A Novel Protac®-Modified Thrombin Generation Assay To Identify Individuals at Higher Thrombotic Risk.

There is a need for good laboratory prothrombotic markers to identify individuals at high risk of venous thromboembolism (VTE). Thrombin generation (TG) estimation is an attractive test since thrombin is a vital enzyme in the clotting cascade, influenced by the interplay of nearly all the clotting factors. However, unmodified thrombinography is not sensitive enough to changes in the protein C (PC) pathway including deficiencies of PC, PS and FVLeiden (FVL) and may lack sensitivity to prothrombotic states. So far studies have focused on the addition of exogenous activated PC (APC) or thrombomodulin (TM) to the TG assay to investigate the contribution of the natural anticoagulant pathway. However, TM is still a relatively expensive research reagent and APC bypasses the activation step of endogenous PC and hence may be less sensitive to conditions like PC and PS deficiency.

We studied the effect of Protac® on TG. Protac® is a purified snake venom-extract that rapidly activates endogenous PC to APC.

We used the CAT™ to measure TG in a group of patients with inherited thrombophilia, their first degree family members with no detectable defect (‘normal’ FM’s) and control subjects. All analyses were done on platelet poor plasma using a tissue factor concentration of 5pM and 4μM phospholipids. The endogenous thrombin potential (ETP; or area under the curve) was calculated using the Thrombinoscope® software. All samples were tested with and without Protac®. The percentage (ETP%) was calculated using the formula: ETP + Protac / ETP − Protac x 100.

Protac® has a dose dependent inhibitory effect on the ETP. Initially we selected a concentration of 0.3U/ml Protac® to test a group of normal females and males (n=17 and 20 respectively). Males are more sensitive to Protac® than females (mean ± SEM 11.8±1.5% for males & 33.2±3.7% for females;P<0.0001). Therefore we analysed the thrombophilic cohort using 0.3U/ml Protac® for women and 0.2U/ml for men. We detected a clear difference between female controls (n=13) vs ‘normal’ FMs (n=13;P<0.05), and ‘normal’ FMs vs women with FVL (n=19;P<0.01). The ETP% was higher in ‘normal’ male FMs(mean 19.9%;n=19) when compared to their controls (mean 8.7%;n=7). The differences were significant between male controls and those with FVL (n=18;P <0.001). We observed a wide range in ETP% for both the FVL groups and the ‘normal’ FMs (eg in the male FVL group range = 24.8–76.3% and for their ‘normal’ FMs 2.2–48.6%).

We correlated the ETP&ETP% with the different standard thrombophilia tests. The ETP% correlates inversely with PC, PS, APCR and age, whereas there is a positive correlation between the ETP and FVIII, fibrinogen and age. The APCR test only showed a weak correlation with FVIII. Both the ETP% and the APCR tests predicted well for FVL (95%CI 10.4–27.9, n=47 ETP% and CI −1.3 – −0.9, n=43 APCR). We could also detect a weak correlation between FVIII and the ETP + Protac which was lost when the ETP was expressed as a ratio.

Unlike the APCR test, the Protac®-TG assay is sensitive to all the constituents of the PC pathway. Our results are consistent with reports showing that a positive family history of VTE is a risk factor for thrombosis despite negative standard thrombophilia tests. It may also help discriminate between individuals with FVL who have a higher prothrombotic risk than others with the same defect. Finally, this new assay is relatively cheap and easy to perform and is ideal for large studies.