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34 The Influence of Antiphospholipid Antibodies on the Protein C Pathway

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on the Protein C Pathway

Philip G. de Groot and Ronald H. W. M. Derksen

Introduction

Blood coagulation is the mechanism that maintains the integrity of the high pres- sure closed circulatory system of blood. To prevent extravasations of the blood after injury, the hemostatic mechanism, which includes platelets, coagulation, and fibrinolytic proteins in plasma and endothelial cells, is activated. A platelet plug will be formed that prevents further blood loss. Subsequently, the coagulation cascade replaces the unstable platelet plug by the stable fibrin clot. An essential feature of the hemostatic reaction is that platelet deposition and fibrin formation is localized and limited to the immediate area of the injury. Therefore, it is essential that differ- ent natural anticoagulant mechanisms are operative to regulate coagulation. When the natural anticoagulant mechanisms do not function optimally, this will lead to thrombotic complications. One of the most important natural anticoagulant systems is the protein C pathway [1, 2]. The high number of patients that have been described with heterozygous protein C or protein S deficiency and familial throm- bophilia highlight the clinical importance of the anticoagulant properties of protein C and protein S. Complete protein C deficiency represents a potentially lethal condi- tion. Thrombotic complications can be controlled with protein C replacement therapy [3, 4].

Antiphospholipid antibodies (aPL) are a heterogeneous group of autoantibodies defined by 2 very distinct assay methods. One group, called lupus anticoagulant (LA), is defined as antibodies that inhibit in vitro phospholipid dependent coagula- tion assays. The second group, anticardiolipin antibodies (aCL), is defined by their ability to bind to negatively charged phospholipids in an enzyme-linked immunosorbent assay (ELISA) [5]. Paradoxically, the presence of aPL in plasma is a major risk factor for the development of arterial and venous thrombosis and is not associated with a bleeding diathesis, as would be expected when clotting times are prolonged [6].

The pathophysiology that underlies the relation between the aPL in plasma and the risk for thrombo-embolic complications is still unexplained [7] but an attractive hypothesis is that the aPL interfere with (one of) the natural anticoagulant path- ways in the body. In this chapter we will discuss one of these possibilities: a link between aPL and the protein C system.

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Protein C axis

In the early 1980s, a phospholipid dependent antithrombotic pathway was described that soon turned out to be one of the body’s major defense mechanisms to uncontrolled coagulation. Vascular endothelium expresses a membrane bound receptor on its surface, thrombomodulin, which binds thrombin and thereby alters its substrate specificity. Thrombin bound to thrombomodulin is no longer able to activate platelets or to convert fibrinogen into fibrin, but it converts a vitamin K dependent protein, protein C, into activated protein C (APC) [8]. APC is a physio- logical anticoagulant via its potential to inactivate clotting factors Va and VIIIa, which results in inhibition of further thrombin formation (Fig. 34.1). Protein C acti- vation by thrombin–thrombomodulin complex is further enhanced about 20-fold when protein C is bound to the endothelial cell protein C receptor (EPCR).

Thrombomodulin also influences fibrinolysis. Thrombin bound to thrombomod- ulin activates TAFI (thrombin inducible fibrinolysis inhibitor, carboxypeptidase B).

TAFI removes carboxyterminal lysine residues from fibrin, thereby preventing the binding of tissue plasminogen activator (tPA) and plasmin(ogen) to fibrin. TAFI thus reduces fibrinolysis. Activation of TAFI is thought essential for the stability of a fibrin clot [9].

The role of the protein C axis extends beyond hemostasis. Activated protein C has potent anti-inflammatory properties and administration of human activated protein C significantly decreases mortality in patients with severe sepsis [10].

Furthermore protein C has a role in cell survival and cell proliferation [11].

Protein C is a vitamin K dependent glycoprotein with a molecular weight of 62 kDa. In blood it circulates as an inactive zymogen, mostly in the form of a two chain molecule [12]. The thrombin–thrombomodulin complex activates protein C by

Figure 34.1. The protein C pathway.

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splitting off a 12 amino acid activation peptide after cleavage at position Arg 169.

The plasma concentration of protein C is 4 µg/mL (65 nM) and the circulating level of APC in healthy subjects is about 2.2 ng/mL [13]. The biological half-life of protein C is about 8 hours. In plasma, APC is neutralized by forming complexes with the APC inhibitor (PCI or PAI-3), α1-antiproteinase (α1-antitrypsin) and α2- macroglobulin. The inactivation of APC by PCI is accelerated by heparin [14].

Protein S, another vitamin K dependent protein, amplifies the activity of APC.

Protein S is a single chain plasma glycoprotein with a molecular weight of 70 kDa [15]. The total plasma concentration of protein S is about 25 µg/mL (350 nM). The biological half-life of protein S is 42.5 hours. Protein S forms a 1:1 complex with APC on phospholipid surfaces. Two independent processes regulate the APC co- factor activity of protein S. First, protein S is cleaved by thrombin, resulting is a molecule that has lost its APC co-factor function [16]. The second inhibition of APC co-factor activity of protein S is the result of its ability to form a 1:1 complex with C4b binding protein (C4BP). Approximately 60% of plasma protein S circulates in complex with C4BP. Only the free form of protein S has APC co-factor activity [17].

The complex between protein S and C4BP arises by a non-covalent association between protein S and the β-chain of C4BP. About 80% of C4BP in the circulation is composed of 7 α-chains and 1 β-chain, joined together by inter-chain disulfide bridges in the C-terminal region. The remaining C4BP contains only α-chains.

During acute-phase reactions the plasma levels of C4BP increases dramatically.

However, due to the preferential synthesis of the α-chain, only β-chain-free C4BP is synthesized. As β-chain-free C4BP cannot bind protein S, the levels of free protein S remain stable during inflammation.

Thrombomodulin is an endothelial transmembrane protein consisting of a lectin domain, 6 EGF domains, a carbohydrate rich domain, a transmembrane domain, and a 36 amino acid long intracellular tail [18]. The thrombin-binding region is located in EGF domains 5 and 6 but for protein C activation, the presence of EGF domain 4 is also essential. Thrombomodulin expression is not restricted to the cell membrane. There also exists a soluble form in plasma. Plasma concentrations of soluble thrombomodulin are around 25 ng/mL. In many vascular disorders (includ- ing diabetes mellitus) soluble thrombomodulin levels are increased [19].

Endothelial cell protein C receptor is a transmembrane protein homologous to the major histocompatibility complex class 1 family members [20]. Human EPCR is palmitoylated on the terminal cysteine, suggesting that ECPR is located in the cave- olae of the cells. Large vessel endothelial cells predominantly express EPCR. Its function is to concentrate protein C near the surface of the vessel wall and to increase APC formation through mass action effects. When APC is generated, it remains bound to EPCR for a short while. As long as APC is bound to EPCR, it cannot inactivate factors Va and VIIIa. However, it can cleave the protease activat- able receptor PAR-1, initiating responses in PAR-1 bearing cells.

The discovery of the concept of activated protein C resistance followed by the

explanation of the resistance by a mutation in clotting factor V further emphasizes

the importance of the protein C-axis as an antithrombotic pathway [21, 22]. During

blood coagulation factor V is converted into factor Va. Factor Va serves as a non-

enzymatic co-factor in the prothrombinase complex. The presence of factor Va

tremendously accelerates further thrombin formation [23]. Factor Va is inactivated

by proteolytic degradation of its heavy chain by APC. The inactivation is a sequen-

tial event; the first cleavage takes place at Arg 506, followed by cleavages at Arg 306

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and Arg 679. The first cleavage results in a partly inactivated form of factor Va with 30% residual activity, cleavage at Arg 306 results in a completely inactive molecule [24]. In 1993, Dahlback and co-workers described patients with thrombophilia whose plasma was resistant to APC [21]. With this APC resistance test large popula- tions of thrombophilic patients could be characterized. In 1994, a point mutation in factor V gene was identified as the genetic risk factor that described the patients with APC resistance [22]. The point mutation is a GA transition of nucleotide 1691, which predicts the synthesis of a variant factor V molecule with an Arg506Gln mutation. Replacement of Arg 506 by Gln will prevent cleavage of factor Va by APC, which results in a delay in the inactivation of factor Va and thus in sustained throm- bin formation.

Specificity of aPL

Research over the last 15 years has shown that the subset of aPL that are related to the risk for thrombo-embolic complications do not recognize phospholipids alone. In conventional aCL and LA assays the antibodies are primarily directed towards different phospholipid binding proteins, most notably β

2

-glycoprotein I and prothrombin [25, 26]. Both β

2

-glycoprotein I and prothrombin bind to nega- tively charged phospholipids. When bivalent complexes between 2 target mole- cules and 1 antibody molecule are formed, the affinity for a phospholipid surface of β

2

-glycoprotein I and prothrombin increases about 100 times, which subse- quently favors the binding of the complexes to phospholipids over the binding of other phospholipid binding proteins present in plasma, such as clotting factors [27].

