Discovery and characterization of an antibody directed against exosite I of thrombin

Abstract

Essentials• An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype.• This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk.• Characterization of the antibody showed that it specifically binds to thrombin exosite I.• A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. Summary. Background:We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L À1 . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. Objectives: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. Methods: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. Results: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. Conclusions: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L À1 , suggesting that the presence of a highaffinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.

Upstream versus downstream thrombin inhibition

“…They found an IgA paraprotein with antithrombin activity in a patient identified during screening for coagulation abnormalities [71]. Further characterization of this antibody showed that it specifically binds to thrombin exosite I and is not associated with a severe bleeding phenotype.…”

Thrombolysis, the only Optimally Rapid Reperfusion Treatment

“…In the presence of venous thromboembolism, the D-dimer level goes up to ≥5,000 ng/ mL, showing that the system has a signi icant reserve capacity. Only in a patient with an unusually potent auto-antibody to thrombin has the normal D-dimer concentration been reported to be nearer zero (12-32 ng/mL) [14], a inding that also con irms that fresh intravascular ibrin, whose formation was suppressed by the antibody, is the D-dimer's source.…”

An anticoagulant that does not cause bleeding – an abrupt stop on the road to the Holy Grail

“…A completely different approach for potential safe anticoagulation was identified by chance in a patient who presented with a traumatic subdural hemorrhage and greatly prolonged global plasma coagulation test results (prothrombin time, activated partial thromboplastin time, and thrombin time) due to an anti‐thrombin immunoglobulin A paraprotein . Testing of the antibody revealed a specific and high‐affinity interaction with the fibrinogen recognition site (exosite I) of thrombin.…”

“…With the promising in vitro and animal data, the failure of JNJ‐9375 in the TEXT‐TKR study comes as a surprise. Although it is not known what the explanation for this observation is, several reasons may be possible.…”

“…It may be that in situations with high tissue factor, JNJ‐9375 is less potent in inhibition of fibrin formation. An indication may have already been that the prothrombin time, a test initiated with a high concentration of tissue factor, was much less prolonged in the patient with the paraprotein than the thrombin time or activated partial thromboplastin time . The rat arteriovenous shunt model that demonstrated efficacy of JNJ‐9375 is a model mainly driven by contact activation, and may therefore not be representative of the situation during knee replacement.…”