue repair. Thrombin is generated, on the surface of activated cells, from its inactive precursor prothrombin by activated factor X, as part of the prothrombinase complex. Thrombin acts as procoagulant by cleaving fibrinogen to fibrin and interacts with protease-activated receptors to activate various procoagulant cells. Thrombin binds glycoprotein (GP) Ib–IX–V complexes on the surface of platelets to promote platelet aggregation and activation. On the other hand, thrombin behaves as anticoagulant on binding to thrombomodulin to favor activation of protein C. The participation of β2GPI in thrombin generation has been demonstrated by the significant reduction of in vitro ability to generate thrombin observed in plasma from β2GPI-null mice. β2GPI directly binds to thrombin, and β2GPI/thrombin interaction may interfere not only with the coagulation system but also with many of the biological functions in which thrombin participates.

The tissue factor pathway inhibitor (TFPI) is a natural anticoagulant that regulates tissue factor (TF)-induced blood coagulation. Anti-TFPI activity identified in the IgG fraction of patients with APS was associated with increased in vitro TF-induced thrombin generation. Inhibitors of TFPI or interference with TFPI activity could upregulate the TF pathway and contribute to the hypercoagulability in APS.

aPL and the fibrinolytic system

Fibrinolysis is a tightly regulated process by which fibrin-rich thrombus is remodeled and degraded. The fibrinolytic system involves the conversion of plasminogen to plasmin by the tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator, and the hydrolytic cleavage of fibrin to fibrin degradation products by plasmin.

The regulation of plasmin generation and activity is highly important for maintaining the homeostatic balance in vivo. Inhibition of the fibrinolytic system may occur at the level of plasminogen activator by specific plasminogen activator inhibitors (PAI-1 and PAI-2) or at the level of plasmin by α2-macroglobulin and α2-plasmin inhibitors.

Impairment of fibrinolysis due to aPL may predispose to thrombosis. Upregulation of PAI-I levels and decreased tPA release were reported in patients with primary APS and venous thrombosis, suggesting that tPA/PAI-1 balance is crucial for developing thrombosis.

Several studies demonstrated that β2GPI and anti-β2GPI antibodies interact with components of the fibrinolytic system. Both the intrinsic and the extrinsic fibrinolysis pathways may be inhibited by aPL. β2GPI blocks the neutralization of tPA by PA-1 in a concentration-dependent manner. The addition of monoclonal aCL blocked the effect of β2GPI, that is, these monoclonal antibodies inhibited fibrinolysis by an elevation in PAI-1 activity. Monoclonal anti-β2GPI antibodies significantly suppressed the intrinsic fibrinolytic activity in vitro; this inhibition was attributed to a reduced contact activation reaction initiated by activated factor XII. Impaired activated factor XII-dependent activation of fibrinolysis was observed in pregnant women with APS with late-pregnancy complications. Antibodies interacting with the catalytic domain of tPA have been detected in APS patients and could represent a cause of hypofibrinolysis.

β2GPI could regulate the fibrinolysis by direct interaction with plasminogen. The fifth domain in β2GPI, phospholipid-binding domain, can be proteolytically cleaved by plasmin to create “nicked” β2GPI, which is unable to bind to phospholipids. Nicked β2GPI binds to plasminogen and blocks its conversion to plasmin by tPA controling the extrinsic fibrinolysis through a negative feedback pathway. On the other hand, intact β2GPI, in the presence of plasminogen and tPA, may promote plasmin generation. Therefore, intact β2GPI may stimulate fibrinolysis, but following plasminogen activation and cleavage, nicked β2GPI could inhibit further plasmin generation.

Moreover, β2GPI interacts with tPA and stimulates fibrinolysis in plasma. Monoclonal aCL with anti-β2GPI activity, obtained from APS patients, could impair activated factor XII-dependent activation of fibrinolysis during pregnancy.

Finally, patients with APS had elevated levels of lipoprotein (a) [Lp (a)]. Patients with maximal elevation of Lp (a) had a reduced fibrinolytic activity, estimated by low D-dimer and PAI-1. Lp(a) competes with plasminogen for binding sites, leading to reduced fibrinolysis. Lp(a) also increases PAI-1 expression by endothelial cells and could interact with cellular plasminogen receptors. This behavior confers Lp(a) a prothrombotic potential.

Annexin 2, an endothelial cell receptor for β2GPI, stimulates fibrinolysis through binding to tPA and plasminogen. Antibodies against annexin 2 found in patients with APS correlated with a history of thrombosis. These antibodies could inhibit cell surface fibrinolysis through direct interaction with endothelial cell annexin 2.

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