Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X.

P.F Neuenschwander,J.H Morrissey

doi:10.1016/s0021-9258(17)37152-1

Abstract

The roles of the putative membrane-interactive regions of factor VIIa (fVIIa) and tissue factor (TF) have been examined. Enzymatic removal of the 4-carboxyglutamic acid (Gla) domain of fVIIa had no effect on hydrolysis of a tripeptidyl chromogenic substrate in the absence or presence of TF. Additionally, Gla-domainless fVIIa (GdVIIa) was similar to native fVIIa in activating factor X in the absence of TF and phospholipid. However, GdVIIa in complex with recombinant soluble TF (sTF) was 76-fold less efficient in factor X activation than was fVIIa.sTF. The difference increased to 740-fold using TF relipidated in vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine (TF/PCPS). While Gla domain deletion produced a 10(3)-fold increase in the Kd for binding to TF/PCPS, the Kd for binding to TF/PC increased only 20-fold, and that for sTF in the absence of phospholipid increased 10-fold. Kd values for GdVIIa binding to TF/PCPS, TF/PC, or sTF were nearly identical. Thus, most of the binding energy required for formation of the fVIIa.TF complex was present even after Gla domain deletion. Both fVIIa and GdVIIa were capable of binding sTF in the presence of excess divalent metal-ion chelator, suggesting Ca(2+)-independent binding or the presence of a novel very high affinity Ca2+ binding site in fVIIa. The results demonstrate that the effect of the Gla domain on the Kd is apparent only in the presence of PS, and that interactions involving the fVIIa Gla domain and phospholipid are critical for efficient proteolysis of factor X on a membrane surface.

Highlights

THE FACTOR VIIa Gla DOMAIN IS DISPENSABLE FOR BINDING TO TISSUE FACTOR BUT IMPORTANT FOR ACTIVATION OF FACTOR X*
It has been demonstrated that the factor VIIa (fVIIa) (GdVIIa) wassimilar to native fVIIa in activating fVIIa.tissue factor (TF) complex requires a phospholipid surface for optimal factor X in the absence of TF and phospholipid
Roles of the membrane-interactive regions of fVIIa and TF were examined with respect to their effects on (i) amidolytic activity of fVIIa and fVIIa.TF, (ii) proteolytic activity of M I a and fVIIa.TF, (iii) bindingof fVIIa to TF, andCa2+ binding properties of M I a and MIa.TF

Summary

MembrIannteeractions of FaVcItoIar and Tissue Factor

Using solution-phase kinetic methodws,e report thatmost of described in detail elsewhere. Reported concentrationsof relipidated the binding energy in association of fVIIa with TF is derived wild-type TF arefrom values obtainedby saturation of relipidated TF from protein-protein interactionsratherthan from proteinphospholipid interactions. Values for a and p were determined from plots of extracellular domainof TF, amino acids219 as numberedby Monis- amidolyticactivity versus enzymeconcentration for freeandbound sey et al.;Ref. 27) was expressed in bacteria and purified as described enzyme, the latter in thperesence of saturating amountsof T F or sTF. This ensured complete removalof Gla domain-contain- complexes were inferred from fits of activity data with a quadratic ing material. MIa between residues and Ser (as numberedby Hagen et al.; where M I a , and TF, are the initial added levels of M I a and TF, Ref. 33) to generate GdVIIa. Determination of ProteinConcentrations-Concentrations of Ma, (either M I a or TF; see "Results("F)o.r clarity, square brackets denoting. MIa, and GdVIIa were determined by titrating active sites with p - nitrophenyl-p'-guanidinobenzoate as described [34, 35].

SCHEMI E

RESULTS

Membrane Znteractions of Factor VIZa and Tissue Factor

DISCUSSION