New studies on the correlation between clinical manifestations and the presence of certain sub-populations of aPL suggested that specially those anti–β

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-glycopro- tein I antibodies that are able to prolong clotting times are clinical relevant [28].

The recognition that β

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-glycoprotein I is the real target for aPL logically led to the deduction that the pathogenesis of thrombosis is the result of interference of anti–β

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-glycoprotein I antibodies with the biological function of β

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-glycoprotein I.

Despite different types of functions related to β

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-glycoprotein I in in vitro assays, individuals with complete deficiency of β

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-glycoprotein I do not have an increased thrombotic risk [29]. Also knockout mice for β

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-glycoprtein I do not show an increased occurrence of thrombo-embolic complications [30]. Apparently β

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-glyco- protein I is not involved in regulation of pro-or antithrombotic pathways. However, in the presence of anti–β

2

-glycoprotein I antibodies, the situation may be different.

Due to the increased affinity of β

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-glycoprotein I antibody complexes for phospho- lipids, β

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-glycoprotein I may now be able to interfere with the function of proteins such as protein C and protein S, that only can express their biological activity in the presence of negatively charged phospholipids.

Whether anti– β

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-glycoprotein I antibodies are the only pathological subset of

autoantibodies is not known. A large number of antibodies directed to other phos-

pholipid binding proteins have been identified. The most interesting antibodies

found are antibodies directed against prothrombin, protein C, protein S, and

thrombomodulin, because the presence of these antibodies could be immediately

linked to thrombo-embolic complications [31, 32].

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aPL and the Protein C axis

The assembly of coagulation complexes on negatively charged phospholipid sur- faces is a prerequisite for their activity: no exposure of anionic phospholipids, no binding, no activity. Prolongation of clotting times in coagulation assays by aPL is the result of competition between antibody–protein complexes and clotting factors for the available catalytic surface. Also, assembly of the APC–protein S complexes on anionic phospholipid surfaces is essential for the catalytic activity. Thus, it is logical to assume that when aPL inhibit the binding of the clotting factors, they also inhibit the binding of protein C and protein S and thereby their activity. Extensive investigations, however, have shown that there are more interactions between aPL, β

2

-glycoprotein I, and proteins of the protein C system. Long before the discovery of the role of co-factors in the aPL syndrome, Canfield and Kisiel isolated an APC inhibitor with a N-terminal amino acid sequence identical to β

2

-glycoprotein I [33].

This original and forgotten observation has been repeated and extended to charac- terize aPL/β

2

-glycoprotein I as an inhibitor to the protein C system. The antiphos- pholipid antibodies and/or β

2

-glycoprotein I can interfere with the protein C system in different ways [34].

1. The antibodies inhibit the formation of thrombin, the activator of protein C (the thrombin paradox)

2. The antibodies inhibit the activation of protein C via interference with thrombo- modulin (anti-thrombomodulin antibodies).

3. The antibodies inhibit APC activity (acquired APC resistance). This can be achieved:

(a) via inhibition of the assembly of the proteins on the anionic surface:

(b) via direct inhibition of APC activity;

(c) via antibodies against the cofactors Va and VIIIa.

4. The antibodies interfere with the level of protein C and/or S (acquired protein C/S deficiency).

The Thrombin Paradox

At first glance it is not easy to understand that inhibition of thrombin formation

could cause thrombosis. In 1993, the group of Hanson et al made an interesting

observation [35]. They infused 2 U/kg/min thrombin in baboons that were con-

nected with an ex vivo thrombosis model. The low doses of thrombin reduced

platelet deposition and fibrin incorporation in the ex vivo thrombus. Circulating

APC levels increased significantly. This suggests that low doses of thrombin prefer-

entially activate protein C. These and other observations have led to the develop-

ment of the so-called thrombin paradox [36]: thrombin exerts both anti-and

prothrombotic properties (Table 34.1) and, as a consequence, thrombin is the key

factor in the regulation of hemostasis. When thrombin has so many different activi-

ties, its substrate specificity should be well regulated. One of the fundamental ideas

on thrombin activity is that, due to the affinity of thrombin for its substrates and

receptors, the concentration of the formed thrombin is one of the main factors that

determine its substrate specificity (Fig. 34.2). When only low concentrations of

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thrombin are formed protein C is preferentially activated and thrombin acts as an antithrombotic agent. However, when more thrombin is formed, fibrinogen is con- verted into fibrin and factors V and VIII are activated. Thrombin now expresses prothrombotic properties. When large amounts of thrombin are formed, TAFI and factor XIII are activated resulting in an antifibrinolytic response.

