GDKV-Induced Antiphospholipid Antibodies Enhance Thrombosis and Activate Endothelial Cells In Vivo and In Vitro

Abstract

AbstractAntiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). β2-Glycoprotein I (β2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with β2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of β2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274–Cys288 in the fifth domain of human β2GPI, which represents the phospholipid-binding site of β2GPI. The Abs generated had aPL and anti-β2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of β2GPI are thrombogenic and activate endothelial cells.