Silvia S. Pierangeli

A Retrospective Review of 61 Patients With Antiphospholipid Syndrome Analysis of Factors Influencing Recurrent Thrombosis

BACKGROUND Antiphospholipid syndrome (APS) is a disorder of recurrent venous or arterial thrombosis, pregnancy losses, and thrombocytopenia. Recurrent thrombosis has particularly adverse effects on patients prognosis. The factors that influence recurrence and management techniques that prevent these events remain controversial. To add further insight regarding predisposing factors and the prevention of thrombotic recurrence, 61 well-characterized patients with APS were followed up for a median time of 77 months. METHODS A retrospective cohort study was conducted in which the following factors were examined to determine their influence on thrombotic recurrence: primary vs secondary syndrome; the presence of hypertension, hyperlipidemia, diabetes, or smoking; patient age, sex, and race; pregnancy and oral contraceptives use; and treatment with warfarin sodium, warfarin plus aspirin, aspirin alone, prednisone, or no treatment. RESULTS There was no difference between patients with primary and secondary APS with respect to recurrent arterial (55% vs 38%, respectively) or recurrent venous (47% vs 50%, respectively) thrombotic events. In all patients with APS, white race (P = .02) was associated with recurrent arterial events. Venous thrombosis occurred during pregnancy or in the postpartum period in 16 (30%) of 53 women and in 8 women taking oral contraceptives. Recurrent arterial and venous thromboses were significantly decreased with prophylactic warfarin use when compared with prednisone use or no treatment. Recurrences were infrequent in patients with prothrombin ratios of 1.5 to 2.0. CONCLUSIONS Treatment with warfarin was most effective in preventing recurrent arterial and venous thrombosis. Pregnancy and the use of oral contraceptives or prednisone may also influence recurrence.

Thrombogenic Effects of Antiphospholipid Antibodies Are Mediated by Intercellular Cell Adhesion Molecule-1, Vascular Cell Adhesion Molecule-1, and P-Selectin

Abstract — Recent studies have shown that antiphospholipid (aPL) enhances expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on endothelial cells (ECs) and that these effects are correlated with increased adhesion of leukocytes to endothelium in cremaster muscle in vivo and with thrombosis in a mouse model. Activation of ECs by aPL may create a hypercoagulable state that precedes and contributes to thrombosis in patients with aPL syndrome (APS). This study proposed to examine whether this in vivo activation of ECs and enhanced thrombosis by aPL are mediated by ICAM-1, P-selectin, or VCAM-1. The dynamics of thrombus formation and the number of adhering leukocytes were studied in ICAM-1–deficient (ICAM-1−/−) mice or ICAM-1–/P-selectin–deficient (ICAM-1−/−/P-selectin−/−) mice treated with affinity-purified aPL antibodies (ap IgG-APS) or with control IgG and compared with wild-type mice treated in a similar fashion. In another set of experiments, the adhesion of leukocytes to cremaster muscle and the dynamics of thrombus formation were studied in CD1 mice treated with aPL or control IgG before and 30 minutes after intravenous infusion with 100 &mgr;g monoclonal antibody anti–VCAM-1. The results indicate that the enhanced adhesion of leukocytes to endothelium in wild-type mice was significantly reduced in ICAM-1−/− and completely abrogated in ICAM-1−/−/P-selectin−/− mice treated with ap IgG-APS compared with wild-type mice treated with ap IgG-APS (6.9±2.3, 0.4±0.4 versus 35±12, respectively). More importantly, this correlated with a significant reduction in thrombus size compared with wild-type mice treated with ap IgG-APS (895±259 &mgr;m2, 859±243 &mgr;m2 versus 3816±672 &mgr;m2, respectively). Infusion of the mice with anti–VCAM-1 antibodies significantly reversed the enhanced adhesion of leukocytes (14.9±3 to 11.3±2.1) and thrombus size 3830±1008 &mgr;m2 versus 876±548 &mgr;m2) in mice treated with ap IgG-APS. The data indicate that ICAM-1, P-selectin, and VCAM-1 expression are important in thrombotic complications by aPL antibodies and may provide novel targets for therapy in patients with APS.

