Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation.

Abstract 3652

A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin.

The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site.

We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin.

Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms.