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

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.

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.

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.

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.

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.