Impact of Natural Variation in Coagulation Factor Zymogens on Contact Pathway-Initiated Thrombin Generation

Contact pathway inhibitors are emerging as potential therapies for venous thromboembolism with favorable bleeding risk compared to conventional agents. However, the relative importance of individual coagulation zymogens to contact pathway-initiated coagulation is unknown. Prior studies have been limited to evaluating associations between contact and intrinsic pathway zymogen levels with activated partial thromboplastin times (aPTTs). The utility of findings from aPTT-based measurements is likely limited by the reliance of this assay on high concentrations of trigger reagent and a single functional output. Calibrated automated thrombography (CAT) has emerged as a highly sensitive and titratable technique with which to evaluate contact pathway-initiated thrombin generation (TG). The aim of this study was to determine the impact of natural variation in contact pathway zymogen levels on both aPTT and CAT-based measurements of coagulation. Citrated plasma was obtained from healthy donors (n=60). Plasma levels of PK, FXII, FXI, FIX, FVIII and C1INH antigen were measured by commercial enzyme linked immunosorbent assays validated using congenitally deficient or immunodepleted plasmas. TG was measured in the healthy donor plasma by CAT using a submaximal concentration of silica nanospheres as a trigger. aPTT was measured using an automated hematology analyzer. In additional experiments, plasma levels of individual contact and intrinsic pathway factors were varied by reconstituting congenitally deficient plasmas with increasing levels of purified zymogen (0-400% of normal) with contact pathway-initiated TG measured as described above. In healthy donor plasmas, significant associations between aPTT and zymogen levels of PK (r = -0.35, P<0.01), FXII (r = -0.27, P<0.05), and FXI (r = -0.34, P<0.01) but not FVIII, FIX or C1INH were observed. Interestingly, significant associations between contact pathway-initiated TG parameters and levels of all zymogens analyzed, except for PK, were observed. In particular, C1INH was significantly associated with peak TG (r = -0.32, P<0.05) and endogenous thrombin potential (ETP) (r = -0.4 P<0.01). Significant associations were also observed between TG and zymogens for C1INH targets FXIIa and FXIa. In particular, FXII was correlated with peak (r=0.42, p<0.001), velocity (r=0.44, p<0.001) and ETP (r=0.3, p<0.01). FXI was correlated with lag time (r=-0.25, p<0.05), peak (r=0.35, p<0.01), time to peak (r=-0.27, p<0.05), and velocity (r=0.4, p<0.01). To further evaluate the impact of variation in a single zymogen, additional reconstitution experiments were conducted. As observed in the healthy donor plasmas, varying levels of PK had no significant effect on contact pathway-initiated TG. In contrast, a strong dose-dependent relationship between plasma levels of FXII, FXI, FIX, and FVIII and contact pathway-initiated TG were observed. Half maximal effective concentration (EC50) values for ETP were 22.5% for FXII, 5.5% for FXI, 12.4% for FIX, and 25.7% for FVIII. EC50 values for velocity were 25.3% for FXII, 9.3% for FXI, 30.9% for FIX, and 68.2% for FVIII. Our findings indicate that CAT-based measurements of TG have improved sensitivity over the aPTT when evaluating associations with coagulation factor zymogen levels in healthy individuals. Notably, this is the first study to report a significant association between levels of FXII, FXI and their endogenous inhibitor C1INH and TG in the general population. Further, from the CAT-based measurements it is apparent that variation in individual zymogen levels differentially affects contact pathway-initiated TG.