Quantitative Protein Mass Spectrometry to Assess Plasma Proteins Involved in Coagulation and Fibrinolysis

Half of all patients referred to a tertiary center for suspected bleeding disorders can’t be diagnosed with current diagnostic assays, which reveals the limitations of these assays. Hemostasis laboratories use screening assays that measure coagulation factor levels (APTT, PT, fibrinogen), but these aren’t precise enough to predict the interindividual variability in bleeding phenotype. Different tests produce inconsistent results and it’s unclear which one accurately reflects the clinical bleeding phenotype. This creates the risk of underdiagnosis or overdiagnosis. There is a need to enhance existing assays and create new tests to accurately diagnose bleeding disorders.

Quantitative protein mass-spectrometry (QPMS) is a new technology that characterizes and quantifies proteins in plasma. However, it’s complex and requires multiple optimization steps for clinical use. In collaboration with the University of Victoria (UVic), we’ve explored QPMS for the relative quantification of multiple coagulation and fibrinolysis proteins in plasma samples from healthy individuals and thrombosis patients. However, QPMS assays developed at UVic only cover part of the known coagulation and fibrinolysis proteins, and some clinically relevant proteins like VWF and FVIII produce poor results. The LUMC departments of Thrombosis & Hemostasis and Clinical Chemistry are now collaborating to improve QPMS assays and enable their clinical use. We’ve developed a test for antithrombin, and now aim to create a test that quantifies all coagulation and fibrinolysis-related proteins in one assay. We’ll integrate existing assays and develop novel tests for proteins that haven’t been included yet. The test will be analytically and clinically validated using plasma samples from healthy volunteers and patients with unexplained bleeding tendencies. We hope that this novel QPMS test, once validated, will be used as a second step in the diagnostic process for bleeding disorders and lead to a more precise and rapid diagnosis.