Journal of thrombosis and haemostasis : JTH · 2025
The factor (F)VIII K1693N mutation (FVIII-Nara) in a patient with moderate hemophilia A confers resistance to thrombin-catalyzed cleavage at Arg1689 involving P4' position.
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Furukawa Shoko, Baatartsogt Nemekhbayar, Kawamura Takeshi, Horiuchi Kaoru, Doi Masaaki, Kashiwakura Yuji, Ohmori Tsukasa, Nogami Keiji
Аннотация
BACKGROUND: Some patients with nonsevere hemophilia A (HA) are diagnosed with reduced factor (F)VIII activity (FVIII:C) relative to FVIII antigen (FVIII:Ag) and are classified as cross-reactive material-positive. We previously found a novel FVIII-Lys1693Asn (K1693N) natural mutation in a patient with HA with moderate phenotype (FVIII:C/FVIII:Ag = 1.9 IU/dL/124 IU/dL).
OBJECTIVE: To examine the mechanism(s) involving the P4' site on thrombin cleavage site at Arg1689 in a cross-reactive material-positive phenotype.
METHODS: FVIII-K1693N and its comparison variant K1693A were analyzed for t thrombin-mediated activation. FVIII-K376N, listed in the HA database due to a similar Lys-to-Asn substitution, was examined with K376A as its comparison. All mutants were expressed in BHK cells.
RESULTS: FVIII-K1693N showed delayed cleavage at Arg1689 and low peak FVIII:C, while FVIII-K1693A showed mildly reduced FVIII:C after thrombin activation. Mildly reduced peak FVIII:C and similar cleavage to wild-type was illustrated, however, with the FVIII-K1693A mutation after thrombin activation. These findings indicated that the conversion to Asn at P4' site mediated thrombin resistance. FVIII specific activities of FVIII-K376N and FVIII-K376A were 6.1%/3.1% (one-stage assay) of wild-type, respectively. Thrombin-catalyzed cleavage at Arg372 was comparable to wild-type with both mutants, but thrombin-catalyzed FVIII activation was severely-depressed. Spontaneous decay rates of FVIIIa activity with FVIII-K376N (k = 0.53) and FVIII-K376A (k = 0.99) were greater compared to wild-type (k = 0.32), suggesting that cleavage of P4' site following cleavage at Arg372contributed to A1-A2 domainal interaction.
CONCLUSION: Lys1693 and Lys376, both located at P4' sites, are suggested to influence FVIII function via distinct mechanisms, indicating that patient-derived mutations can reveal functional aspects of FVIII activation.
OBJECTIVE: To examine the mechanism(s) involving the P4' site on thrombin cleavage site at Arg1689 in a cross-reactive material-positive phenotype.
METHODS: FVIII-K1693N and its comparison variant K1693A were analyzed for t thrombin-mediated activation. FVIII-K376N, listed in the HA database due to a similar Lys-to-Asn substitution, was examined with K376A as its comparison. All mutants were expressed in BHK cells.
RESULTS: FVIII-K1693N showed delayed cleavage at Arg1689 and low peak FVIII:C, while FVIII-K1693A showed mildly reduced FVIII:C after thrombin activation. Mildly reduced peak FVIII:C and similar cleavage to wild-type was illustrated, however, with the FVIII-K1693A mutation after thrombin activation. These findings indicated that the conversion to Asn at P4' site mediated thrombin resistance. FVIII specific activities of FVIII-K376N and FVIII-K376A were 6.1%/3.1% (one-stage assay) of wild-type, respectively. Thrombin-catalyzed cleavage at Arg372 was comparable to wild-type with both mutants, but thrombin-catalyzed FVIII activation was severely-depressed. Spontaneous decay rates of FVIIIa activity with FVIII-K376N (k = 0.53) and FVIII-K376A (k = 0.99) were greater compared to wild-type (k = 0.32), suggesting that cleavage of P4' site following cleavage at Arg372contributed to A1-A2 domainal interaction.
CONCLUSION: Lys1693 and Lys376, both located at P4' sites, are suggested to influence FVIII function via distinct mechanisms, indicating that patient-derived mutations can reveal functional aspects of FVIII activation.