Factor XI Activation In Tissue Factor-Initiated Blood Coagulation

In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa>TF>FIXa>FXa>α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (<0.01% zymogen activation) increases the amount of FIXa available during the activation of FVIII, yielding higher concentrations of intrinsic tenase earlier in the initiation phase. The second consequence was increased FIXa accumulation as the reaction proceeds. This prediction was confirmed in two ways: 1) Western blot analysis of SCP reactions containing FXI showed ∼20% consumption of FIX over 20 min compared to no detectable FIX consumption in reactions without FXI; and 2) Resupply of TF-initiated SCP reactions (± 30 nM FXI) demonstrated that both the FVIII dependence and the long-term stability of the observed re-initiation of thrombin generation depended on the presence of FXI. Resupply studies were then performed in contact pathway inhibited blood (N=4 individuals) by using an inhibitory anti-FXI antibody to negate FXI contributions to both the TF-initiated and resupply dynamics. No effect of blocking FXI function on the time courses of αTAT formation was observed in the TF initiated phase. Two of the four individuals showed attenuated αTAT formation upon resupply when FXI function was blocked from the onset of TF initiation. Conclusions. FXIa is a potent initiator of coagulation, and the computational and SCP analyses predict the potential for feedback activation by thrombin after TF initiation to somewhat enhance the propagation phase of thrombin generation and more dramatically enhance the resupply response. However in contact pathway inhibited blood, FXI contributions to Tf-initiated thrombin generation are not discernible and the effect on the resupply response is variable between individuals suggesting additional mechanisms suppressing FXI activation in blood.

Mann:Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.