Background. The very first case of TTP reported by Eli Moschcowitz in 1924, was a 16 year old girl, presenting with fever and petechia (hematoma). Within a week she developed paralysis of her left arm and leg and died. Autopsy revealed multiple small thrombi, the hallmark of TTP in arterioles and capillaries in heart, kidneys, liver and spleen. Today mortality rate is still 20% with a high relapse rate of 40-50%. TTP is the result of a severe deficiency of ADAMTS13 - the vonWillebrand Factor (VWF) cleaving protease, causing the accumulation of ultralarge VWF multimers in the microciruclation leading to the observed blood clots and ultimatively to organ ischemia and dysfunction. There are two forms of TTP: (1) hereditary with homozygous or double heterozygous ADAMTS13 gene mutations, and (2) acquired due to anti-ADAMTS13 autoantibodies neutralizing enzymatic activity. Plasma exchange with fresh frozen plasma with the goal to administer ADAMTS13 and to remove neutralizing anti-ADAMTS13 antibodies is the therapy of choice for this life-threatening disease.
Aims. Studying the pathophysiological mechanisms underlying the severely deficient(Part I) or moderately reduced ADAMTS13 activity (Part II) linked to systemic thrombosis in TTP and beyond.
Experimental set-up. (Part I) We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing acute TTP in order to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary genetic and functional characterization of the patient’s entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells, revealed that the spleen harbors a distinct set of B-cells producing neutralizing anti-ADAMTS13 antibodies, explaining the success of splenectomy, as both patients are in remission for more than 6 years. Detailed sequence analysis most extraordinarily revealed that the ADAMTS13-binding sites (Fab) of the 29 monoclonal antibodies generated from both patients, are characterized by seven unique and novel antigen-binding motifs (Complementarity determinig region 3, CDR3). Strikingly, four of these motifs were shared by both patients. As both patients are neither genetically nor geographically related and taking into account that the probability of two B-cells to produce very similar specific antibodies is less than 10-9, our findings strongly suggest that these motifs are part of the key antigen-binding sites of neutralizing anti-ADAMTS13 antibodies present in relapsing acquired TTP. My goals are twofold, first I aim to confirm these findings in the spleen of two additional relapsing acquired TTP patients and second, to use these anti-ADAMTS13 antibodies as tools to screen a small protein library (DARPin) for high specific binders blocking the neutralizing antibodies.
(Part II) Clinicial consequences include myocardial infaction, stroke or pregnancy related complications such as preeclampsia/HELLP, which are more prevalent in the general population compared to rare TTP disease. In these conditions, there is often an inverse correlation between VWF and ADAMTS13, with elevated VWF levels and midly to moderately reduced ADAMTS13 activites. My goal is to better understand how ADAMTS13 and VWF contribute to these disease using more comprehensive near-physiological systems and ADAMTS13 genotyping of suitable patient groups to evaluate their risk to develop cardiovascular diseases.
Expected impact. Our preliminary findings regarding the unique set of CDR3 signatures that characterize the pathological relevant, neutralizing antibodies paves the road to define their triggering ADAMTS13 epitope(s). Identification of small molecules that neutralize pathogenic anti-ADAMTS13 antibodies are ideal tools to develop new diagnostic tests and therapeutic treatments for acute TTP. Moerever, I hypothesize that unraveling the pathophysiological mechanisms leading to ADAMTS13 deficiency can serve as a model to understand micro- and macrovascular thrombosis in general, beyond the TTP setting.