Rolf T. Urbanus

Antiphospholipid antibodies and risk of myocardial infarction and ischaemic stroke in young women in the RATIO study: a case-control study

BACKGROUND Arterial thrombosis is a major clinical manifestation of the antiphospholipid syndrome, which is an autoimmune disease found mostly in young women. Although the presence of circulating antiphospholipid antibodies in individuals who have a thrombotic event is a prerequisite for the diagnosis of the antiphospholipid syndrome, the risk of arterial thrombosis associated with antiphospholipid antibodies in the general population is unclear. METHODS In RATIO (Risk of Arterial Thrombosis In relation to Oral contraceptives), a large multicentre population-based case-control study, we enrolled women aged under 50 years who were admitted to hospital at 16 centres with first ischaemic stroke or myocardial infarction between January, 1990, and October, 1995. An additional 59 women who presented with ischaemic stroke at the University Medical Centre Utrecht between 1996 and 2001 were also enrolled. Information on cardiovascular risk factors (such as oral contraceptive use, smoking, and hypertension) were assessed with a standard questionnaire. During the second phase (1998-2002), blood samples were taken to measure antiphospholipid antibody profiles (lupus anticoagulant, anticardiolipin IgG, anti-beta(2)-glycoprotein I IgG, and antiprothrombin IgG) and to determine genetic prothrombotic risk factors (factor V G1691A variant, prothrombin G20210A variant, and factor XIII 204Phe allele). FINDINGS 175 patients with ischaemic stroke, 203 patients with myocardial infarction, and 628 healthy controls were included. Patients were frequency matched with controls for age, residence area, and index year. Lupus anticoagulant was found in 30 (17%) patients with ischaemic stroke, six (3%) patients with myocardial infarction, and four (0.7%) in the control group. The odds ratio for myocardial infarction was 5.3 (95% CI 1.4-20.8), which increased to 21.6 (1.9-242.0) in women who used oral contraceptives and 33.7 (6.0-189.0) in those who smoked. The odds ratio for ischaemic stroke was 43.1 (12.2-152.0), which increased to 201.0 (22.1-1828.0) in women who used oral contraceptives and 87.0 (14.5-523.0) in those who smoked. In women who had anti-beta(2)-glycoprotein I antibodies, the risk of ischaemic stroke was 2.3 (1.4-3.7), but the risk of myocardial infarction was not increased (0.9, 0.5-1.6). Neither anticardiolipin nor antiprothrombin antibodies affected the risk of myocardial infarction or ischaemic stroke. INTERPRETATION Our results suggest that lupus anticoagulant is a major risk factor for arterial thrombotic events in young women, and the presence of other cardiovascular risk factors increases the risk even further. FUNDING Netherlands Heart Foundation and Leducq Foundation.

Antiphospholipid antibodies promote leukocyte–endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APCs signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3β (GSK3β), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3β, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent Kd, ≈30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APCs ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3β, thereby contributing to APCs beneficial effects on cells.

Platelet activation by dimeric β2‐glycoprotein I requires signaling via both glycoprotein Ibα and apolipoprotein E receptor 2′

Summary.  Background: Dimerization of β2‐glycoprotein I (β2‐GPI) by autoantibodies is thought to trigger the clinical manifestations observed in the antiphospholipid syndrome. Arterial thrombosis, a frequently occurring clinical manifestation of the antiphospholipid syndrome, is a process in which platelets play a crucial role. Previous work has shown that binding of dimeric β2‐GPI to the platelet receptors apolipoprotein E receptor 2′ (ApoER2′) and glycoprotein Ibα (GPIbα) mediates increased platelet activation in an in vitro thrombosis model. Objective: The individual roles of ApoER2′ and GPIbα in mediating platelet activation by dimeric β2‐GPI has hitherto been unclear. In this study, we have determined the roles of either receptor in platelet activation by dimeric β2‐GPI. Methods: Platelet activation by dimeric β2‐GPI was studied under conditions of flow. Intracellular signaling induced by dimeric β2‐GPI was subsequently analyzed by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis. Results: The increase in platelet deposition onto a fibronectin surface under conditions of flow by dimeric β2‐GPI was completely abolished by inhibition of the interaction of dimeric β2‐GPI with either GPIbα or ApoER2′. Upon platelet stimulation with dimeric β2‐GPI, GPIbα translocated to the cytoskeleton via the scaffold protein 14‐3‐3ζ. Concomitantly, ApoER2′ dissociated from the adapter protein Disabled1, presumably through phosphorylation of the cytoplasmic tail. Inhibition of one process could not inhibit the other. Conclusion: We show that dimeric β2‐GPI signals via two distinct pathways in platelets, both of which are required for platelet activation. Abrogation of either signal results in loss of activation.

Platelet adhesion to dimeric β2‐glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibα and apolipoprotein E receptor 2′

Summary.  Background: The major antigen implicated in the antiphospholipid syndrome is beta2‐glycoprotein I (β2GPI). Dimerized β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized β2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized β2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized β2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized β2GPI was completely inhibited by the addition of soluble forms of both apoER2′ and GPIbα, and the addition of receptor‐associated protein and the removal of GPIbα from the platelet surface. GPIbα co‐precipitated with apoER2′, suggesting the presence of complexes between GPIbα and apoER2′ on platelet membranes. The interaction between GPIbα and dimeric β2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+‐dependent. Deletion of domain V from dimeric β2GPI strongly reduced its binding to both GPIbα and apoER2′. Antibodies that inhibit the binding of thrombin to GPIbα inhibited platelet adhesion to dimeric β2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbα had no effect. Dimeric β2GPI showed reduced binding to low‐sulfated GPIbα compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric β2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbα and apoER2′. These receptors are present in a complex on the platelet surface.

