Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor

Matthew Ndonwi,George J Broze,Amy E Schmidt,Sayeh Agah,S Paul Bajaj

doi:10.1074/jbc.m701908200

Abstract

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Highlights

Factor X (FX)2 and factor IX (FIX) are vitamin K-dependent glycoproteins that play key roles in blood coagulation [1, 2]
We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)induced coagulation
In FX and protein C, the Gla and the two EGF domains make up the light chain, whereas the serine protease domain makes up the heavy chain of the molecule [1]

Summary

EXPERIMENTAL PROCEDURES

Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) and H-DIle-Pro-Arg-p-nitroanilide (S-2288) were obtained from Diapharma Inc. (West Chester, OH). Primer pairs D/F and E/F were used for PCR of the FIX fragments for the construction of FIXPCGla and FIXPCGla/EGF1, respectively, from pRc/CMV vector containing FIXWT cDNA. The chimeric DNA constructs with either the Gla or Gla/EGF1 domains of FIX replaced with those of protein C were digested with HindIII and XbaI and ligated into pRc/ CMV expression vector. The apparent Ki for interaction of each FX or FIX protein to the FVIIa/relipidated TF (or to FXIa) was determined from its ability to inhibit the activation of [3H]FIX (prepared using plasma-derived FIX) as measured by a 3H-labeled activation peptide release assay. Experimental mixtures contained 50 nM [3H]FIX, 1 nM FVIIa, 100 pM relipidated TF, 50 ␮M PL, and various concentrations of the competing FX or FIX proteins in TBS/BSA/Ca2ϩ. We used the structure of the FVIIa/sTF complex at 1.6 Å resolution, in which these residues are well ordered and are in the electron density. Figures were generated using the Pymol molecular graphics system [45]

RESULTS

D36 N33 PC

DISCUSSION