Prothrombotic PROC variant rebalancing hemostasis in severe hemophilia A with attenuated bleeding risk

Severe hemophilia A (HA), defined by a clotting factor [F]VIII level of <1%, leads to a severe bleeding tendency. A pathogenic F8 variant p.His300Lysfs∗41 accounted for <1% FVIII levels in a patient with HA; however, he exhibited a discordantly mild-to-moderate bleeding phenotype.

To identify and characterize the hemostatic impact of co-inherited genetic modifiers in a HA patient exhibiting a clinical phenotype discordant with their FVIII level.

Multigene sequencing to detect possible genetic modifiers identified a heterozygous PROC variant (p.Trp414Arg). Plasma protein C (PC) activity and antigen were measured. Recombinant wild-type and mutant PC were produced using Expi293F cells with function and quantity measured by chromogenic activity assays and competitive ELISA. Protein structure was predicted with AlphaFold 3. Thrombin generation assays in FVIII-deficient plasma with varying PC concentrations were used to mimic PC deficiency in HA.

PC activity in the patient's plasma was 58% (clotting-based) and 53% (chromogenic), and PC antigenic concentration was 59%. Mutant PC in transfection supernatant demonstrated concordant marked reduction in PC activity (0% vs 90%) and quantity (4% vs 100%) compared with wild-type PC. Thrombin generation assays demonstrated increases in endogenous thrombin potential and peak height upon addition of thrombomodulin in PC-deficient vs PC-replete FVIII-deficient plasma.

The partial quantitative PC deficiency in the patient resulted from a heterozygous pathogenic PROC p.Trp414Arg variant. Quantitative reduction in PC improved thrombin generation in FVIII-deficient plasma upon addition of thrombomodulin. Thus, this PROC variant was predicted to attenuate bleeding severity in HA by improving thrombin generation. Multigene panel sequencing permits the identification of genetic modifiers that result in naturally rebalanced hemostasis in inherited bleeding disorders.