1.  Abstract

Recently, we reported the first example of a monoclonal anti-thrombin IgG in a patient with multiple myeloma.  On several occasions, when the paraprotein concentration reached ~5 g/dl, the patient experienced life-threatening bleeding and had marked prolongation of the thrombin time and the activated partial thromboplastin time (aPTT).  The bleeding subsided after the IgG concentration was reduced to <2 g/dl by plasmapheresis, accompanied by partial correction of the thrombin time without an appreciable change in the aPTT.  We found that the monoclonal IgG bound to exosite II of thrombin and altered the kinetics of hydrolysis of synthetic peptide substrates.  In this study, we investigated the mechanism by which low concentrations of the monoclonal IgG prolong the thrombin time and the aPTT of plasma.  Although the IgG did not affect cleavage of fibrinogen by thrombin, it increased the rate of inhibition of thrombin by purified antithrombin ~3-fold.  Experiments with plasma immunodepleted of antithrombin or heparin cofactor II confirmed that prolongation of the thrombin time required antithrombin.  By contrast, prolongation of the aPTT required neither antithrombin nor heparin cofactor II.  The IgG delayed clotting of plasma initiated by purified factor IXa but had much less of an effect on clotting initiated by factor Xa.  In a purified system, the IgG decreased the rate of activation of factor VIII by thrombin.  These studies indicate that binding of a monoclonal IgG to exosite II prolongs the thrombin time indirectly by accelerating the thrombin-antithrombin reaction.  In addition, the IgG may prolong the aPTT by interfering with activation of factor VIII, thereby diminishing the catalytic activity of the factor IXa/VIIIa complex.  These observations suggest that exosite II of thrombin might be a suitable target for novel anticoagulant agents.