Fluvastatin inhibits up‐regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells

Summary.   Background: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up‐regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF‐α) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL‐induced expression of TF on ECs. Methods: IgGs were purified from four patients with APS (IgG‐APS) and from control sera (IgG‐NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG‐APS or IgG‐NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 µm) with and without mevalonate (100 µm). TF expression on HUVECs was measured by ELISA. Results: PMA and the four IgG‐APS preparations increased the expression of TF on EC significantly (4.9‐, 2.4‐, 4.2‐, 3.5‐ and 3.1‐fold, respectively), in a dose‐dependent fashion. Fluvastatin (10 µm) inhibited the effects of PMA and the four IgG‐APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dose‐dependent. Mevalonate (100 µm) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. Conclusion: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.

E-Selectin mediates pathogenic effects of antiphospholipid antibodies

Summary.  Antiphospholipid (aPL) antibodies, detected in patients with antiphospholipid syndrome (APS) are associated with thrombosis, pregnancy loss and thrombocytopenia. Studies have shown that aPL are thrombogenic in vivo, but the mechanism(s) involved are not completely understood. Several studies have demonstrated that aPL antibodies activate endothelial cells (ECs) in vitro, as determined by up‐regulation of adhesion molecules: E‐selectin (E‐sel); intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1), and in vivo. The objectives of these study were to determine the effects of aPL antibodies on the expression of E‐selectin on ECs, on the adhesion of monocytes to ECs and to study the role of E‐selectin on aPL antibodies enhanced thrombus formation and activation of ECs in vivo. We demonstrated that the surface expression of E‐selectin on HUVEC by ELISA was increased 400‐fold when treated with tumor necrosis factor‐alpha (TNF‐α) and 421‐fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factor‐kappa B (NF‐κB). APL antibodies increased significantly the number of adhering leukocytes to ECs in vivo in C57BL/6 J mice when compared to IgG‐NHS treated mice. This effect was abrogated in E‐selectin‐deficient mice. The thrombus size was significantly increased in C57BL/6 J mice treated with aPL antibodies when compared to mice treated with IgG‐NHS. This enhancement in thrombus size by aPL antibodies was abrogated in E‐selectin‐deficient mice treated with aPL antibodies.

β2-glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies

Summary. Some investigators have reported that a serum protein, β2‐glycoprotein 1 (β2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to β2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to β2GP1 in the absence of cardiolipin.

Antiphospholipid antibodies and the antiphospholipid syndrome: an update on treatment and pathogenic mechanisms.

Purpose of reviewThe antiphospholipid syndrome is a disorder of recurrent thrombosis, pregnancy loss and thrombocytopenia associated with the presence of antiphospholipid antibodies and persistently positive anticardiolipin or lupus anticoagulant positive tests. Since its recognition in the 1980s, growing interest in the field, not only with respect to diagnosis and treatment, but also regarding the pathogenesis of antiphospholipid antibodies, has emerged. Recent findingsFirst, this review addresses the recently updated classification criteria for diagnosis and treatment of the antiphospholipid syndrome. A discussion on the newly described potential beneficial roles of hydroxychloroquine and the statins for the treatment of antiphospholipid syndrome-associated clinical manifestations is included. Importantly, this article analyzes recent data that examine the molecular and intracellular events that antiphospholipid antibodies trigger in target cells, as well as new findings in the identification of the receptors for these antibodies on the membrane of those cells. A separate section discusses novel pathogenic mechanisms of antiphospholipid antibodies, including the activation of complement and their interaction with homologous catalytic domains of several serine proteases of the coagulation system. SummaryUnderstanding the molecular interactions and the intracellular signaling that antiphospholipid antibodies trigger, new therapeutic and targeted strategies to ameliorate clinical manifestations in patients with antiphospholipid syndrome may be established.

