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A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

In vitro studies have documented β2 glycoprotein I (β2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of β2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled β2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The β2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar β2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after β2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that β2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes.

Recombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion.

We monitored the number of intravascular platelet-leukocyte aggregates (PLAs) and thrombotic occlusions (TOs) by intravascular microscopy in the mesentery of rats receiving antiphospholipid (aPL) immunoglobulin G (IgG) purified from the sera of patients with antiphospholipid syndrome. aPL IgG had no procoagulant effect, but it caused rapid endothelial deposition of fibrinogen, followed by PLA and TO in rats receiving an intraperitoneal injection of bacterial lipopolysaccharide 3 hours before IgG infusion. Anti-β2-glycoprotein I-depleted aPL IgG failed to induce PLAs and TOs. C3 and C9 colocalized with aPL IgG on the mesenteric vessels. The number of PLAs and TOs was markedly reduced in C6-deficient rats and in animals treated with anti-C5 miniantibody, suggesting the contribution of the terminal complement (C) complex to the aPL antibody-mediated intravascular thrombosis. In conclusion, our data indicate that antibodies to β2-glycoprotein I trigger coagulation subsequent to a priming proinflammatory factor and that the terminal C complex is the main mediator of the coagulation process.