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Defining the antithrombin measurand: role of proteoforms in harmonisation of diagnostic tests in thrombosis

Periodic Reporting for period 1 - HarmonizATforms (Defining the antithrombin measurand: role of proteoforms in harmonisation of diagnostic tests in thrombosis)

Reporting period: 2019-05-01 to 2021-04-30

Proteins are complex molecules that may exist in multiple molecular forms. Thus far, most clinical chemistry tests target all of the molecular forms, without differentiating the actual ones present in an individual. This holds true for the underappreciated antithrombin (AT) activity test as well. Antithrombin exists in many molecular forms, caused by >350 known genetic variants, as well as heterogeneity in protein glycosylation. Both of these are known to affect AT activity in various ways, resulting in variable phenotypes associated with AT deficiency. However, these phenotypes cannot be distinguished using current activity tests. This results in insufficient clinical sensitivity of the test, an improperly defined target population, and sometimes inconclusive results. Alternative tests that can distinguish molecular forms of AT are needed to provide more clinical insight. To conclude, within this project we developed an alternative test that allows for characterization of AT at the molecular level, and validated the method analytically according to current clinical chemistry practice. We now aim to further assess whether our method can aid in harmonization of current AT tests, but more importantly, will also assess whether our test provides increased clinical sensitivity and/or could identify subgroups of patients with AT deficiency that might benefit from specific therapy.
Within this project, we aimed to develop a clinical chemistry test that characterizes AT at the molecular level, analytically validate the method and assess the level of agreement between activity tests, antigen based tests and our molecular test. Mass spectrometry (MS) is a technology that allows for the quantitation of proteins in a multiplexed manner at the molecular level. We used a so-called bottom up proteomics approach, in which the protein of interest is enzymatically digested into representing peptides. These peptides are then quantified based on their specific mass relative to an internal standard. However, if molecular variation in specific aminoacids exists, this is also reflected in their mass, and thus observable by the MS technology. We were able to develop a method that allows for 1. quantitation of AT in molar units, 2. identification of most of the important mutations, 3. distinction of the alpha and beta form of AT based on the glycosylation, and 4. generally altered AT glycosylation. The method was then thoroughly analytically validated according to clinical chemistry guidelines for its precision, linear range, LoD and LoQ, stability and specificity. To assess the potential for harmonization of AT activity tests using our method, AT standards were characterized using intact protein mass spectrometry. However, the standards proved not to be suitable for our intended purpose. We are currently collaborating with the EQA organizer ECAT to develop alternative standards and samples to assess the behaviour of specific well-characterized AT materials in a number of CE-IVD marked AT tests.

Based on this work we were able to present to researchers and clinicians at several (online) conferences, including the annual symposium of the American Society for Mass Spectrmetry, The Dutch Society for Clinical Chemistry and in July 2021 the International Society for Thrombosis and Hemostasis. Moreover, based on our work, we have initiated collaborations with the University of Murcia on the characterization of AT in clinical samples, and within the LUMC, the department of internal medicine, with whom we collaborate on the application of the development strategy established in our work on other proteins in the coagulation cascade. The work has thus far resulted in 2 peer reviewed publications, and we were also able to guide 3 bachelor and master students during their final internships. Due to the COVID pandemic, and the not yet clinical applicability of the current results, the outreach of to the general public has been limited.
The aim of the project was to develop a novel test to quantify and characterize AT. This test would then first be used to aid in harmonization of already existing tests. We envision that this will allow for the characterization of materials to be used as EQA materials, so that we will be able to understand the causes of currently sometimes inconclusive results. However, gynaecologists at our university medical center knew of our development, and we were already able to provide insight in a clinical case of a patient with recurrent pregnancy loss. We therefore believe that a second line of research should already focus on the clinical validation of our test and how the use of this test as an add-on test could provide benefit for patients (e.g. those suffering recurrent pregnancy loss). As such, the current clinical pathway for AT testing is analytically not sensitive enough, and screening of AT deficiency in individuals with a family history of VTE or who have suffered a VTE is therefore not clinically effective. We envision that our molecular definition of AT will provide a more sensitive test, which hopefully will allow screening for AT deficiency in the previously mentioned populations.
Besides the direct impact on antithrombin testing, we are now also involved in collaborations addressing the biochemical functionality of antithrombin as well as the expansion of the developed method to other proteins in the coagulation cascade and fibrinolytic system.
Overview of accomplishments and future aims and impact