Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

Abstract

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.

Fig. 1.

FV(a) is a cofactor for the activation of FXI by thrombin. (A) FXI (30 nM) was activated with thrombin (10 nM) in the presence 10 μM PS:PC:PE and vehicle, FVa (30 nM), FVIIIa (30 nM), HK (630 nM), or Protein S (150 nM) for 10 min at 37 °C. Reactions were stopped with hirudin (10 U/mL). The amount of formed FXIa was measured with Pefachrome XIa3371 as described in Materials and Methods. (B) Factor XI (30 nM) was activated with thrombin (10 nM) in the presence of 10 μM PS:PC:PE and vehicle, FV, or FVa. Samples were taken in time and reactions were stopped with hirudin (10 U/mL). The amount of formed FXIa was determined with Pefachrome XIa3371. (C) Factor XI (30 nM) was activated with thrombin (10 nM) in the presence of 10 μM PS:PC:PE and increasing amounts of FVa for 10 min at 37 °C. Reactions were stopped with hirudin (10 U/mL). The amount of formed FXIa was determined with Pefachrome XIa3371. (D) Factor XI (30 nM) was activated with thrombin (10 nM) in the presence or absence of FV (30 nM) and increasing concentrations of phospholipid vesicles for 10 min at 37 °C. The amount of formed FXIa was determined with Pefachrome XIa3371. (E) FXI (30 nM) was activated with thrombin (10 nM) in the presence of 10 μM PS:PC:PE and vehicle, FVa (30 nM), HK (630 nM), or PK (580 nM) for 20 min at 37 °C, as indicated. Reactions were stopped with hirudin (10 U/mL) and amounts of formed FXIa were determined indirectly, as described in Materials and Methods. Data are shown as mean ± SD for data from at least three experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

The FV(a)-dependent increase in the activation of FXI by thrombin enhances the coagulation reaction. FXI (30 nM) was activated with thrombin in the presence of 10 μM PS:PC:PE and vehicle or FVa (30 nM) for 10 min at 37 °C, after which reactions were stopped with hirudin (10 U/mL). Thrombin and FVa alone served as control. (A) Coagulation was initiated with diluted samples as described in Materials and Methods. Data are shown as mean ± SD for data from at least three experiments. (B) Thrombin generation was measured with the Technothrombin TGA kit as described in Materials and Methods. Graph shows a representative experiment. *P < 0.05.

Fig. 3.

Factor XI binds to factor V(a) with high affinity via multiple binding sites. Surface plasmon resonance studies were performed with either FV or FVa immobilized on C1 sensor chips. Binding of FXI (0, 3.1, 6.3, 12.5, 25, 50, 100 and 200 nM) to immobilized FV (A) or FVa (C) was assessed at a flow rate of 50 μL/ min at 25 °C. No binding of FXIa (100 nM) to either FV or FVa was observed (insets). Domains responsible for the interaction between FXI and FV (B) or FVa (D) were mapped with individual tPA-tagged apple domains of FXI (25 nM), designated A1FXI-tPa to A4FXI-tPA. Binding of constructs to immobilized FV or FVa was assessed as described in Materials and Methods. PK and its tPA-tagged apple4 domain (A4PK-tPA) served as controls. Data are expressed as mean ± SD in fmol bound analyte (FXI, PK, or construct) per fmol immobilized ligand (FV or FVa). Inset shows the obtained association (k_a) and dissociation (k_d) rates ± SD, as well as the affinity constants (K_D) ± SD for the interaction between FXI and FV or FVa. All binding experiments were performed at least three times. Representative SPR traces are shown.

Fig. 4.

FVa influences feedback activation of FXI in plasma. The role of FV in the activation of FXI by thrombin in plasma was investigated after depletion of prothrombin to prevent feedback activation. All plasmas were activated with 20 nM thrombin in the presence of 10 μM PS:PC:PE vesicles. Samples were taken in time and reactions were stopped with 10 U/mL hirudin. Factor Xa-AT complexes were measured as described in Materials and Methods. The generation of FXa-AT complexes after 120 s in control plasma was normalized to 100%. Under these conditions, 100% represents the formation of 0.27 ± 0.12% of the FXa-AT complex levels present in serum obtained after the full coagulation of recalcified normal plasma in glass tubes. The results displayed are the combined data of four separate experiments, each performed in triplicate. (A) Prothrombin-depleted pooled normal plasma and factor XI deficient plasma were activated with thrombin. (B) The role of FV was investigated in pooled normal plasma that was depleted of either protein S (ΔPS) or factor V (ΔFV). (C) FXI (30 nM) was activated with thrombin (10 nM) in the presence of vehicle, FVa (30 nM), or APC (5 nM) as indicated for 20 min at 37 °C. The amount of formed FXIa was determined indirectly as described in Materials and Methods. (D) Prothrombin-depleted pooled normal plasma was activated with thrombin in the presence or absence of APC (20 nM). (E) Prothrombin-depleted plasma of an individual homozygous for the G1691A mutation in the FV gene was activated with thrombin in the presence or absence of APC (20 nM). Data are depicted as mean ± SD. ***P < 0.001.