Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

The Biochemical Journal
Fatbardha VarfajPhilip J Fay

Abstract

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 f...Continue Reading

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Citations

Mar 14, 2012·The Journal of Biological Chemistry·Jennifer P DeAngelisPhilip J Fay
Dec 12, 2012·Thrombosis and Haemostasis·Masahiro TakeyamaPhilip J Fay
Dec 17, 2009·Journal of Thrombosis and Haemostasis : JTH·E Castoldi, J Rosing
Oct 31, 2009·Journal of Thrombosis and Haemostasis : JTH·J L PlantierC Négrier
Jun 16, 2010·Haemophilia : the Official Journal of the World Federation of Hemophilia·M L S ChristiansenM Kjalke
Jun 8, 2007·The Journal of Biological Chemistry·Andrew J GaleJohn H Griffin
Sep 14, 2013·The Journal of Biological Chemistry·Hironao Wakabayashi, Philip J Fay
Jun 28, 2007·The Journal of Biological Chemistry·Jennifer L Newell, Philip J Fay
Aug 26, 2010·Journal of Thrombosis and Haemostasis : JTH·T SoedaM Shima

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