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

Abstract

AbstractBackgroundAntibodies to β2-glycoprotein I (β2GPI) cause thrombosis in antiphospholipid syndrome, however the role of β2GPI itself in regulation of coagulation pathwaysin vivois not well understood.MethodsWe developed β2GPI-deficient mice(Apoh-/-)by deleting exon 2 and 3 ofApohusing CRISPR/Cas9 and compared the propensity of wild-type (WT) andApoh-/-mice to develop thrombosis 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 assessed platelet activation in WT andApoh-/-mice in the absence and presence of exogenous β2GPI.ResultsCompared to WT littermates,Apoh-/-mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl3, and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC ofApoh-/-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 fromApoh-/-and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding inApoh-/-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 notApoh-/-platelets, as well as the ability of β2GPI to restore the activation response ofApoh-/-platelets to thrombin. β2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP.ConclusionsIn 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 Pointsβ2GPI 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.