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A Multiplexed Targeted Proteomics Test to Quantitatively Assess Plasma Proteins Involved in Coagulation and Fibrinolysis

In half of all patients, referred to a tertiary center because of a suspected bleeding disorder, it is not possible to establish a diagnosis using current diagnostic assays, underlining the incompleteness of these assays. Currently, hemostasis laboratories use screening assays to assess hemostasis based on coagulation factor levels (APTT, PT, fibrinogen), these are however not sensitive enough to predict interindividual heterogeneity in bleeding phenotype. Various assays (one stage, chromogenic) give discrepant results and it is unknown which test reflects clinical bleeding phenotype best. This holds the risks of underdiagnosis (and undertreatment) or overdiagnosis (and overtreatment). There is an unmet need to improve available assays and to develop novel tests for more precise diagnosis of bleeding disorders.

Mass spectrometry-based quantitative targeted proteomics (MS-QTP) is a novel technology for the characterization and absolute quantitation of proteins (and their proteoforms) in plasma. MS-QTP relies on enzymatic digestion of proteins of interest into peptides, which are subsequently quantified in a targeted manner. However, MS-QTP is complex and requires multiple steps of optimization to allow for clinical chemistry application. In collaboration with the University of Victoria (UVic), we started exploring the use MS-QTP at a discovery level. Using plasmas from healthy individuals and thrombosis patients, we demonstrated multiplexed relative quantification for 12 coagulation and fibrinolysis proteins in one single run from <5µL plasma (Mohammed et al. 2017). With UVic we now apply MS-QTP for multiplexed relative quantitation of large protein panels (proteins from multiple pathways) in our ‘discovery’ cohort studies (Tilburg et al. 2020, Mohammed et al. 2021). While useful for unbiased evaluation of plasma proteins, MS-QTP assays developed at UVic cover only part (~50%) of known coagulation and fibrinolysis proteins. Moreover, absence of, or a poor signal with high analytical imprecision is encountered for some of the proteins, notably the clinically relevant VWF and FVIII. Thus, the full potential of the powerful MS-QTP technology for the analysis of coagulation and fibrinolysis proteins is not utilized yet. Recently, the LUMC-depts Thrombosis & Hemostasis and Clinical Chemistry initiated a collaboration to analytically improve the current MS-QTP assays, to enable absolute quantitation, and importantly, clinical application. Thus far, an in-depth MS-QTP test was developed for antithrombin enabling quantitation of the protein and its clinically relevant proteoforms (Ruhaak et al. 2018, Kruijt et al. 2021). We now follow a comparable in-depth approach to create a test that permits rapid quantitation of all coagulation- and fibrinolysis-related proteins in one assay that ultimately allows for clinical chemistry application. Hereto, we will integrate and improve the existing assays, with novel valid assays for VWF and FVIII, as well as proteins that were not included yet in the original panel (>20 proteins). Analytical and initial clinical validation of the test is performed using plasma samples from healthy volunteers and bio-banked samples from patients suffering from (unexplained) bleeding tendency. We envision, that this novel MS-QTP test, upon validation, could be used as a second step assay in the current diagnostic work-up of bleeding disorders and ultimately allows for rapid and more precise diagnosis.