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Z. Hao et al.
AB
CD
Figure 3. Contribution of propeptide’s conserved and non-conserved residues to reporter-protein carboxylation. Effect of conserved residues at -6 and -10 of the propeptide of bone Gla protein (BGP) (A) and factor X (FX) (C) on the carboxylation of reporter-proteins as evaluated by cell-based assay. Chimeric reporter-proteins with wild-type or mutant propeptides were transiently expressed in HEK293 cells and the carboxylated reporter-protein was determined by ELISA, as described in B. (B) Propeptide sequence of FX and the replacement of part of its sequence with that of protein C (PC) (highlighted in red). Highly conserved residues are highlighted in yellow. (D) Carboxylation of the reporter-protein directed by chimeric propeptide sequences of FX and PC as indicated in (B).
Effect of naturally occurring propeptide mutations on coagulation factor carboxylation
Naturally occurring mutations have been identified at positions -1, -4, -9 and -1025-30 in the propeptide of coagu- lation factors. Patients bearing mutations at positions -9 and -10 of FIX propeptide possess near-normal levels of active FIX; but, these levels are reduced to <1% of normal during anticoagulation therapy (referred to as warfarin hypersensitivity).27,28,31-33 Meanwhile, other VKD clotting factor levels only decreased to 30-40%. To examine the effect of these mutations on coagulation factor carboxyla- tion in a cellular milieu, we introduced these mutations to FIX propeptide in our reporter-protein for cell-based func- tional study. Our results show that, despite significant dif- ferences in GGCX affinity in vitro,25 these mutations have only a moderate effect on reporter-protein carboxylation in a cellular environment (Figure 4A), which is consistent with clinical observations of patients bearing these muta- tions that have near-normal levels of active FIX.
To clarify the warfarin hypersensitivity phenotype dur- ing anticoagulation therapy of patients carrying these mutations, we examined the response of the mutant
reporter-proteins’ carboxylation to increasing concentra- tions of warfarin. We stably expressed the individual mutant or wild-type reporter-protein in HEK293 cells and examined their carboxylation efficiency under different warfarin or vitamin K concentrations. Results showed that, compared with the wild-type reporter-protein, the N-9K mutant has a moderate effect on warfarin inhibition, while the warfarin response curve of the A-10T and A- 10V mutants significantly shifts to lower concentrations of warfarin (Figure 4B), suggesting that they are more sensi- tive to warfarin inhibition. The half-maximal inhibition concentration (IC50) of warfarin for the A-10T and A-10V mutants decreased 11.6-fold and 4.5-fold, respectively (Figure 4C), which is consistent with a recent similar cell- based study.26 As warfarin blocks the vitamin K recycling, we also examined the effect of vitamin K on the carboxy- lation of these mutant reporter-proteins. We determined the half-maximal effective concentration (EC50) of vitamin K for the carboxylation of these mutant reporter-proteins. Compared to the wild-type reporter-protein, the A-10T and A-10V mutants required a significantly higher concen- tration of vitamin K (5.8-fold and 2.9-fold, respectively) to
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haematologica | 2020; 105(8)