ADAMTS13 structure and the molecular basis of VWF recognition and cleavage

Blood clotting occurs in response to blood vessel damage. This requires the specific recruitment of platelets (specialised blood cells) to the site of injury as one of the first (of many) events that prevents bleeding. This process is highly dependent upon a protein known as von Willebrand factor (VWF) that circulates in blood. The ability of VWF to perform this task is regulated by an enzyme that is also present in the blood, ADAMTS13, and that, under very single substrate specific circumstances, cleaves VWF into smaller forms that are less capable of recruiting platelets.
Clinically, deficiency in VWF is the most common inherited bleeding disorder, whereas people with ADAMTS13 deficiency suffer from a life-threatening thrombotic disorder with a ~90% mortality rate. More subtle differences in the blood levels/function of VWF and ADAMTS13 are also important determinants of an individual's risk of bleeding and thrombosis, and also influence the likelihood of both heart attack and stroke.
ADAMTS13 is a very highly specific proteolytic enzyme that cleaves only one protein (VWF) and does so at just a single site, and even then, only under very specific conditions of blood flow. ADAMTS13 is made up of multiple domains. The metalloprotease domain of this enzyme contains the active site that cleaves VWF, whereas the other variably contribute to the binding of ADAMTS13 to VWF. Despite this knowledge, how ADAMTS13 recognises and cleaves VWF so specifically remains unclear. To understand this at a molecular level, we will ascertain the structure of different domains fragments of ADAMTS13, both in free forms and in stabilising complexes with specific antibody fragments that can aid in determining structures. In addition, we will also elucidate the structure of ADAMTS13 fragments whilst bound to the corresponding fragments of VWF. We will characterise the binding and cleavage of these VWF fragments by ADAMTS13 and also explore the influence of calcium binding to this process.
The information from this project will provide important insights into how ADAMTS13 functions at a molecular level its unique single substrate specific cleavage of VWF.
This data will provide the opportunity to rationally engineer ADAMTS13 to improve its efficacy as a therapeutic agent, for which it is currently under development as a more specific clotbuster for the treatment of thrombotic disease.

Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that is critical for platelet recruitment to sites of blood vessel damage. VWF platelet-tethering function is highly dependent upon its multimeric size, with larger VWF multimers being more haemostatically competent. VWF plasma multimeric size, and consequently its platelet-tethering function, is regulated in the blood by proteolytic processing by the plasma metalloprotease, ADAMTS13. Deficiency in VWF causes the most common inherited bleeding disorder, whereas people with ADAMTS13 deficiency suffer from life-threatening thrombotic thrombocytopenic purpura. Increasing VWF and decreasing ADAMTS13 levels in plasma are risk factors for both myocardial infarction and stroke.
ADAMTS13 is very highly specific as it cleaves only one known substrate protein, VWF, and does so at just a single site - between Tyr1605 and Met1606 in the VWF A2 domain. Physiologically, proteolysis can only occur when VWF is unravelled by shear as the scissile bond and cryptic exosites in VWF are only revealed when VWF unravels.
The holy grail of research into ADAMTS13 and VWF is to understand the complex mechanism of VWF force-induced VWF unfolding and highly single substrate specific cleavage by ADAMTS13. To address this, we will express, purify and characterise a panel of ADAMTS13 domain fragments to understand their interaction with and proteolysis of VWF and the importance of Ca2+ binding in this process. We will also determine the crystal structures of these fragments, in isolation, in complex with anti-ADAMTS13 Fab fragments and in complex with VWF. The results from these studies will not only provide detailed information upon ADAMTS13 function, it will provide insight in to thrombotic disease, provide insight into the structure and function of other ADAMTS family members, and perhaps more importantly provide the opportunity to rationally modify/improve ADAMTS13 for use as a therapeutic agent.

The beneficiaries of the research, beyond the immediate field of haemostasis research include;
1. Pharmaceutical companies involved in production of thrombolytic agents for the treatments of cardiovascular disease (Boehringer Ingelheim, Chiesa, Genetech) and also companies that produce recombinant plasma proteins (Biogen Idec, Novo Nordisk, Bayer, Baxter and CSL Behring) may also be interested in ADAMTS13. The pharmaceutical industry will benefit through the potential to rationally modify and develop ADAMTS13 as a therapeutic agent. Numerous haemostatic proteins have been used or trialled in the setting of disease. Those that have proved efficacious have benefited appreciably through rational modification to improve their production, function, bioavailability, stability. These include, factor VIII (haemophilia A), factor IX (haemophilia B), factor VIIa (bleeding), activated protein C (sepsis and stroke), t-PA (stroke, MI, PE), VWF (VWD). To rationally modify a protein, it is essential to understand its structure and function. To know which parts of the molecule are important (or redundant), and what the limiting steps in its production and function are central to facilitating this. Baxter is currently beginning trials of recombinant ADAMTS13 in the setting of TTP. Work from this proposal will provide a molecular structure of ADAMTS13 in isolation and in complex with its substrate. Importantly, this will provide the opportunity to improve the wild-type molecule through targeted addition of glycans to sites not involved in its function to reduce immunogenicity in inherited TTP, reduce inhibition and clearance in acquired TTP, and to reduce ADAMTS13 clearance and so prolong plasma half-life (improving bioavailability, reducing dosing/frequency of dosing).
2. Patients with TTP, paediatric stroke or ischaemic stroke. A British Heart Foundation project awarded to Dr Crawley and Prof Lane is examining a gain-of-function ADAMTS13 variant that improves its function. That project is exploring the efficacy of the ADAMTS13 variant in the setting of a murine model of ischaemic stroke. Such modifications have the potential to reduce the number and/or frequency of dosing and our results will improve the likelihood of novel therapies coming to market.
3. Clinicians in the field of haemostasis will benefit should ADAMTS13 prove as efficacious in humans as early data from mice/rats suggest through the provision of an additional therapeutic strategy to treat patients with TTP and ischaemic stroke, two serious life-threatening diseases. Both diseases require rapid emergency treatment to ameliorate the symptoms. Current treatments are limited by time to onset of action, as well as potentially serious adverse side effects. Indeed, the vast majority of ischaemic stroke patients are not treated with t-PA due to them missing the therapeutic window for its use and the elevated risk of bleeding. In this way, patients with these diseases will also have to potential to benefit. If ADAMTS13 and/or rationally modified derivatives thereof come into clinical practice, patients will benefit from improved efficacy, reduced/less frequent dosing, and reduced exposure to other therapies with harmful side-effects.
Ultimately, the goal of this research beyond the academic community is to provide benefit for patients. If modification of ADAMTS13 is capable of improving efficacy whilst simultaneously enabling reduced dosing (and in turn reducing costs), this could be of major benefit.
With respect to timescales, we envisage the opportunity to start designing/testing rationally modified ADAMTS13 in vitro within 18 months (the approximate time that we would start consultation with the pharmaceutical industry). Benefits to patients and clinicians will potentially be observed in the longer term, due to the requirement for rigorous testing and trialling prior to the availability of a new therapy. This would likely be a time frame of 10-12 years.