A peptide that mimics the Vth region of β2glycoprotein I reverses antiphospholipid-mediated thrombosis in mice

Antiphospholipid (aPL) antibodies bind to 2glycoprotein I (2GPI) and cause endothelial cell (EC) activation and thrombosis in mice. 2GPI binds to EC through its Vth domain and induces their activation. TIFI is a 20 amino acid synthetic peptide that shares similarity with the Vth domain of 2GPI. Our objectives were to examine the ability of TIFI to affect aPL-mediated thrombosis in mice and the interactions of TIFI, 2GPI with phospholipid surfaces and target cells. CD1 mice were injected with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgGNHS and with either TIFI or with control peptide (VITT). Size of induced thrombi was determined. Inhibition and competition studies were done using aPL antibodies, cardiolipin (CL) liposomes in the presence of varying amounts of TIFI and 2GPI. Binding of fluorescinated 2GPI to human ECs and to murine macrophages in the presence or absence of TIFI, was also examined. TIFI significantly decreased thrombus size in mice injected with IgG-APS. TIFI reverted the 2GPI-dependent binding of aPL antibodies to CL liposomes in a dose-dependent fashion. This effect was abrogated by addition of 2GPI, suggesting that TIFI displaces the binding of 2GPI to phospholipids. TIFI inhibited the binding of fluorescinated 2GPI to human EC and to murine macrophages. The data indicate that TIFI abrogates thrombogenic properties of aPL in mice by competing with 2GPI and preventing its binding to target cells. This may be important in designing new modalities for the treatment of thrombosis in APS.

Identification of an Fcγ receptor–independent mechanism by which intravenous immunoglobulin ameliorates antiphospholipid antibody–induced thrombogenic phenotype

OBJECTIVE Patients with the antiphospholipid antibody syndrome (APS) often experience recurrent arterial and venous thrombosis and pregnancy losses. Intravenous immunoglobulin (IVIG) therapy has prevented pregnancy loss in some women with APS and has reversed fetal resorption rates in murine models of pregnancy loss. Although the basis for these effects is unknown, effector mechanisms of pathogenic antibodies often involve receptors for IgG (Fc gamma receptors [Fc gammaR]). We examined the potential mechanisms of action of WIG in an in vivo murine model of antiphospholipid antibody (aPL)-induced thrombosis and endothelial cell activation. METHODS Mice infused with IgG containing human anticardiolipin antibodies (aCL) were treated with IVIG (36 microg i.v.), saline, or ovalbumin. Surgically induced thrombus formation and in vivo leukocyte adhesion to endothelial cells were measured. Circulating levels of aCL were measured by enzyme-linked immunosorbent assay. To determine whether Fc gammaR are required for the effects of IVIG, we treated mice deficient in stimulatory Fc gammaR. To examine the effects of IVIG on endogenously generated antibody, we treated mice immunized with beta2-glycoprotein I (beta2GPI). RESULTS IVIG treatment inhibited aPL-induced endothelial cell activation and enhancement of thrombosis in mice passively infused with human aPL-containing IgG, and this was associated with a decrease in aPL levels. Similarly, IVIG lowered aPL levels and inhibited thrombogenesis in mice immunized with beta2GPI. The thrombophilic effects of aPL were evident in Fc gammaR-deficient mice. CONCLUSION Treatment with IVIG inhibits the thrombogenic effects of aPL in vivo and reduces the levels of aCL in the circulation. Blockade of stimulatory Fc gammaR on inflammatory cells is not necessary for this effect. The mechanism of action of IVIG is more likely saturation of the IgG transport receptor, leading to accelerated catabolism of pathogenic aPL. These results have implications in the management of thrombosis in APS and may have applications for pregnant patients with a history of APS.

