A) Phospholipids (PL)

      Anionic PL are essential cofactors for the blood coagulation system. APLA are supposed to bind the phosphodiester group of negatively charged PL. The length and composition of the polar head groups as well the saturation of the fatty acid side chains contribute to the antigenicity (Levy et al, 1990). Possible antigenic targets of APLA on EC include negatively charged PL such as cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid.

• Cardiolipin (CL)
  CL has been most often used to study the interaction of APLA with anionic PL. The antibodies that directly bind to CL are characterized by the presence of a larger than average density of positively charged arginine residues in one of antigen binding loops, which mediate the binding to the negatively charged PL.
• Phosphatidylserine (PS), phosphatidylinositol (PI)
  PS and PI may be possible antigenic targets of APLA on EC. PS is a PL occurring in abundance in the inner leaflet of the cytoplasmic membrane and is exposed to the outer surface after cell activation or apoptosis (Mevorach et al, 1998). PS promotes the interaction of coagulation factors. PI derivates are important mediators of cell activation.
• Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA)
  Zwitterionic antigens such PE and PA have also been implicated in APS (Berard et al, 1996). PL have a positively charged substituted group that interacts electronically with the phosphodiester, thus blocking APLA access to the phosphodiester. APLA do not usually bind the zwitterionic molecule PC, but removal of the positively charged choline group results in the formation of negatively charged PA, which could be bound by APLA.
• Other PL antigens
  Other PL can also be involved. Recently, lysobisphosphatidic acid, a lipid related structurally to CL and present in the membranes of late endosomes, has been identified as another immunologic target for APLA. The team of Division of Angiology and Hemostasis observed that APLA could react with lysobisphosphatidic acid of internal membranes of late endosomes, resulting in accumulation of APLA in late endosomes and subsequent EC activation (Kobayashi et al, 1998; Galve-de Rochemonteix et al, 2000, Dunoyer-Geindre et al, 2001). The oxidation of PL may be necessary for epitopes recognition by some APLA. Horkko et al (1997) demonstrated that many APLA bound to CL only after it had been oxidized, but not to a reduced CL analogue that could undergo oxidation. They suggested that the reactive groups of oxidized CL, such as aldehydes, generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with b₂GPI (and other proteins) and that they are epitopes for APLA.

      Whether this broad response of APLA indicates polyclonality or crossreactivity could not be determined using sera as a source of antibody. It has been shown that a human monoclonal IgM LA is able to bind to PA, PI and PS. This provided evidence for the polyspecificity of at least some APLA (Thiagarajan et al, 1980).

      Binding of APLA to PL is also influenced by the physical state of the PL. Mice immunized with hexagonal versus lamellar phase PL are more likely to develop APLA (Rauch et al, 1990). Protein cofactors like b₂GPI may shift the orientation of some PL from the lamellar to the hexagonal phase.

B) Proteins

Beta₂ glycoprotein I (b₂GPI)

      b₂GPI, also called apolipoprotein H, is a major protein constituent of human plasma, where its concentration is about 200 mg/L. It is the main factor for the recognition of anionic PL by APLA.

      - Structure

      b₂GPI is a single-chain, 5 kDa protein consisting of 326 amino acids. It contains large numbers of Pro and Cys residues, and is highly glycosylated (Figure 1). The protein is a member of the complement control protein or short consensus repeat (SCR) superfamily, characterized by repeating stretches of about 60 amino acid residues, 'Sushi domains', each with a set of 16 conserved residues and 2 fully conserved disulfide bonds. It has five repeating SCR domains. The first four homologous repeat regions consist of about 60 amino acids with 2 disulfide bonds in each domain. The fifth domain contains 80 amino acids and 3 disulfide bonds. The protein binds to phospholipid membranes via the cationic portion of its fifth SCR domain. b₂GPI has a weak affinity for anionic PL but APLA, binding to domains I and V, increase this affinity, due to the bivalency of the antibodies and to conformational changes induced in the b₂GPI protein.

      

Fig. 1 : Structural representations of human blood plasma b₂GPI revealing the extended chain of the five SCR domains

(A) Ribbon drawing of b2GPI with consecutive domains labelled I-V. N-linked glycans, as well as the position of the putative O-linked glycan, Thr130, are indicated by a ball-and-stick model. The strands are shown in red and helices in green (B) Topology diagram of b2GPI. The central sheets of all five domains are labelled B2(-B2)-B3-B4(-B5), the N- and C-terminal -sheets are labelled B1'-B2' and B4'-B5', the -helix and the 3/10 helix are denoted A1 and A2 and numbers of residues delimiting secondary structure elements are given. Disulfide bonds are indicated with dashed lines. The positions of N-glycosylation are given by hexagons; a diamond indicates the putative O-glycan. Horizontal dashed lines mark domain boundaries (C) Ribbon representation of domain III of b2GPI with labelled secondary structure elements. The two fully conserved disulfide bonds are shown in yellow (D) Ribbon representation of domain V of b2GPI with labelled secondary structure elements. The three disulfide bonds are indicated with yellow lines. The aberrant face, which contains the membrane-binding site, is located on the right-hand side.

      - Functions

      It was found that b₂GPI interacts specifically with lipoprotein(a) and the endothelial cell protein annexin II (Ma et al, 2000). Although the physiologic importance of b₂GPI anticoagulant activity is still unclear, b₂GPI acts at least in vitro as an inhibitor of the intrinsic blood coagulation pathway due to its ability to interact with negatively charged surfaces, which in turn are necessary for the activation of factor XII (Schousboe et al, 1985). Interestingly Mori et al (1996) have shown that b₂GPI can inhibit the anticoagulant activity of activated protein C. Thus, it remains unclear whether b₂GPI in vivo has anticoagulant or procoagulant properties. b₂GPI has been reported to inhibit adenosine diphosphate-mediated platelet aggregation and the prothrombinase activity of activated platelets (Shi et al, 1993). b₂GPI binds to cells undergoing apoptosis and may be involved in the rapid, noninflammatory clearance of these cells by phagocytes. Recent study showed that the opsonization of apoptotic cells with anti-b₂GPI antibodies may be a proinflammatory event, stimulating the presentation of apoptotic cell antigens by dendritic cells. Thus, anti-b₂GPI antibodies might possibly contribute to an autoimmune response (Rovere et al, 1999). In addition, b₂GPI binds to the cell surfaces by binding to negatively charged molecules such as anionic PL, heparan sulfate proteoglycans and preferentially to oxidized low density lipoproteins. This property has been proposed to be clinically relevant providing a link between anti-b₂GPI antibodies and atherogenesis (Matsuura et al, 1998).

A) Immunoglobulin classes

      Previous data have shown that the strongest associations of ACA and anti- b₂GPI antibodies with clinical manifestations of APS involve antibodies of the IgG isotype. Cohen et al (1993) determined in vivo that immunisation of mice with pathogenic IgG and IgM ACA, isolated from the serum of a patient with APS, induce the production by the mice of anti-ACA with ACA activity. The mice developed overt APS. IgG ACA were found to have higher pathogenic potential than IgM ACA. However, several works suggest that IgM as well as IgA may also be associated with the disease, although to a lesser extent. Anti-b₂GPI antibodies of IgG, A, and M classes have been reported in 84.8%, 59.3% and 51.5% of patients with primary APS, respectively (Lacos et al, 1999). Amoroso et al (2003) evaluated the prevalence of IgG and IgM antibodies to various antigens in sera from 87 patients affected by SLE. IgG ACA, IgG anti-PA, IgG anti-PI, IgG anti-PS, and IgG anti- b₂GPI were found in 53%, 37%, 32%, 38%, and 24% of patients, respectively. IgM-ACA, IgM anti-PA, IgM anti-PI, IgM anti-PS, and IgM anti- b₂GPI were detected in 15%, 17%, 18%, 14%, and 16%, respectively.

• IgG
  The association of thrombotic risk with high-titer IgG ACA or anti- b₂GPI antibodies, often together with LA, has been confirmed in several studies (Gattorno et al, 1995; Silver et al, 1996). Sammaritano et al (1997) investigated whether the presence of ACA of a specific IgG subclass is associated with clinical complications of APS. They found that IgG₂ was the predominant subclass of ACA, detected in 75% of the patients, and it was significantly associated with thrombotic complications. The IgG subclass distribution of anti- b₂GPI and ACA in patients with primary APS and secondary APS was studied recently (Samarkos et al, 2001). Mean values for anti- b₂GPI antibodies were as follows: IgG₁- 24.4%, IgG₂- 70.2%, IgG₃- 5.0%, IgG₄- 0.4%. The reported IgG subclass distribution for ACA was: IgG₁- 40.1%, IgG₂- 32.8%, IgG₃- 23.7%, IgG₄- 3.3%. Comparing the ranking of IgG subclasses of anti- b₂GPI antibodies (IgG₂ >IgG₁ >IgG₃ >IgG₄ ) and ACA (IgG₁ >IgG₂ >IgG₃ >IgG₄ ), IgG₂ was the most prevalent subclass for anti- b₂GPI antibodies whereas for anti-CL antibodies IgG₁, IgG₂ and IgG₃ were all frequently elevated. An association between IgG₂ and IgG₃ anti-b₂GPI antibodies and venous thrombosis has been shown, while IgG₂ and IgG₃ ACA were found to be more specifically associated with arterial thrombosis.
• IgM
  In a study determining the distribution of ACA in primary and secondary APS, IgM were found in 26% of secondary APS cases and in 15.8% of primary APS group (Vianna et al, 1994). The antibodies of IgM isotype were related mainly to thrombocytopenia and heart valve disease (Diri et al, 1999). APLA, associated with infections, typically are IgM isotypes and are usually transient (Jaeger et al, 1992).
• IgA
  Evidence that IgA antibodies may be important continues to accumulate (Lacos et al, 1999). A significant relationship has been demonstrated between increased IgA levels and a history of venous thrombosis, thrombocytopenia, heart valve disease, livedo reticularis, and epilepsy. Interestingly, black patients with primary and secondary APS appear to have a higher frequency of IgA anti-b₂GPI antibodies, suggesting genetic predisposition for these antibodies (Diri et al, 1999).
• Coexistence of different classes of APLA in the same patient
  It has been recently reported that the concurrent presence of IgG, IgM, and IgA appears to increase the frequency of recurrent spontaneous abortions as compared with the presence of a single isotype among the autoantibodies (Guglielmone et al, 1999). Vogel et al (1991) determined APLA in an unselected group of 63 SLE patients. They found APLA in 50.8% of the patients and the simultaneous presence of LA and ACA was associated with an increased of arterial thromboembolic events.

      In summary, various studies have demonstrated that APLA are a large and heterogeneous family of immunoglobulins from IgG, IgM, and IgA classes and that the simultaneous presence of APLA increases the risk of clinical complications.

A) Screening assays for LA (Table 2)

      

Table 2 : Main screening assays for LA
Test	Coagulation pathway
aPTT (activated Partial Thromboplastin Time) daPTT KCT (Kaolin Clotting Time) dRVVT (dilute Russell's Viper Venom Time) dPT (dilute Prothrombin Time) Textarin time	contact activation of the intrinsic pathway contact activation of the intrinsic pathway contact activation of the intrinsic pathway final common pathway (RVV directly activates FX to FXa and to a lesser extent FIX to FIXa VIIa TF activation of FIX, FIXa activation of FX, VIIa TF activation of FX and the prothrombinase reaction Pt activator (in the presence of FV, calcium and PL)

      The presence of LA is often first suspected when the aPTT is abnormal and fails to correct with normal plasma. Among screening coagulation tests, the aPTT is more sensitive than PT, probably because of the lower PL content of the reagent used in aPTT. A common concept is that the amount of PL in the test system is a critical determinant of sensitivity. Test systems with reduced amount of PL such as daPTT, KCT, dRVVT, dPT thus might offer the possibility of increased sensitivity due to their low PL concentration (Working Group on Haemostasis of the "Société Française De Biologie Clinique", 1993). The Textarin time was found to be the most sensitive screening test for LA when compared with the other test systems. Data show that the PL concentration is not the only determinant of assay sensitivity to LA. Altering the incubation time of aPTT, rather than the PL concentration, was a sensitive way of detecting LA (Robert et al, 1994) but it does function with few cephaloplastins. Furthermore, for partly unknown reasons, some assays appear to be more sensitive to certain subgroups of LA. Therefore, at least two different types screening assays must be performed before the presence of LA can be ruled out. The presence of platelets or platelet fragments in plasma after centrifugation can affect the results of coagulation tests, particularly when plasmas are frozen before testing, and the use of twice centrifuged plasma, or filtration through a 0.2-mm filter, has been advocated to avoid this problem (Ames et al, 1996). This is particularly important for assays without added PL (KCT) or with very low amount of PL (dPT). The residual amount should not exceed 10 G/l (Exner et al, 2000).

      The protein dependence of LA may affect the tests differently. For example, it has been shown that KCT is most abnormal in the presence of Pt-dependent antibodies (Galli et al, 1995), whereas the dRVVT is mainly abnormal in the presence of b₂GPI-dependent antibodies.

B) Mixing studies for LA

      The presence of an inhibitor is usually documented by mixing patient plasma with normal plasma and by demonstrating a persistence of an abnormal clotting time. The sensitivity of LA testing depends on the ratio of patient plasma to normal plasma used to detect the anticoagulant effect, and these variations have not been standardized. The most common ratio of patient to normal plasma, used in mixing studies, is 1:1. However, Clyne et al (1993) showed a relatively high incidence of negative mixing studies using a 1:1 ratio in aPTT system. Some authors have suggested 4:1 mixture for evaluation of mildly prolonged aPTT (McNeil et al, 1991). Other groups have proposed a 1:4 mixture for some cases (Petri et al, 1997).

      The problem with negative mixing studies may be related to the observation that some LA show time-dependent inhibition in clotting assays. Up to 15% of LA may show correction of the prolonged clotting time if tested immediately after mixing but a lack of correction if the clotting time is repeated after incubation of patient and normal plasma. These data have been challenged by Exner (2000), who demonstrated that upon buffering the mixture, the time dependence is abolished.

      There is a lack of uniform criteria for the evaluation of mixing studies. One approach is to determine an index of correction. The basic formula for this index is:

Index= 100x(b-c)/a, where a= clotting time of patient plasma, b= clotting time of patient + normal plasma mixture, and c= clotting time of normal plasma.

C) Confirmatory assays for LA

      A number of confirmatory assays based on the principle of adding to or altering the PL content of the test system have now been described (Table 3). They can be grouped according to the screening assay on which they are based. In the diagnosis of LA, it is important to use a confirmatory study that corresponds to the screening test which is abnormal.

      

Table 3 : Confirmatory assays for LA
Screening assay	Source of PL
aPTT based assays dRVVT KCT dPT	Platelet neutralisation procedure, PL dilutions, hexagonal phase PL Platelet vesicles, platelets Platelet vesicles, liposomes PL dilutions

      The basis of these assays is to determine the effect of altering the PL content of the assay system, by adding platelets, platelet vesicles or hexagonal phase PL. The majority of test systems that are now employed to demonstrate PL dependence use increased concentrations of PL or frozen thawed platelets. The increased PL or platelet membranes 'neutralize' or 'bypass' LA. The platelet neutralisation procedure uses washed frozen and thawed platelets as a source of PL and is most commonly used in aPTT (Triplett et al, 1983). The hexagonal phase PL test is based on the finding that PE is capable of supporting coagulation test and can effectively neutralize LA activity. Comparing these 2 tests, platelet neutralisation procedure and hexagonal phase PL in systemic lupus erythematosus plasmas, Reber et al (1994) found that platelet neutralisation procedure gave a higher rate of detection than hexagonal phase PL and other tests of detection. Addition of platelets may also shorten the aPTT in the presence of anti-F VIII antibodies.

      As shown in Figure 4, LA and ACA may occur independently or may coexist. LA and ACA activities may be due to the same antibody, or the activities may be physically separable (Champley, 1991). There is another family of antibodies, called reagin antibodies, which are observed in patients with APS. This group of antibodies reacts with the Venereal Disease Research Laboratory (VDRL) reagent. The VDRL reagent denotes a mixture of antigens, including CL, cholesterol and lecithin (Singh et al, 2001).

A) APLA in healthy population

      Estimates of the prevalence of APLA in healthy populations vary depending on the criteria used. It has been reported that ACA exist in approximately 5% of normal individuals, although less than 2% showed persistently elevated levels (Vila et al, 1994).

B) APLA in patients with no thrombosis or pregnancy morbidity, but with infections, drugs, auto-immune disorders

      APLA are commonly found after certain acute or chronic infections. They develop in as many as 30% of children after viral infections. A high proportion of children and adults infected with mycobacteria, malaria, Q fever, hepatitis C, parvovirus B19, cytomegalovirus, etc. develop APLA (McNeil et al, 1991; Mengarelli et al, 2000). In HIV-positive patients, Constans et al (1998) found a 41% frequency of IgG APLA; IgM APLA were positive in 7% and IgG anti-b₂GPI were rare (3-4%). In Geneva University Hospital, ACA were determined by an ELISA assay in 116 HIV-1-infected patients and positive test was found in 23.3% of the patients with a predominance of IgG ACA isotype (Bernard et al, 1990). Another study at Geneva University Hospital included 43 HIV-positive and 29 HIV-negative heavily transfused haemophiliacs. The presence of ACA was detected in 10 patients, all of them infected by HIV (Naimi et al, 1990).

      The increase of APLA in patients infected with HIV may be due to disruption of the cell membranes which leads to the exposure of normally "hidden" PL during apoptosis (Clements et al, 1995). APLA from HIV-infected individuals tend to recognize various PL, most commonly PS (Petrovas et al, 1999). In addition, ACA in patients infected with HIV recognize oxidized CL more strongly than reduced CL. This finding, in conjunction with the increased oxidative stress found in these patients, may further explain the generation of APLA as a result of neoepitope formation by oxidized PL (Tzavara et al, 1997).

      Some medications associated with the development of APLA include neuroleptics, quinidine and procainamide (Merrill et al, 1997). APLA could be induced also by phenothiazines, chlorthiazide, ethosuximide, oral contraceptives or alpha-interferon (Kutteh et al, 1997). The duration of APLA after infection or discontinuation of drug exposure is not well established, and the risk of thrombosis is variable.

      LA have been identified in 10% to 20% of patients with well-established SLE, and ACA- in 30% to 50% of the individuals with SLE (Sammaritano et al, 1990) who present the majority of cases of secondary APS.

      APLA also occur in patients with other autoimmune disorders like Sjögren's syndrome, mixed connective tissue disease, rheumatoid arthritis, systemic sclerosis, ankylosing spondylitis, vasculitis, idiopathic thrombocytopenic purpura, etc. APLA are found also in patients with diabetes mellitus, Crohn's disease and autoimmune thyroid disease. APLA can be detected in some patients with malignancies such as thymoma; carcinoma of the lung, kidney, ovary, cervix uteri, prostate; lymphoma, leukemia and various myeloproliferative disorders (Kutteh et al, 1997).

C) APLA in patients with thrombosis and/or pregnancy morbidity / primary APS /

      According to the Sappporo's criteria for APS, all individuals with APS have by definition ACA and/or LA. Recent data show that LA are the strongest risk factor for thromboembolic events in patients with primary APS and secondary APS (Galli et al, 2000). No clear results have been reported showing that the measurement of ACA defines the patient's thrombotic risk. It has been found that ACA do not recognize anionic PL but are directed against plasma proteins bound to suitable anionic (PL and other) surfaces. Among them, b₂GPI and Pt are the most common antigen targets. b₂GPI is required by the great majority of ACA to react with CL in immunoassays (Galli et al, 1990). A recent study examined the distribution of APLAs among patients with SLE. The most prevalent APLA were found to be anti-b₂GPI antibodies. They were observed in 36.8% of all patients with SLE, being present in 40.4% of SLE patients with secondary APS and 34.9% in SLE patients without clinical features of APS (Bruce et al, 2000).

A) Vascular thrombosis

      A retrospective analysis of 100 patients with primary APS and secondary APS (Munoz-Rodriguez et al, 1999) reported one or more thrombotic episodes in 53% of the patients: 40% had venous thrombosis, 53% had arterial thrombosis, and 7% had combined arterial and vein thrombosis (Table 4).

      

Table 4 : Clinical manifestations of vascular thrombosis
Venous thrombosis	deep venous thrombosis superficial thrombophlebitis pulmonary embolism unusual sites- hepatic, mesenteric, axillary, pulmonary, renal veins
Arterial thrombosis	coronary, carotid, aorta and peripheral artery thrombosis cerebrovascular thrombosis retinal vessel thrombosis
Thrombotic complications	cardiovascular manifestations central nervous system manifestations osteoarticular and cutaneous manifestations

B) Pregnancy morbidity

      Pregnancy loss is a defining criterion for APS and occurs with a particularly high frequency in SLE patients. In addition to embryonic losses (before 10 weeks gestation) or foetal losses (after 10 weeks), APS is associated with a number of potential serious obstetric complications, including thrombosis, severe preeclampsia, utero-placental insufficiency, foetal distress and iatrogenic preterm birth (Table 5). These complications have significant maternal consequences, and they also may contribute to foetal loss. They may be associated also with other thrombophilic disorders such as factor V Leiden or G20210A mutations.

      

Table 5 : Obstetrical complications in APS
Recurrent pregnancy losses Unexplained second or third trimester loss Foetal death Intrauterine growth retardation Premature birth Severe preeclampsia Utero-placental insufficiency Pregnancy-related thrombosis (venous or arterial)

      Cervera et al (2002) analysed 590 women with APS who had 1 or more pregnancies: 74% of them succeeded in having 1 or more live births. The most common obstetrical complications in the mothers were preeclampsia (9.5% of pregnant women), eclampsia (4.4%) and abruptio placentae (2%). The most common foetal complications were embryonic loss in 34.5% of the pregnancies, foetal loss in 16.9% of the pregnancies, and premature birth in 10.6% of life births.

      In women with SLE, a previous adverse outcome was identified as the most important risk factor for another miscarriage in a subsequent pregnancy (Finazzi et al, 1996). Faden et al (1997) observed that the presence of anti-b₂GPI antibodies correlates well with some obstetrical complications, mainly eclampsia and preeclampsia. Anti-IgM anti-b₂GPI antibodies correlated well with a history of pregnancy loss (Forastiero et al, 1998).

      In 1996, Oshiro et al performed a retrospective study of 366 women with two or more consecutive pregnancy losses, where 79 of them were noted to have LA or ACA, and 290 did not. Both groups had similar rates of pregnancy loss (84%). However, those with APS had 50% foetal deaths compared with 15% foetal deaths in those without APS. Approximately 80% of those with APS had at least one foetal death compared with less than 25% in those without APS. Branch et al (1997) studied 147 women with recurrent pregnancy loss, negative for LA and with medium-to-high levels of IgG ACA, 104 healthy, fertile controls of similar age and gravidity, and 43 women with well-characterized APS. Twenty-six (18%) women with recurrent pregnancy loss and nine (9%) controls tested positive (above the 99th percentile) for APLA. Sera from five (3.4%) women with recurrent pregnancy loss and four (3.8%) controls demonstrated binding to PL antigens other than CL.

A) Thrombocytopenia

      Thrombocytopenia was observed in 30-52% of patients with APS (Munoz-Rodriguez et al, 1999; Cervera et al, 2002). It was found in 21% of the cases with primary APS and in 38% of cases with secondary APS. Thrombocytopenia in secondary APS patients was significantly associated with the presence of ACA at medium-high titer (Amoroso et al, 2003). It was usually mild (platelet count above 90 G/l) and, except in very severe cases, no bleeding was observed. Similarly to immune thrombocytopenias, pathogenic antibodies were directed towards epitopes on platelet membrane glycoproteins and were distinct from 'antiphospholipid' antibodies (Godeau et al, 1997). Galli et al (1994) measured in 68 patients with APLA also anti-GPIb/IX and GPIIb/IIIa IgG, directed against platelet membrane-associated glycoproteins. Increased plasma levels of these anti-glycoprotein antibodies were found in 40% of cases. Furthermore, APLA have been reported in around 30% of subjects with typical immune thrombocytopenias.

B) Atherosclerosis

      Recent studies demonstrated that EC activation induced by APLA might accelerate atherosclerosis associated with APS (Ross et al, 1999). Several studies have shown a close link between an atherosclerotic process and APS (Harats et al, 1999; Bruce et al, 2000 (b)).

C) Catastrophic APS

      Catastrophic APS is a rare, accelerated form of APS. The patients with catastrophic APS present with wide spread noninflammatory thrombi involving the kidneys, lungs, heart, gastrointestinal tract, liver, the central nervous system as well as adrenal glands, in various combinations. Individuals presented with a clinical picture of acute multi-organ thrombosis with a 60% mortality due to myocardial infarction, acute respiratory distress syndrome, renal failure, or stroke (Asherson et al, 1996; Triplett et al, 2000). In the study of Asherson et al (2001), a total of 80 patients with catastrophic APS were analyzed. The most important manifestations found at the onset of the episode were cardiopulmonary (25%), neurologic (22%), abdominal (22%), and renal (14%) with a very high mortality (48%).

A) Endothelial cells

      Endothelium is a metabolically active interface between the blood and extravascular tissues. Resting EC exert anticoagulant and antithrombotic properties by preventing contact of blood with prothrombotic underlying tissues and by producing and presenting on the EC surface molecules that aid in this function (Pearson et al, 2000). When EC are activated by different stimulus such as inflammatory cytokines (TNFa, IL-1), bacterial lipopolysaccharide (endotoxin), viral infections or hypoxia, they express both procoagulant and proinflammatory properties. It is now known that APLA can bind to and also activate EC in a similar manner, thus provoking a procoagulant phenotype in EC. In the next paragraphs we will review the main properties of resting endothelium and the changes after its activation (Table 7).

Activated EC
Endothelial activation leads to loss of anticoagulant properties (Table 7) and is characterised by several modifications.

      Diminution of NO

      Recent studies in hypercholesterolaemic animals and humans show that the deficiency in agonist-induced nitric oxide synthesis can be improved by elevating the circulating levels of arginine. This suggests that the intrinsic levels of nitric oxide synthase are preserved but that some aspects of the coupling between agonist receptor and nitric oxide synthesis are disturbed (Maxwell et al, 1998). In contrast with nitric oxide decrease, PGI₂ release is enhanced in patients with atherosclerosis. This has been attributed as a consequence of excessive platelet reactivity.

      Tissue factor expression

      Tissue factor is a transmembrane protein expressed by stimulated EC. It is the physiological trigger of normal coagulation and a major initiator of clotting in thrombotic disease (Figure 5). Tissue factor initiates the extrinsic pathway of coagulation by serving as a cofactor and receptor for factor VIIa to efficiently cleave its substrates, factor IX and factor X, to their active forms (Roubey et al, 2000).

      

Fig. 5 : Model of blood coagulation

TF-VIIa: tissue factor-FVIIa complex TFPI: tissue factor pathway inhibitor APC: activated protein C PS: protein S T: thrombomodulin TAFI: thrombin-activatable fibrinolysis inhibitor t-PA: tissue-type plasminogen activator

      An uninterrupted line indicates activation, while an interrupted line indicates inactivation. The uninterrupted line between Xa and TFPI indicates that FXa has to form a complex with TFPI, and that this complex then inhibits TF-FVIIa.

      Increase in cell surface anionic PL

      Activated EC have increased exposure of surface anionic PL, which are cofactors for the coagulation system.

      Diminution of TM

      Activated EC down-regulate the synthesis and cell surface expression of TM and consequently reduce the formation of the thrombin-TM complex, thereby limiting thrombin generation (Laszik et al, 2001).

      Secretion of plasminogen activator inhibitor type I (PAI-I)

      PAI-I is synthesized and secreted by activated EC. It is the major plasma inhibitor of t-PA, involved in the regulation of fibrinolysis, degradation of the extracellular matrix and angiogenesis.

      Release of Von Willebrand factor (vWF)

      Activated EC release vWF from storage granules (Weibel-Palade bodies) in response to thrombin, which participates in platelet adhesion.

      Cell surface protease receptors expression

      Activated EC serve as a site of protease by expressing cell surface protease receptors. Thus EC facilitate the formation of enzyme complexes involved in the regulation of coagulation and fibrinolysis.

      Increased adhesion molecule expression

      Activated EC express adhesion molecules for leucocytes such as E-selectin, intracellular adhesion molecule (ICAM-1 and -2), vascular cell adhesion molecule (VCAM-1).

