Vitamin K-dependent coagulation factors deficiency 1(VKCFD1)is a rare autosomal recessive bleeding disorder caused by mutations in the gamma-glutamyl carboxylase(GGCX)gene.Currently,the main treatment for VKCFD1 is vitamin K1,but there are significant variations in clinical outcomes due to its indirect targeting of GGCX.It is essential to identify drugs that target GGCX directly,and a crucial step in this process is to establish a drug evaluation system for GGCX.In this study,we utilized a cell line(DKO HEK293)with the vitamin K epoxide reductase(VKOR)gene knocked out and further knocked out endogenous GGCX using CRISPR/Cas9 technology(GCKO cell line).We then created a reporter cell line by stably expressing both the pathogenic GGCX mutant(I532T)and either coagulation factor X(FX)or anticoagulant protein C(PC).Based on this cell line,a preliminary screening of drugs targeting GGCX was conducted by measuring the carboxylation efficiency of FX and PC.The results showed that sanguinarine and homoharringtonine hindered cell proliferation and nearly completely blocked the carboxylation of FX and PC.Protopine,allantoin,and cis-aconitate significantly increased it and without affecting cell proliferation.In addition,genipin,diosmin,sennoside A,and(+)-catechin hydrate significantly enhanced FX carboxylation activity,while 2-phenylacetamide and thiamine nitrate notably boosted PC carboxylation levels.It demonstrates that the screening system for bleeding or clotting drugs targeting GGCX established in this study can provide efficient and timely feedback on the effects of the carboxylation of FX and PC,providing powerful tool and reference for screening drugs for the treatment of VKCFD1.
VKCFD1γ-glutamyl carboxylaseDrug screeningELISABleeding or clotting disorders
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