Plasmin to the rescue in thrombotic thrombocytopenic purpura (LSBR 1520)
Project leader: Dr. Coen Maas, Laboratory for Clinical Chemistry and Haematology, UMCUtrecht
Postdoc researcher: Steven de Maat (Nov. 2016 – Dec. 2019)
Thrombotic thrombocytopenic purpura (TTP) is a rare but devastating autoimmune disease. It is caused by antibodies that neutralize ADAMTS13. This enzyme prevents thrombosis of the microvasculature by continuously “trimming” the multimeric protein von Willebrand Factor (VWF). In its absence (as occurs in TTP), VWF multimers are larger than normal and more thrombogenic as a result.
During TTP attacks, blood platelets clump together with VWF and form microthrombi that occlude the microvasculature. These small blood vessels are needed to provide oxygen and nutrients to important organs of the body, including the heart, brain and kidneys.
The golden-standard treatment of these acute attacks is plasma exchange with the aim to restore ADAMTS13 levels and activity. The problem is that the antibodies in the blood of patients also render the ADAMTS13 in the donor plasma ineffective. As a consequence, lengthy plasma transfusion therapy is required to alleviate symptoms. But in the mean time, the longer an attack persists, the more tissues become damaged: time is tissue.
An exciting new development is a therapeutic antibody fragment that blocks the binding of blood platelets to VWF. Although this agent prevent formation of new microthrombi, it was not developed to disrupt microthrombi that are already jamming the microvasculature.
We previously identified that besides ADAMTS13, another enzyme is capable of cleaving VWF. This enzyme is called plasmin and we identified that stimulating this action shows promise in preclinical experimental models for TTP. We discovered that it is active during TTP attacks, but apparently insufficiently capable of fully preventing disease. Taken together, we interpret this role of plasmin as a backup protector against VWF-driven thrombosis when ADAMTS13 is not completely able to fulfil its role.
In this project, we further explored the role of plasmin, with the ultimate aim of developing a therapy to disrupt microthrombi for rapid restoration of blood flow to essential organs. We first identified that that the role of endogenous plasmin during TTP is protective in nature. In preclinical experimental models for TTP, we found that blocking the function or activation of plasmin worsened disease symptoms, while stimulating its activation improved the outcomes.
We next reasoned that the natural role for plasmin to degrade VWF may have an impact on patient diagnostics: in diagnostic tests for TTP, a chemical substance (FRETS-VWF73) is used that resembles VWF and used to detect ADAMTS13 activity. We found that not only ADAMTS13, but also plasmin is able to react with this substance. This means that when plasmin is present in plasma, it could interfere with detection of ADAMTS13. We subsequently designed a solution to this diagnostic problem. During this research, we surprisingly identified that there is a link between plasmin and ADAMTS13. When both are present in plasma, plasmin stimulates ADAMTS13 to do a better job. This may have impact on our understanding of the clinical picture of patients during TTP attacks. The relationship between ADAMTS13 activity and disease severity is not 1:1. The identification of an additional molecular player sheds a new light on this topic.
Finally, we set out to develop a therapeutic protein molecule for the sole purpose of quickly disrupting microthrombi that jam the microvasculature. We call this protein Microlyse. Its design is shown to the left; a first part (nanobody) has the function to stick to VWF. It is connected via a linker to an enzyme that triggers plasmin to become active. In this way, plasmin activity is generated in a concentrated manner at the site where microthrombi occlude the vasculature and cause disease. We have done the full biochemical analyses, and confirmed the function of this agent in test tube (in vitro) models for TTP. We next went on to confirm its activity in preclinical experimental (in vivo) studies. This work is nearing completion and we hope to bring this invention to the next stage of clinical development.