The significance of autoantibodies against β2-glycoprotein I.

The antiphospholipid syndrome (APS) is defined by the persistent presence of antiphospholipid antibodies in patients with a history of thrombosis and/or pregnancy morbidity, including fetal loss. APS is an autoimmune disease with a confusing name because the pathologic auto-antibodies are shown to be directed against the plasma protein β(2)-glycoprotein I and not against phospholipids. In fact, auto-antibodies that recognize phospholipids themselves are not associated with thrombosis but with infectious diseases. One of the intriguing questions is why autoantibodies against β(2)-glycoprotein I are so commonly found in both patients and the healthy. Several potential mechanisms have been suggested to explain the increased thrombotic risk in patients with these autoantibodies. In this overview, we will summarize our knowledge on the etiology of the autoantibodies, and we will discuss the evidence that identify autoantibodies against β(2)-glycoprotein I as the culprit of APS.

beta(2)-Glycoprotein I: a novel component of innate immunity

Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the hosts immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that β₂-glycoprotein I (β₂GPI) is a scavenger of LPS. In vitro, β₂GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of β₂GPI to LPS caused a conformational change in β₂GPI that led to binding of the β₂GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of β₂GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that β₂GPI is involved in the neutralization and clearance of LPS and identify β₂GPI as a component of innate immunity.

Interaction of ß2-glycoprotein I with members of the low density lipoprotein receptor family

Summary.  The antiphospholipid syndrome (APS) is a non‐inflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2‐glycoprotein I (β2GPI) bound to phospholipids. We have previously demonstrated that dimerization of β2GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2’ (apoER2′) a receptor belonging to the low‐density lipoprotein receptor (LDL‐R) family. Here, we show that dimeric β2GPI, but not monomeric β2GPI, interacts with four other LDL‐R family members: the LDL‐R related protein (LRP), megalin, the LDL‐R and the very‐low density lipoprotein receptor (VLDL‐R). Interaction between dimeric β2GPI and LDL‐R, apoER2′ and VLDL‐R was best described with a one‐site binding model (half‐maximal binding; ∼20 nm for apoER2′ and VLDL‐R and ∼300 nm for LDL‐R), whereas the interaction between dimeric β2GPI and LRP or megalin was best described with a two‐site binding model, representing a high‐ (∼3 nm) and a low‐affinity site (∼0.2 μm). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of β2GPI, which is known to disrupt the phospholipid binding site of β2GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric β2GPI can interact with different LDL‐R family members. This interaction is dependent on a binding site within domain V of β2GPI, which does not overlap with the phospholipid‐binding site within domain V.

The binding site in beta(2)-glycoprotein I for ApoER2 ' on platelets is located in domain V

The antiphospholipid syndrome is caused by autoantibodies directed against β2-glycoprotein I (β2GPI). Dimerization of β2GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2′ (apoER2′), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric β2GPI. To identify which domain of dimeric β2GPI interacts with apoER2′, we have constructed domain deletion mutants of dimeric β2GPI, lacking domain I (ΔI), II (ΔII), or V (ΔV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. ΔI and ΔII prolonged the clotting time, as did full-length dimeric β2GPI; ΔV had no effect on the clotting time. Second, ΔI and ΔII bound to anionic PL, comparable with full-length dimeric β2GPI. ΔV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric β2GPI, ΔI, or ΔII (mean increase 150%) were added to whole blood. No increase was found with plasma β2GPI, ΔV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric β2GPI, ΔI, ΔII, and the W316S mutant can interact with apoER2′ on platelets. ΔV did not associate with apoER2′. We conclude that domain V is involved in both binding β2GPI to anionic PL and in interaction with apoER2′ and subsequent activation of platelets. The binding site in β2GPI for interaction with apoER2′ does not overlap with the hydrophobic insertion loop in domain V.

Immune responses against domain I of β2-glycoprotein I are driven by conformational changes: Domain I of β2-glycoprotein I harbors a cryptic immunogenic epitope

OBJECTIVE The presence of autoantibodies against a cryptic epitope in domain I of β(2)-glycoprotein I (β(2)GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of β(2)GPI. Therefore, we investigated whether immune responses against β(2)GPI are related to its conformation. METHODS Conformational changes in β(2)GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of β(2)GPI in different conformations as well as the individual domains of β(2)GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of β(2)GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. RESULTS We observed that the interaction of β(2)GPI with cardiolipin induced a conformational change in β(2)GPI: electron microscopy revealed that β(2)GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine β(2)GPI in mice. Both human and murine β(2)GPI combined with cardiolipin and misfolded β(2)GPI triggered antibody formation against the native protein as well as against domain I of β(2)GPI, while native β(2)GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of β(2)GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russells viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. CONCLUSION The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in β(2)GPI can induce autoantibody formation.