PEGylated Domain I of Beta-2-Glycoprotein I Inhibits Thrombosis in a Chronic Mouse Model of the Antiphospholipid Syndrome

Abstract

Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS.

Keywords: PEGylation; antiphospholipid syndrome; beta-2-glycoprotein I; domain I; thrombosis.

Figure 1

Antiphospholipid antibody development in chronic mouse model of APS disease. CD1 mice immunized with β₂GPI produced significant titers of anti-β₂GPI (A) and aCL (B) over the 4-week period compared to corresponding OA immunized negative controls. At the time of thrombus surgery at week 4, aCL and anti-β₂GPI titers in positive control β₂GPI-immunized mice were similar to those in β₂GPI-immunized mice treated with non-PEG-DI, 20kDa PEG-DI and 40kDa PEG-DI prior to surgery.

Figure 2

Thrombus area, aortic and peritoneal macrophage tissue factor levels in chronic APS mouse model. Both thrombus area (A) and aortic TF (B) levels decreased significantly in β₂GPI-immunized mice treated with non-PEG-DI, 20kDa PEG-DI and 40kDa PEG-DI compared to β₂GPI-immunized positive control mice. The abrogated levels of thrombus development and aortic TF were similar to that seen in negative control mice. While there was a trend of decreased peritoneal macrophage TF (C) levels in all domain I treated mice, the noted reductions were not statistically significant. (*p<0.01, **p<0.001).