Identification of Protein C system-associated Thrombophilia Using an Endothelial Cell-based Thrombin Generation Assay

Abstract

Background: Defects in the Protein C (PC) system are associated with an increased risk of venous thrombosis, due to a failure to regulate thrombin production. The thrombin generation assay (TGA) may therefore be a useful tool to demonstrate the potential hypercoagulable phenotype of patients with such conditions. We previously showed that the introduction of a surrogate endothelial monolayer to the TGA allows the assessment of the endothelial-dependent PC pathway. Thus, by measuring thrombin generation (TG) kinetics ± these cells, the contribution of the PC anticoagulant system to TG can be assessed in a physiologically relevant manner. PC- or S-depleted (PCd, PSd), as well as factor V Leiden (FVL) plasmas all show an abnormal TG profile in this endothelial-based TG assay (EcTGA). Aims: We investigated whether the EcTGA was capable of detecting a TG abnormality in clinical samples having PC/PS deficiency or FVL. Methods: Residual plasma samples of 151 in-hospital patients tested for thrombophilic abnormalities in our Hemostasis and Thrombosis Clinical Lab were subjected to EcTGA. Results were expressed as % reduction in TG in the presence of endothelial cells. Normal (n=69), FVL, and PCd/PSd plasmas served as abnormal controls. Results: The use of therapeutic anticoagulation prior to sample collection precluded the acquisition of meaningful thrombograms in 125 samples. Of the remaining 26, 18 (69%) had a PC system-related defect. However, other hemostatic abnormalities (commonly an elevated FVIII) were also present, and only 4 (15%) had a unique defect. Abnormal TG profile was observed in 11 of 18 (61%) patients based on a % reduction of TG parameters (PT & ETP) falling below the 10th percentile cut-off derived from normals. Conclusions: EcTGA may be a useful global assay to screen for the presence of PC system-associated thrombophilic abnormalities. However, the frequency of anticoagulation and the “acute-phase” status of hospitalized patients could preclude analysis or affect assay sensitivity in this population.