An attractive hypothesis to explain the prothrombotic action of aPL relies on its well-known inhibitory effect on thrombin formation. Low concentrations of APC circulate in normal individuals. This suggests a continuous activation of protein C and thus a continuous low-level formation of thrombin. It can be hypothesized that

Table 34.1. Substrates for thrombin.

Substrate Product Activity

Fibrinogen Fibrin Prothrombotic

Factor V Factor Va Prothrombotic

Factor VIII Factor VIIIa Prothrombotic

Factor XI Factor XIa Prothrombotic

Platelets Aggregation Prothrombotic

Protein S Inactivation Prothrombotic

Protein C APC Antithrombotic

Endothelial cells NO & PGI

2

production Antithrombotic Carboxypeptidase B (TAFI) Activation Antifibrinolytic

Urokinase Inactivation Antifibrinolytic

Factor XIII Factor XIIIa Antifibrinolytic

Figure 34.2. The thrombin paradox.

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the presence of aPL inhibits this low-level thrombin formation and so decreases cir- culating APC levels. After damage of a vessel, there now will be insufficient circulat- ing APC to prevent uncontrolled thrombin formation and a thrombus is formed. In this way aPL might shift the hemostatic balance to a more prothrombotic state. To test this hypothesis, we measured levels of APC in patients with SLE and related the levels to presence or absence of aPL. We found that levels of circulating APC were significantly lower in patients with systemic lupus erythematosus compared to normal controls, but, in contrast to what was expected, the lower levels of APC were not correlated with the presence of antiphospholipid antibodies or anti–β

2

-glyco- protein I antibodies [37]. We did find a correlation between APC levels and levels of plasma prothromin and anti-prothrombin antibodies. Therefore, it can be specu- lated that the presence of antiprothrombin antibodies results in lower prothrombin levels and subsequently in lower circulating APC levels. Further studies are neces- sary to validate this hypothesis.

Anti–thrombomodulin or Anti–EPCR Antibodies

In 1983, Comp et al described in an abstract that 2 out of 7 IgGs isolated from LA- positive plasmas inhibited protein C activation by thrombin [38]. They claimed that the antibodies were directed towards thrombomodulin. Two French groups using purified thrombomodulin or endothelial cells as source of thrombomodulin extended these observations [39, 40]. However, other groups did not support these observations [41–43]. Potzsch et al tested IgGs of 46 different patients with LA and found only 2 cases with reduced rates of APC formation [44]. The contradictory results are not easy to explain. There is no doubt that in some patients antibodies towards thrombomodulin can be detected. Whether these antibodies always inhibit the functional activity of thrombomodulin is not known. Thrombomodulin activity is regulated by the phospholipid composition in which it is incorporated. Therefore, differences in assay conditions might explain the contradictory results. However, as the frequency of antithrombomodulin antibodies in the populations tested is very low, it cannot be a general mechanism that can explain the majority of the aPL- related thrombotic events. A few publications showed raised levels of thrombomod- ulin in the circulation of patients with the aPL syndrome [45]. Raised levels of thrombomodulin point to an altered endothelial cell metabolism of thrombomod- ulin, for example, an increased synthesis or a preferential secretion of newly synthe- sized molecules. It is not necessarily associated with a decreased expression of thrombomodulin on the endothelial cell surface. Moreover, circulating thrombo- modulin might add to APC formation and be protective against thrombosis.

In a recent paper Hurtado et al [46] showed in a cohort of 43 patients with APS

syndrome and in a cohort of 87 patients with a first episode of unexplained fetal

death that the presence of anti-EPCR IgG and IgM are independent risk factors for

fetal loss with odds ratios (OR) of 23.0 and 6.8, respectively. In this respect it is

interesting to note that in women with unexplained fetal loss the presence of muta-

tions in the EPCR gene are more prevalent [47] and that disruption of the EPCR

gene in mice causes placental thrombosis and early embryonic lethality [48]. Most

likely, EPCR plays a key role in preventing thrombosis at the maternal–embryonic

interface and antibodies that inhibit EPCR activity might be a cause of the increased

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pregnancy morbidity in APS. More studies are necessary to follow this interesting lead.

