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Objective— Recent evidence suggests involvement of coagulation factor XIa (FXIa) in thrombotic event development. This study was conducted to explore possible synergies between tissue factor (TF) and exogenous FXIa (E-FXIa) in thrombin generation. Approach and Results— In thrombin generation assays, for increasing concentrations of E-FXIa with low, but not with high TF concentrations, peak thrombin significantly increased whereas lag time and time to peak significantly decreased. Similar dependencies of lag times and rates of thrombin generation were found in mathematical model simulations. In both in vitro and in silico experiments that included E-FXIa, thrombin bursts were seen for TF levels much lower than those required without E-FXIa. For in silico thrombin bursts initiated by the synergistic action of TF and E-FXIa, the mechanisms leading to the burst differed substantially from those for bursts initiated by high TF alone. For the synergistic case, sustained activation of platelet-bound FIX by E-FXIa, along with the feedback-enhanced activation of platelet-bound FVIIIa and FXa, was needed to elicit a thrombin burst. Furthermore, the initiation of thrombin bursts by high TF levels relied on different platelet FIX/FIXa binding sites than those involved in bursts initiated by low TF levels with E-FXIa. Conclusions— Low concentrations of TF and exogenous FXIa, each too low to elicit a burst in thrombin production alone, act synergistically when in combination to cause substantial thrombin production. The observation about FIX/FIXa binding sites may have therapeutic implications.

Hypercoagulability resulting from elevated prothrombin is associated with increased thrombosis risk. Patients with elevated prothrombin include those with the G20210A mutation that represents the 2nd most common risk factor for venous thrombosis in European Caucasians. We hypothesized that the commercial thrombin generation assay (TGA), which measures the rate of synthetic thrombin substrate consumption, may detect procoagulant condition caused by elevated prothrombin. Normal pooled plasma was treated with prothrombin or other coagulation factors to increase their levels to 200% or 400% of normal. TGA was performed using the Calibrated Automated Thrombogram (CAT) platform (Stago, USA) and analyzed with and without correction for substrate consumption. As expected, addition of elevated coagulation factors resulted in elevated TGA as measured by thrombin peak height (TPH) and endogenous thrombin potential (ETP). ETP allowed detection of up to 83% of spiked samples with 200% prothrombin but TPH has failed to distinguish these samples from the procoagulant samples obtained with other coagulation factors. For samples with 400% prothrombin, CAT failed to return values for TPH for 40% of samples and ETP for 100% of samples but the remaining 60% of TPH values were detected as the procoagulant samples. By analyzing the raw fluorescence data directly, we found that elevated prothrombin, but not other coagulation factors, resulted in thrombin substrate depletion which allowed us to correctly distinguish all samples within our dataset regarding whether or not they had added prothrombin. We conclude that the commercially available TGA has limited utility in distinguishing samples with elevated prothrombin but additional analysis of data can address this problem. With this additional analysis, TGA may be developable into new point-of-care test to diagnose patients with hypercoagulant conditions undetectable by currently used clotting assays. Disclaimer: This is an informal communication and represents authors’ best judgment. These comments do not bind or obligate FDA.