The role of the factor X activation peptide: a deletion mutagenesis approach.
To understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fX (des-137-183) and fX(des-137-183;N191A), which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell's viper venom (RVV-X(1)). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 +/- 0.004 micro M(-1) s(-1)). Both mutants were also found to be activated by fVIIa (0.31 +/- 0.03 micro M(-1) s(-1)), fIXa (0.30 +/- 0.03 micro M(-1) s(-1)), and thrombin (0.00078 +/- 0.00015 micro M(-1) s(-1)). In all cases, the rate of activation was faster for fX(des-137-183;N191A) as compared to fX(des-137-183). We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.
Rudolph
Amy E
AE
Department of Pathology, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. amy.e.rudolph@pharmacia.com
Mullane
Michael P
MP
Porche-Sorbet
Rhonda
R
Daust
Heather A
HA
Miletich
Joseph P
JP
D000595:Amino Acid Sequence; D002241:Carbohydrates; D002384:Catalysis; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015951:Factor Xa / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D013925:Thromboplastin / Q000378:metabolism
D002241:Carbohydrates; D008081:Liposomes; D010455:Peptides; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa