Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.