OC 15.3 - Multiplex quantitation of coagulation and fibrinolysis markers: an upcoming approach to unravel bleeding and thrombotic disorders

Background: A variety of laboratory tests are needed to define the pathology underlying a bleeding or thrombotic disorder in an individual patient. Despite the diagnostic tools available, the etiology of the disorder remains unexplained in most patients with a mild clinical phenotype.

Aims: To determine levels of plasma proteins involved in coagulation and fibrinolysis to obtain protein signatures underlying bleeding phenotypes.

Methods: A coagulation and fibrinolysis protein biomarker panel was composed based on the Tier 1 diagnostic gene list endorsed at the ISTH 2022, supplemented with common inhibitors and regulators of fibrinolysis and coagulation. Targeted mass spectrometry (MS) was performed with a bottom-up proteomics approach to determine the plasma levels of multiple target proteins in a single assay. Factor-deficient plasmas or plasmas derived from non-human species were used in calibration strategies for absolute protein quantitation in conventional (U/mL) or molar units.

Results: Twenty-eight proteins of the coagulation and fibrinolysis panel were quantified from plasma by parallel reaction monitoring (PRM) of 115 peptides in a single 44 minutes MS run. Peptide measurands for quantitation were selected based on theoretical characteristics (e.g. uniqueness and localization in the protein domain) and empirical data from unbiased plasma proteomics studies (e.g. detectability and inter-peptide agreement). VWF, platelet-derived PF4 and PBP, and the coagulation factors fibrinogen (α,β,γ), prothrombin, FV, FVII, FIX, FX, FXI, FXII, FXIIIA, FXIIIB, were quantified in plasma, whereas FVIII was below the limit of detection (± < 10 nM) (Figure 1). The plasma levels of antithrombin, protein C, protein S, TAFI and plasminogen as representative inhibitors and regulations of coagulation and fibrinolysis were determined by MS, but quantification of ADAMTS13 and TFPI required upfront enrichment from plasma.

Conclusion(s): Targeted proteomics enables multiplex quantitation of coagulation and fibrinolysis markers and can now be applied to study the clinical value of the all-in one diagnostic screening approach.