Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site.
Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (K(m), apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V(max)) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P(1)Arg, and this property likely reflects the severity of the hemophilia A phenotype.
Nogami
Keiji
K
Department of Biochemistry and Biophysics, PO Box 712, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642, USA.
Zhou
Qian
Q
Wakabayashi
Hironao
H
Fay
Philip J
PJ
D019943:Amino Acid Substitution; D001120:Arginine; D002384:Catalysis; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism*; D015951:Factor Xa / Q000096:biosynthesis; D006467:Hemophilia A / Q000235:genetics; D006639:Histidine; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013917:Thrombin / Q000378:metabolism*
D006639:Histidine; D005169:Factor VIII; D001120:Arginine; D013917:Thrombin; D015951:Factor Xa