Acquired APC Resistance

In a group of 175 patients with systemic lupus erythematosus (SLE), Fijnheer et al showed that hereditary APC resistance is not related to aPL [49]. However, a number of publications have shown that aPL inhibit the binding of protein C and protein S to negatively charged phospholipids, thereby inhibiting their activity [see 50–56]. The antibodies induce an “acquired APC resistance.” Also in a cohort of pediatric patients a significant association between LA , acquired APC resistance, and thrombotic events was found [57]. Interestingly not all LA affect APC activity and the effects are not always seen in every test system [58]. Differences in test systems and/or the heterogeneity of the antibody population influences the outcome of the in vitro test systems. It is not clear whether the acquired APC resis- tance is due to the presence of anti– β

2

-glycoprotein I antibodies [59, 60], anti-pro- thrombin antibodies [61, 62], anti-protein S antibodies [55], or by combinations of antibodies.

Smirnov et al showed that aPL require the phospholipid PE to have anti-APC activity [63]. When the assays were performed in the presence of PE, aPL inhibited APC activity more potently than prothrombinase activity. There are enough clinical data that show that a partial reduction of prothrombin activation is not sufficient to induce a bleeding diathesis in patients. However, a partial reduction of APC antico- agulant activity is a major thrombotic risk [64]. The observation that oxidation of PE enhances the anticoagulant activity of APC without influence on plasma clotting times [65] strengthens the hypothesis on an important role of PE because oxidative damage is believed to be involved in the genesis of the antiphospholipid syndrome.

It has been shown that lipid peroxidation has been increased in patients with aPL [66].

In an elegant study, Mori et al showed that purified β

2

-glycoprotein I inhibits the binding of protein C to phospholipids much better than the binding of prothrom- bin, resulting in a prothrombotic effect [67]. aPL recognize protein C only in the presence of β

2

-glycoprotein I [68]. These results suggest that aPL-induced protein C dysfunction is mediated by β

2

-glycoprotein I.

An interesting but insufficiently studied option is that patients may have autoan- tibodies directed against factor Va that protect this coagulation factor against inac- tivation by APC. Kalafatis et al [69] described a patient with such an autoantibody against factor V, almost complete APC resistance, and severe thrombotic manifesta- tions. Whether these antibodies occur often in APS is not known.

Acquired Deficiencies of Protein C and/or Protein S

There are a number of publications describing acquired protein C or S deficiencies

in isolated patients with aPL syndrome [see 70, 71]. Studies in larger populations of

aPL- positive patients failed to show a correlation between decreased protein C

plasma levels and the presence of aPL. In one study, a small but significant decrease

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of plasma protein S levels was found, although protein S levels in the aPL antibody group still were within the normal range [72]. Nojima et al [55] found a correlation between acquired APC resistance and anti-protein S antibodies and suggested a functional relationship, however, this observation was not confirmed in other studies.

Atsumi et al [73] made an interesting observation. They found that β

2

-glycopro- tein I down regulates the binding between protein S and C4Bp significantly and that aPL abolish the β

2

-glycoprotein I inhibitory effect. Thus, aPL increase the affinity of protein S for C4Bp which may result in an acquired free protein S deficiency. These observations warrant further investigations.

In general, no decrease in protein C or protein S plasma levels were found in patients with aPL, but in some individual cases a combination of low levels of protein C or protein S in combination with the presence of aPL may be found. These cases are probably very susceptible for thrombotic complications

Concluding Remarks

aPL are a heterogeneous population of antibodies directed against different phos- pholipid binding proteins. It is not clear which mechanism is responsible for the thrombogenic activity of all aPL. There is, however, an attractive hypothesis that sug- gests that aPL selectively inhibit one of the dominant natural anticoagulant path- ways, the protein C pathway. Most attention has been focused on the induction of an acquired APC resistance, probably due to interference of the antibodies with binding of protein C or S to negatively charged phospholipids It is questionable whether in aPL-related thrombosis, an acquired APC resistance is responsible for both arterial and venous events. Although exogenous APC could prevent arterial thrombosis in a number of animal models, deficiencies in the protein C axis are correlated with venous thrombosis and not with arterial thrombosis. A safe conclusion is that inter- ference of the protein C pathway can explain a large part of the venous complications in patients with APS. The observations that suggest a correlation between antibodies against EPCR and pregnancy morbidity warrant further studies.

Acknowledgment

Our own studies described in this review were supported by grants from Dutch Organization of Scientific Research (Zon-MW grant no. 902-26-290) and the Netherlands Heart Foundation (grant no. 2003B074).

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