Cofactor‐independent human antiphospholipid antibodies induce venous thrombosis in mice

Abstract

Cofactor‐independent antiphospholipid antibodies (CI‐aPL) are generally considered non‐pathogenic. We analyzed the effects of human monoclonal CI‐aPL in a mouse model of venous thrombosis. As shown in vitro, CI‐aPL induce a procoagulant state in vivo by activation of endosomal NADPH‐oxidase. Contrary to common belief, CI‐aPL induce venous thrombosis in vivo. Summary BackgroundThere is general consensus that the antiphospholipid syndrome (APS) is caused by antiphospholipid antibodies (aPL) with antibodies against β2‐glycoprotein‐I being the most relevant. aPL that bind phospholipids in the absence of protein cofactors are generally considered pathogenetically irrelevant. We showed that cofactor‐independent human monoclonal aPL isolated from APS patients induce proinflammatory and procoagulant cellular responses by activating endosomal NADPH‐oxidase 2 (NOX2). Similar aPL were detected in all IgG fractions from APS patients analyzed. ObjectivesWe aimed to clarify if cofactor‐independent aPL can be thrombogenic in vivo and, if so, whether these effects are mediated via activation of NOX2. MethodsTwo cofactor‐independent human monoclonal aPL, HL5B and RR7F, were tested in a mouse model of venous thrombosis. Genetically modified mice and in vitro assays were used to delineate the mechanisms underlying thrombus induction. ResultsHL5B and RR7F dramatically accelerate thrombus formation in this mouse model. Thrombus formation depends on tissue factor activation. It cannot be induced in NOX2‐deficient mice. Bone marrow chimeras of C57BL/6J mice reconstituted with NOX2‐deficient bone marrow showed that leukocyte activation plays a major role in thrombus formation. Neither TLR4 signaling nor platelet activation by our aPL is required for venous thrombus formation. ConclusionsCofactor‐independent aPL can induce thrombosis in vivo. This effect is mainly mediated by leukocyte activation, which depends on the previously described signal transduction via endosomal NOX2. Because most APS patients have been shown to harbor aPL with similar activity, our data are of general relevance for the APS.

Hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal NADPH oxidase

“…We have shown that activation of endosomal NOX2 by aPL greatly accelerates thrombus formation in this model. While wild type C57BL/6J mice rapidly develop venous thrombi when exposed to aPL, gp91phox-deficient mice are protected 25. We show now that HCQ can also prevent aPL-induced thrombus formation in this in vivo model.…”

“…Indeed, ability of certain aPL to induce venous thrombosis in vivo depends on NOX2 25. In our hands, aPL induced a more rapid and intense activation of endosomal NOX than TNFα or IL-1β reflected in a much stronger ROS signal.…”

“…In this mouse model, HL5B and a similar monoclonal aPL, RR7F, massively accelerate venous thrombus formation. This effect is fully dependent on activation of NOX2 as it is absent in gp91phox-deficient mice 25. Pretreatment of the mice with HCQ significantly reduced HL5B-induced venous thrombus formation (figure 6A, B).…”

“…The in vivo model of thrombus formation in the inferior vena cava (IVC) used in this study has been previously described in detail 25 34. It is based on severe flow reduction in the IVC.…”

Antiphospholipid syndrome: an update for clinicians and scientists

“…On this front, particular attention has been given to the cell-surface TLRs, TLR2 and TLR4. In mouse models, TLR4 deletion protects against venous and arterial thrombosis in some [31–33], but not all [34]*, studies (it is worth pointing out that the latter study utilized cofactor-independent aPL). Studies of obstetric APS have also yielded mixed results with an older study demonstrating no role for TLR4 in an in vivo model of pregnancy loss [35].…”