Background: The positive factor Va/VIIIa/IXa feedback reactions by which thrombin propagates its own production are essential to robust clot formation, which is underscored by the fact that defects or deficiencies therein lead to hemophilia. We have previously demonstrated that these feedback loops are regulated in a highly interdependent manner, as absence of the factor VIII/IX-dependent feedback results in premature inactivation of factor V (FV) by activated protein C (APC). Interestingly, APC-resistant FV was shown to have the potential of counterbalancing this secondary defect by impairing the APC system and correcting thrombin generation.
Aim: Here we examined whether the factor XI (FXI)-mediated feedback loop is also tightly linked to FV regulation by assessing if APC-resistant FV can overcome the attenuated coagulation in FXI deficiency.
Methods: Constitutively active FV-810 (Pro811-Gy1491 deleted) and FV-810-QQ, in which the main APC cleavage sites Arg306 and Arg506 are substituted by Gln, were expressed and purified. Coagulation was initiated by low tissue factor, and thrombin activity and tPA-induced fibrinolysis were assessed.
Results: Consistent with its bleeding phenotype, thrombin generation was reduced in FXI deficiency. Surprisingly, whereas triggering the protein C pathway by adding thrombomodulin (TM, 10 nM) or APC (2 nM) partially reduced thrombin formation in normal plasma, thrombin production became fully abolished in FXI-deficient plasma. This suggests that, similar to deficiencies in the FVIII/FIX-dependent feedback loops, a lack of FXI activity renders the plasma more sensitive to the APC system. APC-resistant FV could counterbalance this defect, as addition of FV-810-QQ (1 U/ml) increased thrombin formation substantially in FXI-deficient plasma, whereas FV-810 had no effect. In a clot lysis assay, addition of increasing TM concentrations (0-10 nM) confirmed enhanced sensitivity to the APC system as clot formation was severely prolonged in FXI-deficient plasma (40 min. vs. 5-6 min. in normal plasma). Even though fibrinolysis was inhibited by TM-mediated thrombin-activatable fibrinolysis inhibitor (TAFI) activation in both plasmas, the maximum inhibition attained in FXI deficiency was much lower as compared to normal, concurring with previous observations. Subsequent supplementation with FV showed that, being constitutively active, both variants normalized the clot time in FXI-deficient plasma. Clot lysis times, on the other hand, were fully restored upon adding FV-810-QQ to plasma lacking FXI activity; addition of FV-810 showed no effect in both plasmas. This implies that APC-resistant FV may contribute to enhanced TAFI activation, thereby bypassing the need for upstream feedback activation reactions. This was supported by assessing the clot lysis time using varying concentrations of a TAFI inhibitor (carboxypeptidase inhibitor), which demonstrated that supplementation of FXI-deficient plasma with FV-810-QQ increased activated TAFI to the level of normal plasma.
Conclusions: These data show that similar to deficiencies in the FVIII/FIX-dependent feedback loop, a defect in the thrombin-mediated FXI activation pathway leads to a dramatic reduction in thrombin formation due to action of the protein C pathway. Whereas constitutively active normal FV can partially restore impaired coagulation, its APC-resistant counterpart has the potential of rescuing both clot formation and stability, thereby providing a potential alternative for hemophilia treatment.