PF796 THE EFFECT OF ANTI‐BETA2‐GLYCOPROTEIN I ANTIBODY UPON THROMBIN GENERATION IN NORMAL, LEIDEN HETEROZYGOUS AND FACTOR DEFICIENT PLASMAS

Abstract

Background:The antiphospholipid syndrome (APS) is an acquired hypercoagulable condition, in which venous or arterial thromboses are caused by the presence of antiphospholipid antibodies (aPL). Anti‐β2glycoprotein I (anti‐β2GPI), highly correlating with the clinical manifestation of the syndrome, is the most significant aPL. Much evidence has uncovered insights into its molecular mechanisms, the exact molecular or cellular events are still not delineated.Aims:Our aim was to examine the effect of anti‐β2GPI antibodies from APS patients on pooled normal plasma (NP), Leiden heterozygous plasma (LP) and factor deficient plasmas (DP).Methods:Serum samples were collected form triple positive (anti‐β2GPI, anticardiolipin, lupus anticoagulant) APS patients with thrombosis in their history. Anti‐β2GPI IgG was purified in a two‐step affinity chromatography by Protein G and β2GPI affinity columns. Purified anti‐β2GPI IgG was added to NP, LP, or DP in thrombin generation test (TGT) up to 125 U/mL concentration in the presence of 1 pM tissue factor and 4 μM phospholipid.Results:In TGT, time (Lag Time) and quantity parameters (Peak and Endogenous Thrombin Potential [ETP]) were assessed. Having lupus anticoagulant effect, anti‐β2GPI showed significant prolongation in Lag Time in pooled NP (n = 9) by 24% (p < 0.001). Despite the fact that anti‐β2GPI delayed thrombin generation in time, it significantly increased ETP by 11% (p < 0.001). We assessed thrombin generation in LP samples without antibodies. Thrombin generation in LP began at the same time as that of the NP, there was no difference in Lag Time, but surprisingly Peak and ETP values were significantly lower than in NP (ETPNP: 1467 nM∗min, ETPLP: 920 nM∗min, p < 0.001), less thrombin was generated in LP. Anti‐β2GPI was able to exert similar effects on LP as in NP. Lag Time was delayed by 36% (p < 0.001), there was a non‐significant increase in ETP, but the Peak thrombin value was elevated by 32% (p < 0.05, n = 6). The effect of anti‐β2GPI was investigated on FVII, FIX, FXI and FXII deficient plasmas. Similar tendency was seen in intrinsic factor deficient plasmas, Lag Time was prolonged, Peak and ETP was elevated, but anti‐β2GPI could not exert significant effect on any of the deficient plasmas. However, the lack of FVII completely prevented the effect of anti‐β2GPI on thrombin generation.Summary/Conclusion:Anti‐β2GPI delayed thrombin generation in time, while it enhanced the amount of thrombin generated, which may in part explain the lupus anticoagulant paradox. In plasma from Leiden heterozygous patients, thrombin generation was also enhanced by anti‐β2GPI. In intrinsic factor (FIX, FXI, FXII) deficient plasmas, similar tendency was seen as in normal or Leiden plasmas, which was non‐significant, but FVII seems a crucial mediator in the prothrombotic effect of anti‐β2GPI antibodies.