In Vivo Models of Thrombosis for the Antiphospholipid Syndrome

Utilizing this unique animal model of thrombosis we demonstrated that human (IgG, IgM or IgA) polyclonal and monoclonal antiphospholipid antibodies derived from APS patients have a significant enhancing effect on thrombus formation. This effect is reversed by treatment of the mice with hydroxychloroquine (plaquenil). In addition murine polyclonal and monoclonal anticardiolipin antibodies induced by active immunization with human β2-GP1 or human anticardiolipin antibodies showed to have thrombogenic properties in CD1 mice. Antibodies with antihuman β2-GP1 activity alone did not seem to affect thrombus formation.

Probing antiphospholipid-mediated thrombosis: the interplay between anticardiolipin antibodies and endothelial cells.

The association of antiphospholipid(aPL) antibodieswith thrombosisin patientswith antiphospholipid syndrome (APS) is well documented in humans and in animal studies. However, the mechanisms by which aPL antibodies induce thrombosis are the subject of much current study. It has been suggested that aPL may activate endothelial cells (ECs), thus creating a hypercoagulablestate that precedes and contributes to thrombosis in patients with APS. Several studies have shown that aPL upregulateECs’ adhesion molecules (CAMs): intercellularcell adhesion molecule-1 (ICAM-1), vascularcell adhesion molecule-1 (VCAM-1) and E-selectin (E-sel) or induce tissue factor (TF) in monocytes in vitro. Similarly, the incubationof EC with antibodiesreactingwith ß2 glycoproteinI (ß2 GPI) has been shown to induce EC activation with concomitant upregulation of CAMs, IL-6 production and alteration of prostaglandin metabolism. Our group has shown that aPL-mediated upregulation of adhesion molecules on ECs correlates with an increased adhesion of leukocytes to endothelium in the microcirculationof mouse cremaster muscle, an indicationof EC activation in vivo, and with enhanced thrombosis in vivo. In another series of studies, investigators have shown that upregulation of expression of adhesion molecules by some murine monoclonal anti-β2 glycoprotein I (anti-β2 GPI) antibodiescorrelatedwith fetal resorptionin mice invivo. More recently, one studyshowedthat the anti-hypercholesterolaemicdrug fluvastatininhibitedthe aPL-mediatedenhancedadhesionof monocytesto ECs in vitro. Data from our laboratoriesindicate that fluvastatin also reversesthrombus formation and activation of EC induced by aPL in an in vivo mouse model. As additional support for the hypothesis that aPL antibodies activate ECs and may create an hypercoagulablestate in APS patients, two recent studiesindicatedthatlevelsof solubleICAM-1 and VCAM-1 were significantlyincreasedin theplasma of patients with APS and recurrent thrombosis. Furthermore, studies utilizing knockout mice and specific monoclonal anti-VCAM-1 antibodies have demonstrated that expression of ICAM-1, P-selectin, E-selectin and VCAM-1 are important in in vivo aPL-mediated thrombosis and EC activation in mice. Recent data suggests that aPL antibodies also induce expressionof TF not only in monocytesbutin ECs. Hence, the interferenceof aPL with the TF mechanismmay be anotherimportant mechanism by which these antibodies create a hypercoagulablestate and prone patients to thrombosis. Specifically, how aPL alters EC activation state and the molecular and intracellular mechanisms involvedhavenotyetbeen defined.APL may interactwith specific cell surfacereceptors(proteinsand/or lipids) induce signals that have consequences downstream, and that ultimately will result in upregulationof cellsurfaceproteins(i.e., CAMs and TF) and subsequentlyinduceEC activation.In that regard, our group recently showed that aPL-mediated upregulation of adhesion molecules in ECs is precededby activationof the nuclearfactor kappaB (NFkB). Other intracellularmechanisms triggered by aPL are notcompletelyunderstoodand are the subjectof currentinvestigation.In conclusion, studies suggestthat activationof ECs by aPL is an importantmechanismthat may precedethrombusformation in patients with APS. Hence, the interplay between aPL antibodies and ECs is important in the pathogenesisof thrombosis in APS.