Protection of β2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation

Abstract

Background: Antibodies to β2-glycoprotein I (β2GPI) cause thrombosis in antiphospholipid syndrome, however the role of β2GPI itself in regulation of coagulation pathways in vivo is not well understood.

Methods: We developed β2GPI-deficient mice (Apoh ^-/- ) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh ^-/- mice to develop thrombosis using rose bengal and FeCl ₃ -induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh ^-/- mice in the absence and presence of exogenous β2GPI.

Results: Compared to WT littermates, Apoh ^-/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl ₃ , and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh ^-/- mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh ^-/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh ^-/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated α2bβ3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous β2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh ^-/- platelets, as well as the ability of β2GPI to restore the activation response of Apoh ^-/- platelets to thrombin. β2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP.

Conclusions: In mice, β2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations.

Key points: β2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis.β2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin.