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

• β2GPI-deficient mice are relatively protected from arterial, venous, and microvascular thrombosis.

• β2GPI-deficient mice display prolonged tail bleeding and reduced PAR3-dependent platelet activation by thrombin, both corrected by β2GPI.

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.