Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa.
APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.
Varfaj
Fatbardha
F
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Neuberg
Julie
J
Jenkins
P Vincent
PV
Wakabayashi
Hironao
H
Fay
Philip J
PJ
D024342:Amino Acids, Acidic / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002478:Cells, Cultured; D006224:Cricetinae; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D024342:Amino Acids, Acidic; D011486:Protein C; D021122:Protein Subunits; D011994:Recombinant Proteins; D015944:Factor VIIIa; D001120:Arginine