• Effects of APLA on endothelial cells
  It has been hypothesized that APLA bind to antigens such as PS, TM and heparan proteoglycan on EC surfaces (Pierangeli et al, 1999) and that b₂GPI could be a cofactor facilitating this interaction with EC (Del Papa et al, 1997; Dueymes et al, 1996). Indeed EC activation by APLA is associated with increased expression of adhesion molecules, increased synthesis and secretion of proinflammatory cytokines, tissue factor expression, increased endothelin-1, induction of apoptosis, and EC migration.

      Increased expression of adhesion molecules

      Activated EC express surface E-selectin, VCAM-1, and ICAM-1 leading to increased monocytes and leucocytes adhesion. (Simantov et al, 1995; Meroni et al, 2001). Pierangeli et al (2000) demonstrated that in a pinch-induced thrombosis model APLA enhance leukocyte adhesion and increase thrombosis. The authors analyzed in vivo leukocyte adhesion to endothelium in venules of exposed murine cremaster muscle. The thrombogenic effects of APLA were reduced in transgenic ICAM-1-deficient mice, ICAM-1/P-selectin-deficient mice and in mice infused with anti-VCAM-1 antibodies.

      Increased synthesis and secretion of proinflammatory cytokines

      APLA increase EC synthesis and secretion of the proinflammatory cytokines IL-1 and IL-6. This could further contribute to cell activation in an autocrine manner because specific antagonists, such as IL-1 receptor antagonist, can inhibit the process (Del Papa et al, 1997; Meroni et al, 2000). Thus anti-b₂GPI antibodies can induce an EC activation either directly or by a cytokine autocrine loop.

      Tissue factor expression

      It has been demonstrated that incubation of cultured EC with anti-b₂GPI antibodies result in an increased production of TF, further supporting the hypothesis that APLA are procoagulant triggers (Branch et al, 1993 ; Kornberg et al, 2000; Dunoyer-Geindre et al, 2001). Amengual et al (1998) demonstrated by reverse-transcription polymerase chain reaction that human monoclonal anti-b₂GPI antibodies upregulate tissue factor mRNA on HUVEC, suggesting that the tissue factor pathway be implicated in the pathogenesis of APLA related thrombosis.

      Increased endothelin-1 (ET-1)

      Significantly increased plasma levels of ET-1, the most potent endothelium derived contracting factor, were found in patients with APS and arterial thombosis (Atsumi et al, 1998). In vitro incubation of EC with human monoclonal anti-b₂GPI antibodies was shown to upregulate the expression of preproendothelin-1 mRNA.

      Induction of apoptosis

      A subset of APLA that recognizes annexin V induces apoptosis in EC (Nakamura et al, 1998; Pittoni et al, 1998). In vivo, apoptosis of EC would lead to de-endothelialization and exposure of the thrombogenic subendothelium (Bombeli et al, 1999). It is hypothesized that APLA could also displace annexin V that covers the anionic PL on EC membranes, thus increasing the net quantity of thrombogenic PL exposed their procoagulant phenotype (Rand et al, 2000).

      EC migration

      APLA may also interfere with EC migration. This activity could potentially interfere with re-endothelialization and prolong the exposure of the thrombogenic subendothelium (Lanir et al, 1998).

B) Platelets

      Platelet binding to activated EC is the next phase in the process of thrombus formation although the precise mechanisms that mediate EC-platelet interaction are not completely clear. It was demonstrated that activated platelets are present in patients with APS but whether platelet activation in patients with APLA is a direct result of APLA or other autoantibodies or a consequence of vascular injury is uncertain (Emmi et al, 1997). A recent study found that APLA in APS have antiplatelet reactivity but there was no evidence for associated direct platelet-activating ability (Ford et al, 1998). Galli et al (1996) reported that the evidence that human APLA either bind to or activate platelets is still uncertain. Lackner et al (2000) also showed that two human monoclonal APLA had no effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with adenosine diphosphate or thrombin receptor activating peptide (TRAP-6). One possible explanation for these observations might be the presence of specific antiplatelet autoantibodies that coexist with APLA in patients with APS (Reverter et al, 2000). However, some investigators have shown that APLA may induce platelet activation and aggregation in the presence of low concentrations of agonists such as thrombin, adenosine diphosphate or collagen (Martinuzzo et al, 1993; Campbell et al, 1995; Nojima et al, 1999). A correlation was found between the IgG level of ACA and the CD62-positive platelet percentage in patients with primary APS and, more significantly, in the patients with primary APS and neurological disorders.

      APLA-containing plasma promoted platelet aggregation in a perfusion model (Escolar et al, 1992). Wiener et al (2001) suggested that the platelet aggregation in APS is induced by an APLA-complex present in patients' plasma. The initial trigger in this thrombotic process is calcium independent, but probably is followed by release, recruitment, and ultimately fibrin formation by the usual metabolic calcium-dependent fibrinogen binding pathway. Potential targets for APLA on platelets include platelet-activating factor (Barquinero et al, 1994), PS (Vazquez-Mellado et al, 1994; Campbell et al, 1995), and platelet glycoprotein IIIa (Tokita et al, 1996). Recently Ferro et al (1999) characterized 11-dehydro-TXB2 as a sensitive marker of platelet activation, and found it significantly higher in patients with SLE and APLA. A statistically significant correlation was found between plasma levels of vWF and tPA and excretion of this thromboxane metabolite. In a recent study, levels of platelet activation were investigated in 20 patients with primary APS and 30 SLE patients (14 of whom had secondary APS) by measuring CD63 expression on platelets and soluble P-selectin levels. Platelet CD63 expression and soluble P-selectin levels were significantly higher in patients with primary APS and SLE patients with/without APS than normal controls (Joseph et al, 2001). Robbins et al (1998) hypothesized that APLA/b₂GPI complexes could activate platelets to produce thromboxane A2 (a proaggregatory prostanoid) which could contribute to the prothrombotic state found in patients with APS. Shechter et al (1999) found that platelet serotonin concentration in APS patients was significantly lower than that found in the platelets of normal controls but the reasons of low serotonin levels are still not clear.

      Thrombocytopenia is a common finding in patients with APS and a potential association with platelet activation could exist. It was hypothesized that thrombocytopenia is due to platelet activation and consumption of platelets on the damaged vascular endothelium (Walenga et al, 1999). George et al (1999) studied 38 SLE patients and found in 26.3% of them high levels of b₂GPI containing immune-complexes. There was a positive correlation between b₂GPI/ immune-complexes levels and the occurrence of thrombocytopenia.

C) Monocytes

      Monocytes are implicated in the pathogenesis of APLA related thrombosis, mainly by stimulated cell surface expression of tissue factor (Kornberg et al, 1994; Cuadrado et al, 1997). Amengual et al (1998) revealed by flow-cytometry that monocytes from a healthy donor displayed higher tissue factor antigen expression when incubated in the presence of APS plasmas than with control plasmas. Stimulation of monocytes from APS patients with b₂GPI induced substantial monocyte TF, whereas no induction was observed with cells from patients having APLA without APS. Tissue factor induction on monocytes by b₂GPI was dose dependent and required circulating type 1 (Th1) CD4+ T lymphocytes and class II Major Histocompatibility Complex (MHC) molecules (Visvanathan et al, 2000). The authors previously reported that at least 44% of patients with APS possess Th1 CD4+ T cells that proliferate and secrete IFN-g when stimulated with b₂GPI in vitro (Visvanathan et al, 1999). F(ab)2 fragments of ACA have been reported to induce monocyte tissue factor expression as well, suggesting the contribution of an Fc-independent component to the mechanism of antibody-mediated tissue factor activity in APS (Kornberg, 1994). Increased levels of tissue factor mRNA have been found in the majority of mononuclear cell samples from patients with APS (Dobado-Berrios et al, 1999).

D) Polymorphonuclear cells

      Arvieux et al (1995) investigated the ability of six murine monoclonal antibodies to b₂GPI to induce polymorphonuclear cell functional responses. The six monoclonal antibodies tested in combination with b₂GPI led to a concentration-dependent activation of human polymorphonuclear cells. The activation of polymorphonuclear cells was estimated by their granule release, H2O2 production, and cytosolic Ca2+ increase. The results showed that the process of polymorphonuclear cell activation depends on monoclonal antibody binding to these cells through both Fab (via b₂GPI) and Fc domains.

A) Interference of APLA with the components of the protein C/S pathway

      The activation of the coagulation and the protein C pathway are shown in Figures 6, 7 and 8. Once activated by the thrombin-TM complex on the surface of EC, activated protein C (APC) exerts an inhibitory effect by cleavage of the factor Va and VIIIa; protein S and factor V are required as cofactors for the APC activity in vivo (Dalhback et al, 1993).

      Section A demonstrates an incomplete scheme of blood coagulation reactions together with the balancing anticoagulant reactions of the protein C pathway. In section B (I-IV), the membrane-bound molecular events of selected reactions are shown in cartoon-like fashion. I: activation of prothrombin (PT) to thrombin (T), a reaction that also generates the F1.2 prothrombin fragment. II: thrombomodulin (TM) and the endothelial protein C receptor (EPCR) are proteins that span the membrane. The role of EPCR is not fully understood, but it has been shown to be able to bind the Gla-domain of protein C, which results in stimulation of protein C activation. III: the degradation of FVa by APC is enhanced by protein S (PS). IV: degradation of FVIIIa by APC is stimulated by the synergistic cofactor activity of protein S and factor V. The large B domain that protrudes from the triangularly arranged A1-A3 domains of FV is in the linear sequence located between A2 and A3 domains.

