Maarten Pennings

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.

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.

Contribution of Classic and Alternative Effector Pathways in Peanut-Induced Anaphylactic Responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.

Optimisation of lupus anticoagulant tests: should test samples always be mixed with normal plasma?

Coagulation factor deficiencies are thought to interfere with the detection of the phospholipid-dependent coagulation inhibitor known as lupus anticoagulant (LA). Treatment with vitamin K antagonists (VKA) in particular, is thought to preclude accurate LA assessment. For this reason, the procedure to detect LA includes a mixing test, in which coagulation factor deficiencies are corrected by mixing samples with an equal volume of normal plasma. Despite these mixing tests, interpretation of LA test results is considered difficult in patients receiving high intensity VKA treatment. As a result, VKA treatment is often temporarily discontinued to allow LA assessment. However, whether coagulation factor deficiencies influence LA test results is unclear. We found that neither deficiency of a single coagulation factor, nor a functional coagulation factor deficiency due to high intensity VKA treatment, resulted in false positive dRVVT- or APTT-based (silica clotting time; SCT) LA test results. LA was readily detected in unmixed samples from VKA-treated LA-positive patients with both dRVVT and SCT reagents. VKA treatment caused an underestimation of the strength of the LA with SCT reagents, but did not lead to misclassification of LA status. Although mixing with normal plasma during both screen and confirm tests allowed more accurate assessment of the strength of the LA with SCT reagents in samples with an international normalised >2.5, the mixing procedure itself lead to misclassification of LA in weakly positive samples from patients not treated with VKA. Based on these findings, we conclude that mixing studies are not necessary during LA-assessment.

Using Cached Functions and Constructors for Incremental Attribute Evaluation

This paper presents a technique for the efficient incremental evaluation of Attribute Grammars. Through its generality, the applied approach may be affective too in the evaluation of Higher order Attribute Grammars. Our approach is an extension of a simpler algorithm for incremental evaluation, where functions, corresponding to visit sequences, are cached. Consequently, attributes are now either found in the cache or they are recomputed, so there is no longer need to represent the attributed tree explicitly. We may share common subtrees, avoiding repeated attribute evaluation, thus solving a typical HAG problem.

Acute‐phase concentrations of soluble fibrinogen inhibit neutrophil adhesion under flow conditions in vitro through interactions with ICAM‐1 and MAC‐1 (CD11b/CD18)

Immobilized fibrinogen and fibrin facilitate leukocyte adhesion, as they are potent ligands for leukocyte MAC‐1 (CD11b/CD18). However, fibrinogen in its soluble form also binds to MAC‐1, albeit with low affinity. The level of soluble fibrinogen is increased during chronic and acute inflammation, but the function of this increase is unknown.

Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2,3 and 6 in a mouse model for peanut allergy

BackgroundThe relative contribution and the relation between individual peanut allergens in peanut allergic responses is still matter of debate. We determined the individual contribution of peanut proteins to B, T cell and allergic effector responses in a mouse model for peanut allergy.MethodsMice were immunized and challenged by oral gavage with peanut protein extract or isolated allergens Ara h 1, 2, 3 and 6 followed by assessment of food allergic manifestations. In addition, T cell responses to the individual proteins were measured by an in vitro dendritic cell-T cell assay.ResultsSensitization with the individual peanut proteins elicited IgE responses with specificity to the allergen used as expected. However, cross reactivity among Ara h 1, 2, 3 and 6 was observed. T cell re-stimulations with peanut extract and individual peanut proteins also showed cross reactivity between Ara h 1, 2, 3 and 6. Despite the cross reactivity at the IgE level, only Ara h 2 and 6 were able to elicit mast cell degranulation after an oral challenge. However, after systemic challenge, Ara h 1, 2 and 6 and to lesser extent Ara h 3 were able to elicit anaphylactic responses.ConclusionsAra h 1, 2, 3 and 6 sensitize via the intra-gastric route, but differ in their capacity to cause allergic effector responses. Interestingly, extensive cross reactivity at T cell and antibody level is observed among Ara h 1, 2, 3 and 6, which may have important implications for the diagnosis and therapy of peanut allergy. Awareness about the relative contribution of individual peanut allergens and cross reactivity between these allergens is of importance for current research in diagnostics and therapeutics for and the mechanism of peanut allergy.