Severe Acquired Bleeding Tendency Secondary To An Anti-Thrombin Antibody In a Patient With Monoclonal Gammapathy Of Unknown Significance: Direct In Vivo evidence
Authors: Proulle Valerie , Desconclois Celine , Lavenu-Bombled Cecile , Goujard Cecile , Sharda Anish V , Turhan Ali G 
Published: October 2019

Abstract

Introduction
Anti-thrombin antibodies are a rare cause of acquired bleeding disorder. We report a case of a patient with a monoclonal gammapathy of unknown significance (MGUS) referred to us for an acquired bleeding tendency. We found that he had an isolated anti-thrombin antibody and showed that this antibody inhibit platelet accumulation and fibrin generation in vivo in a mouse model of arterial thrombus formation.

Patient
A 40-year-old man with no personal or familial history of bleeding was referred to us for multiple bleeding episodes (rectal bleeding, hematoma) for 18 years. He was diagnosed 12 years previously with an IgG Kappa MGUS which was stable. Complete blood counts were normal. Activated partial thromboplastin time (APTT) and thrombin time (TT) were prolonged at 76 seconds (control 35) and > 120 seconds (control 18) respectively. The prolonged TT was observed using human and bovine thrombin. A TT mixing study was performed by measuring the TT of serial dilutions of patient’s plasma in normal pooled plasma. This study confirmed an acquired anti-thrombin activity of patient’s plasma, as TT of the mix was prolonged from dilutions 1:1 to 1:16 (>120 to 25 seconds). Other coagulation factors were normal. No other auto-antibodies were detected except an isolated lupus anticoagulant. Thrombin generation in patient’s platelet rich plasma measured using the Calibrated Automated Thrombogram (CAT) showed a prolonged lag time (15.5 versus 4.7 minutes) associated with a decreased endogenous thrombin potential (ETP, 834 versus 1730 nmol/L min). The diagnosis of an acquired anti-thrombin antibody associated with a MGUS was proposed. The patient received multiple treatments, including immunosuppressive agents, anti-CD20 monoclonal antibody therapy and bortezomib, in order to eradicate the antibody with no success.

Methods
Anti-thrombin IgG antibodies were affinity purified from the patient’s plasma using activated agarose beads covalently coupled to human alpha thrombin (Aminolink, ThermoScientific). A mouse model of thrombosis was used to test the effect of the infusion of the purified anti-thrombin IgG on thrombus formation in vivo. Platelet accumulation and fibrin generation at the site of injury was imaged after laser-induced injury of cremaster arterioles using intravital microscopy in wild type mice. Platelet and fibrin labeling were performed using anti-CD42 antibody conjugated to DyLight 488 (Emfret) and an anti-fibrin antibody conjugated with AlexaFluor 647 (Sekisui).

Results
Immunoblotting using human and bovine alpha-thrombin thrombin confirmed the purified IgG from the patient’s plasma (yield 86 µg/ml) to be anti-thrombin antibodies. At a concentration of 2.45 mg/mL, these anti-thrombin IgG were able to prolong APTT and TT (59 versus 33 seconds and >120 versus 19 seconds, respectively). Control IgG (2.5 mg/mL) purified from healthy individuals had no effect on APTT and TT. Using the mouse model of laser-induced arteriolar injury, we found that patient-derived anti-thrombin IgG (690 microg) caused a significant inhibition of platelet accumulation and fibrin generation of more than 70% at the site of vessel wall injury.

Conclusion
We report a case of severe acquired bleeding tendency secondary to an anti-thrombin antibody in a patient with an IgG Kappa MGUS. Anti-thrombin IgG antibodies purified from the patient’s plasma retain the anti-thrombin activity in vitro. Additionally, infusion of purified anti-thrombin IgG from the patient induced a dramatic decrease in platelet thrombus accumulation and fibrin generation at the site of injury in a mouse model of arterial injury, proving the etiology of patient’s bleeding tendency. This is the first description of the anti-thrombotic effect of anti-thrombin antibodies directly in vivo.

Disclosures:
Turhan: BMS, Novartis: Honoraria, Research Funding.