      Inhibitors that control coagulation are shown in gray boxes above the complex or factor they regulate (AT: antithrombin, ZPI: protein Z-dependent protease inhibitor, PCI: protein C inhibitor). Top panel: FVIIa binds to tissue factor (TF) to activate FX, generatingFXa. FXa then binds to FVa. The complex of FXa-FV converts prothrombin to thrombin (T). Thrombin can then either bind to TM or carry out procoagulant reactions like fibrin formation or platelet activation. When bound to TM, thrombin can activate protein C (PC) to APC. This process is enhanced when protein C is bound to the EPCR. APC bound to EPCR cleaves substrates other than FVa. APC dissociates from EPCR and can then interact with protein S to inactivate FVa. Bottom panel: The FIXa-FVIIIa complex is inactivated by APC. In this case, FV participates with APC and protein S in the inactivation of FVIIIa. For simplicity, the activation of FVII, FV and FVIII are not shown.

      Protein C activation takes place by way of interaction between the thrombomodulin-thrombin complex and the endothelial protein C receptor. Activated protein C, together with its cofactor, protein S, inactivates factors V and VIII to provide negative feedback to the generation of thrombin. Complex 1 comprises TF and coagulation factors VII, IX and X; complex 2 comprises factors IX and X and cofactor VIII; and complex 3 comprises factor X, prothrombin and cofactor V.

      Experimental findings (de Groot et al, 1996) consistently showed that APLA may interfere with protein C axis in multiple ways as described below.

      - Inhibition of APC anticoagulant pathway, directly or via its cofactor protein

      Several recent studies proposed that APLA cause direct inhibition of the APC pathway and investigated the correlation of APLA specificity and APC pathway inhibition. Bokarewa et al (1994) studied the effect of 38 IgG fractions with either ACA alone or both ACA and LA on the response to APC. Five of eight IgG fractions with LA activity showed a tendency to reduce the effect of APC in the aPTT system and to simulate the activated protein C resistance (APC-R) phenomenon. No correlation was found between protein C activity and ACA levels or the extent of clotting time prolongation. In addition, Nojima et al (2002) found that the co-existence of anti-Pt antibodies and LA activity was a significant risk factor in the pathogenesis of APC-R in patients with SLE. Mali et al (2001) reported similar results from 59 unselected children with SLE showing that acquired APC-R was significantly associated with the presence of LA but not ACA. They proposed that acquired APC-R could be a marker identifying LA-positive patients at high risk of thrombosis. Controversially, Martinuzzo et al (1996) found that the acquired APC-R in patients with APS seems to be associated with ACA and anti-b₂GPI rather than an in vitro interference by LA. Atsumi et al (1998) demonstrated in vitro that ACA antibodies (not anti-protein C autoantibodies) can bind protein C via b₂GPI, and suggested that protein C could be a target of APLA by making a complex of protein C with ACA and b₂GPI, leading to protein C dysfunction.

      In addition, patients with APS were often found to have protein S deficiency. Atsumi et al (1997) demonstrated monoclonal ACA binding to protein S in presence of a combination of b₂GPI and CL, with a consequent increase of the affinity of C4b-binding protein for protein S. Protein S could represent one of the targets for ACA when combined with b₂GPI and CL, thus explaining the acquired free protein S deficiency and the attendant risk of thrombosis in patients with ACA. To explore the coagulation/fibrinolytic balance and its relation with free protein S, Ames et al (1996) carried out a cross-sectional study on 18 thrombotic patients with primary APS and 18 apparently healthy subjects with persistence of idiopathic APLA. Low free protein S was found in all non-thrombotic and in 90% of thrombotic patients with defective fibrinolysis. The data are consistent with increased thrombin generation and accelerated fibrin turnover and fibrinolysis abnormalities in asymptomatic subjects with APLA. It indicates also a possible central role for acquired free protein S deficiency in the thrombotic tendency of APS patients.

      - Interference with the activation of protein C by the thrombomodulin-thrombin complex

      Oosting et al (1993) showed that the anti-TM antibodies inhibiting TM and subsequent protein C activation are directed against the regions containing the epidermal growth factor (EGF) domains in SLE patients with a history of thrombotic complications. When TM was incorporated in PL vesicles, no inhibition by these anti-TM antibodies could be demonstrated. In addition, anti-TM antibodies could not inhibit protein C activation mediated by cultured EC. A conclusion was made that anti-TM antibodies inhibit only soluble TM.

      Carson et al (2000) tested 58 patients with LA and found anti-TM antibodies in 30% of the cases. Similar antibodies were found in only 2% of 201 normal controls. Three IgG fractions of the 6 purified IgG from patients with anti-TM antibodies inhibited protein C activation from 40% to 70% compared to no inhibition in 7 healthy controls.

      - Inhibition of thrombin formation

      Prothrombinase, which consists of factor Xa, factor Va, Ca2+, and PL, converts Pt to thrombin. Thrombin is the final protease of the coagulation system and, in turn, activates protein C in the presence of thrombomodulin on endothelial cells. APC completes a negative feedback loop in the blood coagulation pathway by degrading factor Va and factor VIIIa and thereby inhibits prothrombinase activity (Stenflo et al, 1884). Using chromogenic substance assays and human IgM monoclonal ACA, Ieko et al (1999) found that these monoclonal ACA inhibited both thrombin generation and APC activity. Thrombin generation without APC was inhibited as well by adding these antibodies. This inhibition required the presence of b₂GPI and confirmed the data presented previously by Mori et al (1996).

      - Binding to coagulation-activated cofactors Va and VIIIa in a manner that protects them from proteolysis by APC

      Borrell et al (1992) studied the effect of purified IgM and IgG from 21 patients with APLA on factor Va degradation by APC on HUVEC. Thirteen of 14 IgM and 8 of 10 IgG from patients showed an inhibitory effect on factor Va degradation by APC when compared with control Ig. Oosting et al (1993) investigated the effect of 30 IgG fractions APLA on APC-mediated factor Va inactivation in the absence and presence of protein S. Three IgG fractions inhibited APC-mediated factor Va inactivation independent of protein S and four IgG fractions APLA inhibited in the presence of protein S. The anticoagulant response of purified APC, added to LA-containing plasmas of 46 patients, was measured through the amount of factor VIII inactivation and an acquired APC dysfunction was found (Potzsch et al, 1995). Thirteen of 14 patients with recurrent thrombotic events and 10 of 19 patients with one single episode of thrombosis showed an abnormal APC response. In contrast, among 13 patients with LA without symptoms, only one showed an abnormal APC response.

      A subset of APLA reactive with PE was found particularly active in inhibiting the ability of PE to promote APC-mediated inactivation of factor Va. This effect correlated poorly with LA activity and it was suggested that this 'acquired' APC resistance might be a risk factor for thrombosis even in the absence of LA (Smirnov et al, 1995).

      APLA also interfered with the fibrinolytic pathway through TM inducible fibrinolysis inhibitor and by increasing PAI-1 activity. These findings suggested that the impairment of fibrinolytic activity by APLA might be one of the causes of thrombophilic diathesis in APS (Ieko et al, 1999).

      - Interference of APLA with the components of intrinsic pathway

      Recent studies reported the presence of antibodies to factor XII in a significant number of patients with APS suggesting that their presence might lead to acquired factor XII deficiency (Jones et al, 2000). Jone et al (2002) reported that, although factor XII is a member of the family of proteins which include plasminogen and Pt, antibodies to factor XII in patients with APS appear to be distinct from antibodies to Pt. Sugi et al (2001) reported that certain anti-PE antibodies are not specific for PE, but recognize the contact proteins factor XI and prekallikrein independently or in combination with high molecular weight kininogen. The contact proteins such as high molecular weight kininogen, prekallikrein and factor XII have anticoagulant and profibrinolytic functions and their deficiency was found to be associated with recurrent thrombosis. Several studies confirmed the presence of autoantibodies to the contact proteins in patients with SLE, thrombosis, and recurrent pregnancy loss.

      - Inhibition of antithrombin activity by APLA

      The formation of thrombin-antithrombin complexes (TAT) in APS was impaired. Ieko et al (2000) did not find an increase in level of TAT in APS, while the level of prothrombin fragment 1+2 increased. Therefore, free thrombin present in patient blood might contribute to thrombosis in APS.

      It was demonstrated that at least some APLA cross-react with highly polyanionic heparin and heparinoid molecules and inhibit the acceleration of antithrombin activity (Shibata et al, 1994). Purified IgG from seven patients with APS were reactive with heparin by ELISA, whereas none of five controls had antiheparin reactivity. Specificity studies showed that APS IgG antiheparin antibodies were specifically reactive with a disaccharide present in the heparin pentasaccharide that binds antithrombin. Furthermore, these antibodies inhibited heparin-accelerated formation of antithrombin-thrombin complexes.

B) Impairment of fibrinolysis by APLA

      Ieko et al (2000) investigated the effect of both b₂GPI and APLA on the activity of extrinsic fibrinolysis. The authors found that b₂GPI, without PAI-1, did not affect t-PA activity in a chromogenic assay. When PAI-1 was added to t-PA, the remaining t-PA activity was increased up to 60% by the addition of b₂GPI. The effect of b₂GPI did not require PL. Thus, b₂GPI might protect t-PA activity from the inhibition by PAI-1. When monoclonal ACA were further added to the mixture with a diluted PL, the remaining t-PA activity decreased to 50 and 80%. Monoclonal ACA appeared to inhibit the effect of b₂GPI and to elevate PAI-1 activity. Thus, the impairment of fibrinolytic activity by ACA might be one of reasons for the increased incidence of thrombosis in patients with ACA.

C) Additional effects of APLA

      As PL bear structural resemblance to low density lipoprotein (LDL), several studies (Horkko et al, 1997) showed that APLA might show cross reactivity against oxidized LDL. Oxidized low-density lipoprotein is implicated in atherosclerosis by influencing foam cell formation and cell cytotoxicity. The production of anti-oxidized low-density lipoprotein antibodies results in the formation of immune complexes which are taken up at enhanced rate by macrophages, leading to foam cell formation. George et al (1997) demonstrated in mice immunized with ACA that developed APS, that infusion with oxidized low-density lipoproteins aggravated the manifestations of experimental APS. The authors suggested that cross-reactivity of oxidized low-density lipoproteins with PL might lead to significantly more severe form of APS.

      In summary, although many studies were and are still performed to determine how APLA might increase thrombus formation in vitro, their mechanism of action remains unclear. Several studies have proposed that APLA activation of EC, platelets, and monocytes, as well as their effects on fibrinolysis and proteins C and S, may contribute to the prothrombotic state in APS. The large variety of proposed mechanisms makes it difficult to identify either the main primary mechanism. It is possible also that different mechanisms exist or co-exist among patients and even in one single patient.

A) Primary thromboprophylaxis in case of APLA

      Controversial data exist regarding the prophylactic treatment of patients positive for APLA and without history of thrombosis. Incidental detection of low titers of APLA carries a minimal risk of thrombosis. However, there is convincing evidence to suggest a significant risk associated with the presence of medium to high levels of APLA. Prophylactic therapy is not accepted in clinical practice at present, although there is increasing evidence of the risks of high levels of APLA (Finazzi et al, 1996; Khamashta et al, 1999). Recent guidelines on the investigation and management of APS by Greaves et al (2000) suggested the prophylactic use of low-dose aspirin, which was shown to be protective of deep vein thrombosis in high risk groups (Rai et al, 1997). Prophylactic low-intensity oral anticoagulation with warfarin and low-dose aspirin has been shown to significantly reduce the incidence of ischemic heart disease in patients with APS (General Practice Research, 1998). A decision analysis study of patients with SLE, with and without APLA, also supports the prophylactic use of aspirin (Wahl et al, 2000).

B) Thromboprophylaxis with statins in case of APLA

      Recent literature reported that statins are the principal and the most effective class of drugs to reduce serum cholesterol levels. Statins have been also shown to have anti-thrombotic effects in addition to lower cholesterol and to reduce cardiovascular events, including myocardial infarction, stroke, and death in patients with or without coronary artery disease symptoms. The actions of these drugs extend far beyond cholesterol reduction and involve non-lipid-related mechanisms that modify endothelial functions, immunoinflammatory responses, smooth muscle cell activation, proliferation and migration, atherosclerotic plaque stability and thrombus formation. Thus statins may offer an interesting prophylactic therapeutic approach for APS without the risks associated with anticoagulation. The aim of this study was to evaluate whether statins could modify the APLA-induced adhesion molecule expression by endothelial cells.

A) Pleiotropic effects of statins on vascular cells

      Experimental evidence suggest that statins may influence several events in the vessel wall that are relevant for the progression of atherosclerosis (Koh et al, 2000)

      - Statins and endothelial function

      Stimulation of eNOS and inhibition of endothelin-1 (ET-1)

      An important characteristic of endothelial dysfunction is the alteration in either the expression or function of the endothelial vasoactive factors, ET-1 and NO. ET-1 is an isopeptide synthesized by EC, with powerful vasoconstrictive effects among others. Atorvastatin and simvastatin inhibited pre-proET-1 mRNA expression and reduced immunoreactive ET-1 levels (Perera et al, 1998). Seeger et al (2000) demonstrated that fluvastatin significantly enhanced prostacyclin synthesis and significantly reduced ET-1 production in cell cultures of human umbilical endothelial veins. Since prostacyclin is a vasodilator and endothelin a vasoconstrictor, fluvastatin might have a significant effect on hemodynamics by favoring the balance towards vasodilation.

      Endothelial nitric oxide has been shown to mediate vascular relaxation and inhibit platelet aggregation (Radomski et al, 1992), vascular smooth muscle cells (SMC) proliferation and endothelium-leucocyte interactions (Gauthier et al, 1995). Laufs et al (1998) have shown that inhibition of HMG-CoA reductasein vascular EC upregulates the expression and activity of eNOS and prevents their downregulation by ox-LDL. This effect occurs through an increase in eNOS mRNA stability.

      Effects on the EC fibrinolytic activity

      Lovastatin and simvastatin have been shown to induce an increase in the local fibrinolytic activity of EC by stimulating tPA-expression and activity, an effect that was potentiated by the inhibition of PAI-1 expression (Essig et al, 1998).Wiesbauer et al (2002) showed that statins decreased mRNA levels for PAI-1 in EC and SMC and increased mRNA levels for t-PA in SMC.

      Inhibition of TF

      Eto et al (2002) demonstrated that simvastatin prevents the induction of tissue factor by thrombin in human aortic EC and block the increase in tissue factor activity on the cell surface. Simvastatin also prevented the upregulation of tissue factor expression and activity in human aortic SMC. Simvastatin prevents tissue factor induction through inhibition of Rho/Rho-kinase and activation of Akt.

      Statins decrease MHC class II antigen expression

      Simvastatin selectively decreases interferon-gamma (IFN-gamma)-induced MHC class II expression (mRNA and protein) in human primary EC through actions on the CIITA promoter, general regulator of both constitutive and inducible MHC class II expression. In contrast, simvastatin does not affect the expression of MHC class I, pointing to specific actions in the MHC class II signaling cascade. In repressing induction of MHC-II and subsequent T-lymphocyte activation, statins therefore provide a new type of immunomodulation (Kwak et al, 2001).

      Statin effects on angiogenesis

      Urbish et al (2002) have shown a double-edged, dose-dependent effects of statins in angiogenesis signaling by promoting the migration of mature EC and endothelial progenitor cells at low concentrations of 0.01 to 0.1 mmol/L, and antiangiogenic effects at high concentrations (> 0.1 mmol/L). Promigratory and proangiogenic effects of atorvastatin on mature EC were correlated with the activation of the phosphatidylinositol 3-kinase-Akt pathway. Weis et al (2002) also showed biphasic dose-dependent effects of statins on angiogenesis that were lipid independent and associated with alterations in endothelial apoptosis and vascular endothelial growth factor signaling.

      - Statin effects on SMC migration, proliferation and apoptosis

      Kaneider et al (2001) observed that cervistatin inhibited proliferation of SMC and also led to an induction of programmed cell death by increasing caspase-3 activity. Muller et al (1999) demonstrated that lovastatin inhibit SMC proliferation in association with induction of apoptosis. Lovastatin induced arrest of cells in G0/G1 phase of the cell cycle and DNA synthesis was reduced. A significant induction of p21WAF1/Cip1 protein expression was found by western blot analysis. This led to a strong inhibition of cyclin dependent kinases resulting in a cell cycle arrest.

      - Statin effects on platelet adhesion and aggregation

      Platelet activation and aggregation are crucial initial events in the development of cardiovascular disease and aggravate pathological alterations in the vessel wall. Adenosine diphosphate release from activated platelets induces further platelet recruitment, followed by cell aggregation. Adenosine diphosphate causes granule release and thromboxane A2 generation (Daniel et al, 1999). Neutrophils express locomotive activity in response to adenosine diphosphate and adenosine triphosphate released from activated platelets via activation of P2Y4 and P2Y6 receptors (Di Virgilio et al, 2001). Direct interactions of neutrophils and platelets are mediated through P-selectin (CD62P), the expression of which represents the first step in the formation of a leukocyte-platelet thrombus in vivo (Konstantopoulos et al, 1998). Statins have been shown to inhibit platelet function. A potential mechanism might include a reduction in the production of thromboxane A2 (Notarbartolo et al, 1995). Romano et al (2000) reported that statin treatment reduce the expression of P-selectin on platelets. Thrombin-activated platelets decreased their adenosine diphosphate and adenosine triphosphate release by coincubation with statins (Kaneider et al, 2002). Linjen et al (1994) found that pravastatin reduced the membrane cholesterol content of platelets, suggesting that certain properties of these membranes are altered in a manner that renders them less prone to participation in thrombosis.

      - Statin effects on monocyte/macrophages

      Macrophages are important for the development of atherosclerotic plaques. Secretion of proteolytic enzymes, such as matrix metalloproteases (MMPs), by activated macrophages may weaken the fibrous cap of the plaque, leading to plaque instability, rupture and ensuing thrombosis and arterial occlusion (Uzui et al, 2002). Cerivastatin treatment has been shown to decrease macrophage expression of MMP-1, MMP-3, MMP-9 and TF, as well to reduce the number of macrophages expressing histone mRNA, a sensitive marker of cell proliferation (Aikawa et al, 2001).

      Statins decreased the expression of adhesion molecules on monocytes isolated from patients with hypercholesterolemia (Weber et al, 1997) and also decreased significantly monocyte chemoattractant protein-1 (MCP-1), which plays a major role in recruiting monocytes into the vessel wall (Kothe et al, 2000). Lovastatin was found to inhibit bacterial lipopolysaccharide and cytokine-mediated production of nitric oxide and expression of iNOS in macrophages probably by inhibiting farnesylation of p21(ras) or other proteins that regulate the induction of iNOS. Statins also decrease the production of cytokines TNFa, IL-1, IL-6 by activated monocytes (Pahan et al, 1997; Rosenson et al, 1999; Grip et al, 2000).

B) Pleiotropic effects of statins on extravascular system

      Observational and experimental studies have implicated potential benefit from the administration of statins for other noncardiovascular diseases, including osteoporosis and dementia. Statins might have effects on bone formation. Cell culture experiments showed that murine osteoclast formation can be inhibited by lovastatin (Fisher, 1999). Furthermore, Sugiyama et al (2000) described that statins increase the expression of bone morphogenic protein-2, which plays an important role in osteoblast differentiation and bone formation. Jick et al (2000) presented a case-control study of individuals with dementia suggesting that those who were prescribed statins had a substantially lowered risk of developing dementia, independent of the presence or absence of untreated hyperlipidaemia. Recently, the data of clinical trials revealed also the antineoplastic potential of statins, particularly with the use of lovastatin in patients with cancer, anaplastic astrocytoma and glioblastoma multiforme (Larner et al, 1998). Stimulation of apoptosis could be involved in the beneficial effects of statins for these processes (Muller et al, 1998).

      In summary, statins constitute the most powerful class of lipid-lowering drugs. However, the benefits demonstrated in clinical practice with statin therapy appear to be related, at least in part, with effects that are independent of their cholesterol-lowering effects. Extensive research in the last decade suggests that statins have additional beneficial effects, related to an improvement in endothelial function, a reduction in blood thrombogenicity, antiinflammatory properties as well as immunomodulatory actions.

A) Inhibition of dolichol synthesis

      The mevalonate pathway also leads to the formation of dolichols, having an essential role in lipoprotein synthesis, and of ubiquinone, involved in electron transport (Corsini, 1999).

B) Activation of protein kinase Akt

      Phosphorylation of the protein kinase Akt is important in several cellular cascades, including the endothelial nitric oxide synthase activation and endothelial cell survival. Akt is activated by several growth factors including VEGF, fibroblast growth factor as well as shear stress (Edwards et al, 2000). Statins activate the protein kinase Akt via a phosphoinositol-3-kinase dependent pathway resulting in enhanced eNOS activity, inhibition of endothelial apoptosis and increased release of endothelial progenitor cells (Kureishi et al, 2000).

C) Regulation of caveolin

      Caveolae are small plasma membrane invaginations that play an important role in signal transduction and compartmentalization of signaling molecules. For example, caveolin forms an inhibitory complex with endothelial nitric oxide synthase (eNOS). Recently, inhibition of caveolin expression in the presence of statins has been demonstrated, resulting in an upregulation of nitric oxide release (Feron et al, 2001).

D) Statins and HMG-CoA independent effects

      Until recently, all cholesterol-independent effects of statin treatment were shown to be mediated by inhibition of mevalonate synthesis. Recently, Weitz-Schmidt et al (2001) reported that statins bind to a novel allosteric site within the ß2 integrin function antigen-1 (LFA-1), independent of mevalonate production. LFA-1 is important for the adhesion and co-stimulation of lymphocytes. The authors optimised statins for binding to LFA-1 and developed selective and orally active LFA-1 inhibitors that suppressed the inflammatory response in murine model of peritonitis. These findings, independent not only of cholesterol but also of HMG-CoA, open up a new area of research and treatment options.

A) Human umbilical vascular endothelial cells (HUVEC)

      HUVEC were isolated from collagenase-perfused umbilical cord veins as previously described (Jaffe et al, 1973; Galve-de Rochemonteix et al, 2000). Briefly, the veins were rinsed with Krebs-Ringer bicarbonate buffer (120 mM NaCl, 4.75 mM KCl, 1.2 mM KH₂PO_4, 1.2 mM MgSO_4, 2.5 mM CaCl₂, 25 mM NaHCO_3, 5 mM glucose, pH7.4) to remove the red blood cells and incubated for 10 minutes at 37°C with 1 mg/ml collagenase (CLS type 1, Worthington Biochemical, Lakewood NJ, USA) in Krebs-Ringer bicarbonate buffer with 25 mM HEPES. Cells were collected by flushing the vein with 50 ml of RPMI 1640 supplemented with 5 % foetal calf serum (FCS) (Seromed Biochrom, Berlin, Germany). After centrifugation (10 minutes, 1200 rpm), cells were cultured in Petri dishes coated with 0.1 % gelatin at 37°C in a humidified atmosphere containing 5 % CO₂. The culture medium was RPMI 1640 supplemented with 10 % FCS, 15 mg/ml EC growth supplement (Upstate Biotechnology, Lake Placid NY, USA), 10 mM HEPES, 90 mg/ml heparin (Boehringer Ingelheim, Germany), 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco BRL-Life Technologies, Rockville MD, USA). After reaching confluence, HUVEC were harvested with trypsin-EDTA, splited 1:2 and cultured in tissue-culture dishes coated with gelatin. The cells were used at passages two to four for all experiments.

B) Human saphenous vein cells (HSVEC)

      HSVEC were isolated by collagenase treatment and cultured in gelatin coated dishes as previously described (Kwak et al, 2000). Cells were maintained in medium 199 (Gibco BRL-Life Technologies, Rockville MD, USA) supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco BRL-Life Technologies, Rockville MD, USA), 10% foetal calf serum (FCS) (Seromed Biochrom, Berlin, Germany), 100 mg/ml heparin (Boehringer Ingelheim, Germany), 15 mg/ml EC growth supplement (Upstate Biotechnology, Lake Placid NY, USA), and 25 mM HEPES.

A) Purification of patient IgG

      Patients sera and sera from normal subjects were purified by protein A-Sepharose CL-4B affinity chromatography. The binding of IgG was performed using 0.1 M and 0.01 M Tris buffer, pH 8.0 as a binding buffer. The available binding capacity was 20 mg human IgG per ml of drained gel and the binding efficiency was approximately 90%. IgG were thereafter recovered by acid elution with 0.1 M glycine HCl buffer pH 3.0. The protein content of the eluted fractions was evaluated by BCA (bicinchonic acid) protein assay reagent. The purified IgG fractions were found to have bacterial lipopolysaccharide < 0.06 ng / ml as determined by Limulus Amebocyte Lysate Endochrome Assay.

B) Analysis of adhesion molecule expression on EC by flow cytometry (FACS)

      HUVEC and HSVEC were grown in 24-well plates. After confluence, the cells were incubated in complete medium overnight with different concentrations of simvastatin (0.1 - 5 mM), fluvastatin (0.1 - 15 µM) or pravastatin (5 - 15 mM) with or without mevalonate (400 mM), GGPP (15 mM) or FPP (15 mM). Then, the cells were stimulated with TNF-a (10 ng/ml) or bacterial lipopolysaccharide (20 ng/ml) for a period of 4 hours to measure the effect on expression of E-selectin, and for 6 hours to measure the VCAM-1 expression. When the cells were stimulated with purified APLA (0.5 mg/ml), overnight preincubation with these antibodies in complete medium with 20 % FBS and without heparin was performed. For negative controls cells were incubated with medium alone and normal human serum IgG (0.5 mg/ml), respectively. Thereafter, the cells were washed in RPMI and incubated for 1 hour at 4°C with primary mouse monoclonal antibody (anti-E-selectin, anti-VCAM-1) at 10 mg/ml in PBS (Gibco, BRL-Life Technologies, U.K.), 5 % FCS, 0.02 % sodium azide. Cells were washed in PBS-FCS-sodium azide and incubated for 1 hour at 4°C with the secondary antibody, polyclonal FITC-conjugated goat anti-mouse antibody, dissolved in PBS/FCS/sodium azide. Cells were washed in PBS-sodium azide, harvested with trypsin-EDTA and, after centrifugation at 800 g for 5 minutes, fixed in 2.5 % formaldehyde, 2 % glucose, 0.02 % sodium azide in PBS. Propidium iodide (10 ml of 50 mg/ml) was added to each sample. Cell fluorescence was analysed in a Becton Dickinson FACScan flow cytometer (San Jose CA, USA). A total of 10'000 viable cells were analysed per experimental sample. Data were analysed using the CELLQUEST software (Becton Dickinson). The same experiments were performed with HUVEC incubated for 48 hours with GGTI-286 (10 mM) or FTI-277 (0.5- 2.5 mM). Fresh GGTI-286 or FTI-277 solutions were added after 24 hours. The concentration of GGTI-286 (10 mM) was five-fold higher than its IC₅₀ (2 mM) for the inhibition of geranylgeranyltransferase; the concentration of FTI-277 (2.5 mM) was twenty five-fold higher than its IC50 (0.1 mM) for the inhibition of farnesyltransferase and about twenty five fold lower than its IC₅₀ (50 mM) for the inhibition of gerenalygeranyltransferase (information provided by Calbiochem, San Diego, USA). After the preincubation, cells were stimulated with TNF-a (10 ng/ml).

C) Cell Enzyme Linked Immunosorbent Assay (ELISA)

      A modified ELISA was used to measure E-selectin and VCAM-1 content at the cell surface. HUVEC and HSVEC were grown in 96-well coated plates and exposed to simvastatin with or without mevalonate at same concentrations and incubation times as described for FACS analysis. After 4-6 hours incubation with TNF-a (10 ng/ml), cells were washed 3 times with RPMI containing 2.5 % FCS and fixed in 4 % paraformaldehyde for 15 minutes. After 3 washes with RPMI/FCS, cells were incubated for 1 h at room temperature with primary mouse antibody (anti-E-selectin, anti-VCAM-1), diluted 1:1000 in RPMI/FCS. After 3 washes in RPMI / FCS and 1 wash in PBS, cells were incubated with secondary antibody, goat anti-mouse IgG-horseradish peroxidase (HRP)-conjugated, diluted 1:4000 in RPMI / FCS. After 1-hour incubation at room temperature, the wells were washed 4 times in RPMI/FCS and once in PBS. Then 100 ml of 1 mg/ml o-phenylenediamine dihydrochloride (OPD) solution was added and after 10 minutes incubation at room temperature, the color development was stopped by addition of 50 ml of 3M H₂SO₄. The absorbance was read at 490 nm with an ELISA reader.

D) Statistical analysis

      For ELISA assays, the effect of simvastatin on adhesion molecules expression was analyzed with Kruskal-Wallis ANOVA and its reversal by mevalonate with Mann&Witney test.

