Protection of β2GPI-deficient mice from thrombosis reflects a defect in PAR3-facilitated platelet activation

Abstract

Antibodies to β2-glycoprotein I (β2GPI) cause thrombosis in antiphospholipid syndrome; however, the role of β2GPI in coagulation in vivo is not understood. To address this issue, we developed β2GPI-deficient mice (Apoh-/-) by deleting exons 2 and 3 of Apoh using CRISPR/Cas9 and compared the development of thrombosis in wild-type (WT) and Apoh-/- mice using rose bengal- and FeCl3-induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and activation of platelets from WT and Apoh-/- mice in the absence and presence of β2GPI. Apoh-/- mice demonstrated prolonged time to occlusion of the carotid after exposure to rose bengal or FeCl3 and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Significantly smaller thrombi formed in the IVC of Apoh-/- mice 48 hours after IVC occlusion. The activated partial thromboplastin time and prothrombin time, as well as activated partial thromboplastin time reagent and tissue factor-induced thrombin generation times, of Apoh-/- and WT plasma revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh-/- mice and reduced P-selectin expression and fibrinogen binding to activated α2bβ3 on platelets from these mice after stimulation with thrombin; these changes were reversed by β2GPI. A protease activated receptor 3 (PAR3) antibody blocked thrombin-induced activation of WT platelets and the ability of β2GPI to restore thrombin-induced activation of Apoh-/- platelets. These studies demonstrate that β2GPI may promote platelet activation by enhancing the ability of PAR3 to present thrombin to PAR4.