A) Effect of statins on TNF-a activated EC

a1) Simvastatin effect on HUVEC

      ELISA method

      First we investigated the effect of statins on the stimulation of HUVEC with TNF-a. Compared to cells without TNF-a, in TNF-a treated HUVEC we observed a six fold increase in E-selectin expression by cell-based ELISA. Pre-treatment of the cells with different concentrations of simvastatin resulted in a dose dependent increase in their response to TNF-a (Table 9). At concentrations of 1 to 2.5 mM of simvastatin, as compared to non-pretreated cells, a 80% (p<0.001) and a 40% (p<0.001) increase were observed for E-selectin and VCAM-1 expression, respectively. At higher simvastatatin concentrations, cell detachment and cell death were frequently observed. Addition of 400 mM mevalonate to the preincubation solution reversed the effect of simvastatin.

      

Table 9 : Cell ELISA analysis of simvastatin effect on TNF-a induced E-selectin and VCAM-1

Mean values (n=5) for each simvastatin concentration were expressed as a percentage of mean O.D. value of TNF-a 10 ng/ml treated cells without simvastatin * P<0.001 for E-selectin and VCAM-1 expression mediated by simvastatin § P<0.001 for mevalonate

      Flow cytometry analysis

      We investigated E-selectin and VCAM-1 expression by flow cytometry analysis as well. Figure 14A shows that after TNF-a stimulation the expression of E-selectin was strongly increased. Strikingly, a very heterogeneous response was observed, a majority of cells showing a high level of E-selectin expression and a minority a lower expression. A similar heterogeneous response was observed when ten-fold higher TNF-a concentrations (100 ng/ml) were used (data not shown). This implies that the suboptimal response in some cells was not due to insufficient TNF-a concentrations. For the remainder of our work, a TNF-a concentration of 10 ng/ml was used. In cells pre-treated with simvastatin and then with TNF-a, almost all cells exhibited a high level of E-selectin expression. The effect of simvastatin was reversed by pre-treatment with mevalonate (Figure 14B).

      

Fig. 14 : The effect of simvastatin on TNF-a-induced E-selectin expression (A) and its reversal by pretreatment with mevalonate (B)

      Confluent HUVEC were pretreated overnight with 2.5 mM simvastatin (A) or 2.5 mM simvastatin with 400 mM mevalonate (B). TNF-a (10 ng/ml) was added to the medium for 4 hours and E-selectin expression at the cell surface was measured by flow cytometry analysis as described in the Methods section. I represents E-selectin expression in control HUVEC; II represents E-selectin expression after TNF-a treatment; III represents TNF-a induced E-selectin expression after an overnight preincubation with simvastatin; and IV represents the TNF-a induced E-selectin expression after an overnight preincubation with simvastatin and mevalonate.

a2) Simvastatin effect on saphenous vein EC

      Figure 15 shows that simvastatin pre-treatment had similar effects on VCAM-1 expression in response to TNF-a both in HUVEC (A, B) and human saphenous vein EC (C). Table 10 summarises the mean (± SD, N= 10 independent experiments) fluorescence values obtained by flow cytometry for TNF-a treated HUVEC. It shows that E-selectin expression varied between 150% and 223% in simvastatin pre-treated HUVEC as compared to non-pre-treated cells. VCAM-1 expression was 118% to 156% in the simvastatin pre-treated cells as compared to non-pre-treated cells. Addition of 400 µM mevalonate to the preincubation solution reversed the effect of simvastatin.

      

Fig. 15 : The effect of simvastatin on TNF-a-induced VCAM-1 expression on HUVEC (A), or on human saphenous vein EC (C), and the reversal of simvastatin effect by mevalonate on HUVEC (B)

      Confluent HUVEC were pretreated overnight with 1 µM simvastatin (A), or 1 µM simvastatin with 400 µM mevalonate (B). TNF-a (10 ng/ml) was added to the medium for 6 hours and VCAM-1 expression at the cell surface was measured by flow cytometry analysis. The same experiment was performed with human saphenous vein EC (C). I represents VCAM-1 expression in control HUVEC/saphenous vein; II represents VCAM-1 expression after TNF-a treatment; III represents TNF-a induced VCAM-1 expression after an overnight preincubation with simvastatin; and IV represents VCAM-1 expression after TNF-a and an overnight preincubation with simvastatin and mevalonate.

      

Table 10 : Flow cytometry analysis of simvastatin effect on TNF-a induced E-selectin and VCAM-1 expression

      Mean values (n=10) for each simvastatin concentration were expressed as a percentage of mean value of TNF-a 10 ng/ml treated cells without simvastatin.

b) Fluvastatin effect on EC

      Similar results were obtained by flow cytometry with HUVEC preincubated with fluvastatin and stimulated with TNF-a (Figure 16A). At fluvastatin concentrations of 5 M, E-selectin expression was increased (210% in one experiment, 321% in another one) and VCAM-1 expression was increased 143% and 160% (n=2) when compared with HUVEC treated with TNF-a alone. This effect was completely reversed by mevalonate (Figure 16B).

      

Fig. 16 : The effect of fluvastatin on TNF-a mediated E-selectin expression

      Confluent HUVEC were pretreated overnight with 5 µM fluvastatin (A), 5 µM fluvastatin with 400 µM mevalonate (B). TNF-a (10 ng/ml) was added to the medium for 4 hours and surface E-selectin adhesion molecules were measured by flow cytometry analysis as described in the Methods section. I represents E-selectin expression in control HUVEC, II represents E-selectin expression after TNF-a, III- E-selectin expression of fluvastatin pretreated TNF-a stimulated cells, and IVrepresents E-selectin expression after overnight preincubation with fluvastatin + mevalonate and stimulation with TNF-a.

c) Pravastatin effect on EC

      Preincubation of HUVEC with pravastatin, a hydrophilic statin, similarly augmented the TNF-a response of these cells. At pravastatin concentrations of 10 M and 15 M, a further 28 % increase in E-selectin expression was observed by flow cytometry analysis in TNF-a treated HUVEC and this effect was completely reversed by mevalonate (Figure 17). Mean values (n=6) for E-selectin expression were expressed as a percentage of mean value of non-statin pre-treated, TNF-a stimulated cells. The mean percentage values were: cells treated with TNF-a alone: 100 % (control); pravastatin pre-treated and TNF-a stimulated cells: 128 % ± 13 % (p < 0.02 vs control); cells treated with pravastatin, mevalonate and TNF: 97 % ± 11 %. Since the pravastatin effect was weaker than simvastatin, we focused on simvastatin and fluvastatin for the remainder of our work.

      

Fig. 17 : The effect of pravastatin on TNF-a mediated E-selectin expression

      Confluent HUVEC were pretreated overnight with 15 mM pravastatin. TNF-a (10 ng/ml) was added to the medium for 4 hours and surface E-selectin adhesion molecules were measured by flow cytometry analysis as described in the Methods section. I represents E-selectin expression in control HUVEC, II represents E-selectin expression after TNF-a, III- E-selectin expression of pravastatin pretreated TNF-a stimulated cells.

B) Effect of statins on bacterial lipopolysaccharide activated EC

      Simvastatin pre-treatment similarly increased E-selectin expression in cells stimulated with 20 µg/ml bacterial lipopolysaccharide (Figure 18).

      Confluent HUVEC were pretreated overnight with 2.5 µM simvastatin. LPS (20 ng/ml) was added to the medium for 4 hours and surface E-selectin adhesion molecules were measured by flow cytometry analysis. I represents E-selectin expression in control HUVEC, II represents E-selectin expression after LPS, III- E-selectin expression of simvastatin pretreated LPS stimulated cells.

C) Effect of statins on APLA activated EC

      We next performed flow cytometry analysis to study the effects of simvastatin and fluvastatin on HUVEC, activated with patient derived APLA. Contrary to what could be expected from clinical data, we observed an increase in adhesion molecule expression (Figure 19A) and this effect was completely reversed by mevalonate (Figure 19B). Figure 20 summarises the mean fluorescence values obtained by flow cytometry for HUVEC treated with APLA. It shows that APLA induced a 1.5 to 6 fold increase in E-selectin expression when compared to control (non-treated) cells. Fluvastatin pre-treatment gave a further 1.5- 2 fold increase in E-selectin as compared to non-pre-treated HUVEC. VCAM-1 expression was between 3 and 8 fold higher in APLA-activated cells as compared to non-activated cells and pre-treatment with fluvastatin led to another 2 to five fold increase in VCAM-1 expression as compared to non-pre-treated cells. Addition of 400 µM mevalonate to the preincubation solution reversed the effect of fluvastatin. These results suggest that statins induce an additional increase of VCAM-1 and E-selectin in HUVEC activated by human affinity purified IgG APLA.

      Confluent HUVEC were pretreated overnight with 5 mM fluvastatin. Purified patient APLA (500 µg/ml) was added to the medium for 24 hours and surface VCAM-1adhesion molecules were measured by flow cytometry analysis. I represents VCAM-1 expression in control HUVEC, II represents VCAM-1 expression after APLA incubation, III represents VCAM-1 expression of fluvastatin pretreated APLA stimulated cells (the figure represents the results with APLA obtained from serum of patient 1).

      In the absence of stimulation of HUVEC by TNF-a, bacterial lipopolysaccharide or APLA, pretreatment with simvastatin and fluvastatin had no effect on the expression of E-selectin (mean fluorescence 7.5 ± 2 vs 5.5 ± 2 in control cells; n= 6) and VCAM-1 (8 ± 3 vs 6 ± 2). Also, mevalonate pre-treatment had no effect on the basal expression of E-selectin (mean fluorescence 10 ± 3 vs 5.5 ± 2; n= 6) and VCAM-1 (12 ± 3 vs 6 ± 2, n=7).

      Statin effects on APLA-stimulated HUVEC were comparable with statin effects on TNF-a and bacterial lipopolysaccharide activated HUVEC.